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Wound Lavage in Studies on Vital Pulp Therapy of Permanent Teeth with Carious Exposures: A Qualitative Systematic Review. The aim of this study was to systematically review pulp wound lavage in vital pulp therapy (VPT). A search was conducted in six life science databases to identify clinical trials carried out on permanent teeth with a carious pulp exposure and a recall interval of at least six months. Twenty-seven trials of low to moderate risk of bias (RoB-2 and ROBINS-I) were included. Data was extracted and analyzed regarding study characteristics and methods used for pulp wound lavage. The agent used for pulp wound lavage was specified in all included trials. Most of the identified trials (23/27) randomized the pulp capping material. Many (14/27) reported the use of sodium hypochlorite (NaOCl); ten used only saline or water. One trial was identified that compared pulp wound lavage with 2.5% (NaOCl) to saline, another compared 5% glutaraldehyde to water, both in immature molar pulpotomies. Both studies were underpowered. Neither showed a significant difference between treatments. The use of NaOCl was positively correlated to recent year of publication and use of hydraulic calcium silicate cements for pulp capping (p < 0.05). In conclusion, despite a lack of well-designed trials on pulp wound lavage in VPT, a trend towards using NaOCl for this purpose was observed. Introduction Vital pulp therapy (VPT) is the treatment of the exposed pulp of a tooth with the aim of keeping it healthy and free from infection. This is done in teeth that have been damaged by

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caries, trauma or restorative procedures [1,2]. The different types of VPT can be categorized into indirect and direct pulp capping, partial pulpotomy and full (coronal or pulp chamber) pulpotomy. This is done according to the extent of pulp exposure, perceived stage of bacterial infiltration and damage to the dental pulp [3]. It has been shown that as soon as caries infiltrates the enamel an initial reaction of the adjacent pulp tissue can be seen histologically [4]. However, if the softened and carious dentine is excavated completely without exposure of the pulp, healing is usually possible [5]. In cases with pulp exposure, pulp tissue can either be capped directly or partially removed. In such cases, local areas of severe inflammation and micro-abscesses can be present coronally, whilst the apical parts of the pulp remain healthy [6]. From a histological perspective, it is conceivable that better healing should be achieved after partial removal of the infected pulp tissue [6]. Currently used pulp capping materials including mineral trioxide aggregate (MTA), other hydraulic calcium silicate cements, and calcium hydroxide all have antimicrobial properties due to the high pH environment they induce when in contact with tissue fluids [7]. However, if placed on highly inflamed or necrotic and infected pulp tissue, these high-pH materials can also induce further necrosis rather than healing [8]. Higher success rates have been shown for the capping of accidental pulp exposures compared to carious pulp exposures, the two differing mainly in their extent of bacterial penetration into the pulp [9,10]. Unfortunately, neither clinical signs and symptoms

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nor results of the currently available diagnostic tests correlate to the histological state of the exposed pulp [11]. This is the main conundrum in the clinical management of exposed pulps after caries excavation. In this context, one topic appears to not have been addressed in a systematic manner: the effect of soft tissue lavage after pulp exposure. Lavage of the wound was identified as the central means of disinfection for open and potentially infected wounds during the Great War [12]. Sodium hypochlorite (NaOCl) in aqueous solution was identified at that time as the agent of choice to chemo-mechanically clean such injuries. This was because of its unique and somewhat selective effect on necrotized and infected soft tissues [12,13], which also led to its use in the chemo-mechanical debridement of infected root canal systems [14]. Today, sodium hypochlorite solutions are the first choice in pulpectomy procedures, i.e., in full root canal treatments [15]. However, in the context of potentially necrotized and infected pulp wounds encountered during VPT procedures, it is unclear whether NaOCl solutions are of any benefit or even in routine use. In theory, the use of NaOCl could be highly beneficial for this purpose, as it decontaminates the pulp wound chemically and thus, in theory, allows the capping material to exert its effect on healthier tissue. Its use has also been encouraged in recent guidelines [3], yet the scientific basis for this recommendation appears to be elusive. High success rates have been shown for VPT after removal of bacterial contamination using NaOCl in both primary

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and permanent molars [16,17]. Based on these and other findings, it can be concluded that the step of disinfecting the pulp wound by means of a wound lavage and removing microbially infiltrated, necrotic pulp tissue before it is capped may have an impact on survival of the remaining pulp after VPT [18,19]. This qualitative systematic review aimed to identify the types of pulp wound lavage and their influence on the vital pulp therapy of permanent human teeth with carious pulp exposure in all prospective controlled and randomized controlled clinical trials on vital pulp therapy. Moreover, factors that may be associated with the use of sodium hypochlorite in this context were analyzed. Materials and Methods This systematic review was conducted according to a protocol registered at the International Prospective Register of Systematic Reviews. The reporting follows the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines for systematic reviews [20] (Table S1). The reviewed question was: "what is the influence of topical disinfectants on pulp survival in the vital pulp therapy of permanent teeth with a carious pulp exposure?" Based on the PICO (population, intervention, comparison, outcome) format the search was carried out for clinical trials on permanent human teeth with a carious pulp exposure (P) that received vital pulp therapy (I), compared different treatment protocols (C) and reported the pulp survival after intervention with a minimal recall interval of 6 months (O). Data Sources and Literature Search A systematic search was conducted in six life science databases (MEDLINE, Biosis, Cochrane, EMBASE, Scopus and Web of

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Science) in October 2019. The search plan included a combination of keywords and indexing vocabulary (MeSH terms). Table S2 is an example of the search used in MEDLINE. No language restrictions were applied. To ensure the accuracy of the search one reviewer (A.M.) manually screened relevant journals in the field for potentially relevant publications. Study Selection and Inclusion Criteria The titles and abstracts were screened by two independent reviewers (A.M., D.-K.R.) according to pre-defined inclusion and exclusion criteria. Studies were included if they: (a) Were carried out on human permanent teeth with a carious pulp exposure that received a type of VPT (direct pulp capping, partial pulpotomy or full/pulp chamber pulpotomy); (b) Used a prospective randomized or non-randomized clinical trial design including a test and control group [21]; (c) Reported the pulp survival after a recall duration of at least six months after intervention. Excluded were all studies: (a) On animals; (b) On deciduous teeth; (c) On teeth without pulp exposure (e.g., indirect pulp capping); (d) On teeth with experimental, mechanical or traumatic pulp exposures; (e) That were retrospective, such as case reports or case series without a control group; (f) That were histological without clinical assessment of the outcome. All the articles selected by either reviewer were chosen for the full text evaluation which again was carried out by the two reviewers independently. In case of disagreement, consensus was reached by discussion and third-party-arbitration (M.Z.). Data-Extraction and Quality Assessment Data was extracted and managed on electronic files which were piloted and adapted before the final

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assessment of all the selected studies. Details regarding study characteristics were noted such as year of publication, randomized and non-randomized factors, type of environment in which the clinical trials were carried out (single-center, multi-center), number of operators and type of VPT performed (direct pulp capping, partial pulpotomy, full/pulp chamber pulpotomy). Furthermore, information about the subjects was also listed including age of the subjects (mean, median or age range), sample size (N teeth per group), type of teeth (molars, premolars, incisors), stage of root development (mature, immature) and inclusion of spontaneously painful teeth (yes, no). Data regarding wound lavage was extracted in detail, with a focus on the type of irrigant used (NaOCl, saline etc.), application of cotton pellets (yes, no) and the time required to achieve hemostasis (min) if mentioned. Other intervention related factors were also considered such as the use of rubber dam and sterile instruments, type of capping material (hydraulic calcium silicate cements, calcium hydroxide, etc.), final restoration placed immediately after pulp capping or re-entry. In addition, outcome related factors such as recall intervals (months), rate of drop out (percentage) and pulp survival (percentage) at least six months after the intervention were collected. The relevant information was collected and used for further analysis. All the identified trials were analyzed regarding the method used for pulp wound lavage. The clinical trials targeting the effect of pulp wound lavage on the outcome of VPT were also examined separately. The quality of the studies was assessed using RoB-2 and ROBINS-I tools as recommended by the Cochrane Collaboration

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(methods.cochrane.org [22]). RoB-2 was used for RCTs and assesses bias in the domains of randomization, assignment and adhering to intervention, missing data, outcome measurement and selection of the reported result. ROBINS-I is the recommended tool for bias assessment of non-randomized clinical trials in the domains confounding, selection of participants, misclassification of interventions, deviation from the intended intervention, missing outcome data, measurement of the outcome and selection of the reported results. Data Analysis and Statistic Evaluation A descriptive data analysis was carried out and statistics was applied only where appropriate (JMP 10.0.0, SAS Institute, Cary, NC, USA). Cohen's kappa coefficient was calculated to measure inter-rater reliability. Interaction between the use of NaOCl for pulp lavage and year of publication was assessed using Student's t test. The correlation between the use of calcium silicate cement in one of the experimental groups and NaOCl for pulp wound lavage was analyzed using Fisher's exact test. The alpha-type error was set at 5% (p < 0.05). Results The systematic search initially identified 639 potentially eligible articles after duplicate removal ( Figure 1). After screening of the titles and abstracts, 45 articles were selected and assessed in full-text. Eighteen out of these 45 articles were excluded at this stage (Table S3) and the other 27 selected for the qualitative synthesis [17,. Twenty-six of the selected articles were randomized clinical trials, one was non-randomized [47]. One publication [48] was in Chinese and was translated by a native speaker. There was almost perfect agreement between the two reviewers regarding full text evaluation (Cohen's kappa

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= 0.91). J. Clin. Med. 2020, 9, x FOR PEER REVIEW 4 of 12 of the experimental groups and NaOCl for pulp wound lavage was analyzed using Fisher's exact test. The alpha-type error was set at 5% (p < 0.05). Results The systematic search initially identified 639 potentially eligible articles after duplicate removal ( Figure 1). After screening of the titles and abstracts, 45 articles were selected and assessed in fulltext. Eighteen out of these 45 articles were excluded at this stage (Table S3) and the other 27 selected for the qualitative synthesis [17,. Twenty-six of the selected articles were randomized clinical trials, one was non-randomized [47]. One publication [48] was in Chinese and was translated by a native speaker. There was almost perfect agreement between the two reviewers regarding full text evaluation (Cohen's kappa = 0.91). Although all the included studies showed a low to moderate risk of bias (Table S4), a large heterogeneity was found among them ( Table 1). Because of this it was not possible to analyze the outcomes by means of a meta-analysis. Most of the identified trials examined the influence of the capping material (23/27) on pulp vitality in VPT [17,23,25,[27][28][29][30][32][33][34][35][36][37][38][39][40]41,[42][43][44][45][46]48], one of them also randomized the method used for pulp wound lavage [41]. The use of NaOCl solutions for pulp wound lavage was reported in 11 of the 27 included trials [17,25,31,33,34,36,39,41,42,45,46]. However, it was assessed as the randomized factor in just one of these 11 trials [41]. In ten trials the use of saline or water was reported

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[23,[26][27][28][29][30]35,38,40,43]. Three trials mentioned a rinse with NaOCl only for some cases and the data was not stratified according to the type of irrigant used [24,32,37]. Anesthetic without vasoconstrictor was used to control bleeding from the pulp wound in one trial [44] and in another the pulp wound was disinfected with a cotton pellet containing 75% ethanol [48]. Although all the included studies showed a low to moderate risk of bias (Table S4), a large heterogeneity was found among them ( Table 1). Because of this it was not possible to analyze the outcomes by means of a meta-analysis. Most of the identified trials examined the influence of the capping material (23/27) on pulp vitality in VPT [17,23,25,[27][28][29][30][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46]48], one of them also randomized the method used for pulp wound lavage [41]. The use of NaOCl solutions for pulp wound lavage was reported in 11 of the 27 included trials [17,25,31,33,34,36,39,41,42,45,46]. However, it was assessed as the randomized factor in just one of these 11 trials [41]. In ten trials the use of saline or water was reported [23,[26][27][28][29][30]35,38,40,43]. Three trials mentioned a rinse with NaOCl only for some cases and the data was not stratified according to the type of irrigant used [24,32,37]. Anesthetic without vasoconstrictor was used to control bleeding from the pulp wound in one trial [44] and in another the pulp wound was disinfected with a cotton pellet containing 75% ethanol [48]. A clinical trial assessed the effect of glutaraldehyde in VPT in a non-randomized manner [47]. All the trials were carried out

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using conventional rotating instruments, except one in which an Erbium laser was also used in combination with saline lavage [28]. A statistical analysis was carried out to identify possible factors that may be correlated to the use of an NaOCl solution for pulp wound lavage. For this purpose, the three trials that used NaOCl in selected cases only but did not stratify the data were excluded [24,32,37]. A highly significant (p < 0.005) tendency was found for authors to use NaOCl for chemical soft tissue debridement in the more recent studies (Figure 2a). Moreover, the use of NaOCl was significantly (p < 0.05) correlated to the application of a hydraulic calcium silicate cement in the trials (Figure 2b). A statistical analysis was carried out to identify possible factors that may be correlated to the use of an NaOCl solution for pulp wound lavage. For this purpose, the three trials that used NaOCl in selected cases only but did not stratify the data were excluded [24,32,37]. A highly significant (p < 0.005) tendency was found for authors to use NaOCl for chemical soft tissue debridement in the more recent studies (Figure 2A). Moreover, the use of NaOCl was significantly (p < 0.05) correlated to the application of a hydraulic calcium silicate cement in the trials ( Figure 2B). Two of the included 27 trials explicitly compared different methods of pulp wound lavage [41,47]. Ozgur et. al. (2017) randomized the use of 2.5% NaOCl to saline for wound lavage in partial pulpotomy. This study was carried out on

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80 asymptomatic, immature molars. The subjects were between 6 and 13 y of age and were treated by one operator. Besides randomizing the pulp wound lavage with NaOCl or saline, two different capping materials-calcium hydroxide or MTA were also randomized in this trial. Hence, in total, there were four groups of 20 teeth each. The reported pulp survivals after one year were between 94% and 100% with no significant differences between the groups. No significant influence of disinfection with NaOCl could be shown and the results for both capping materials were comparable. A calculation of sample size was not reported. An older clinical trial examined the effect of the use of glutaraldehyde in coronal pulpotomy [47]. This clinical trial was carried out on 20 immature, asymptomatic molars of 8-9 y old patients, divided into two groups. After bleeding was controlled with a wet cotton pellet, in one group a cotton pellet soaked with 5% buffered glutaraldehyde solution was placed on the amputated pulp for 5 min and then pulp capping was carried out using a calcium hydroxide paste mixed with 5% glutaraldehyde. In the other group, the calcium hydroxide paste was mixed with water and no additional chemical was used on the pulp wound before that. This trial had a recall rate of 100% after one year and reported pulp survival rates of 100% for both groups. However, it was carried out on a relatively small sample of only 10 teeth per group. Two of the included 27 trials explicitly compared different methods of pulp wound

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lavage [41,47]. Ozgur et al. (2017) randomized the use of 2.5% NaOCl to saline for wound lavage in partial pulpotomy. This study was carried out on 80 asymptomatic, immature molars. The subjects were between 6 and 13 y of age and were treated by one operator. Besides randomizing the pulp wound lavage with NaOCl or saline, two different capping materials-calcium hydroxide or MTA were also randomized in this trial. Hence, in total, there were four groups of 20 teeth each. The reported pulp survivals after one year were between 94% and 100% with no significant differences between the groups. No significant influence of disinfection with NaOCl could be shown and the results for both capping materials were comparable. A calculation of sample size was not reported. An older clinical trial examined the effect of the use of glutaraldehyde in coronal pulpotomy [47]. This clinical trial was carried out on 20 immature, asymptomatic molars of 8-9 y old patients, divided into two groups. After bleeding was controlled with a wet cotton pellet, in one group a cotton pellet soaked with 5% buffered glutaraldehyde solution was placed on the amputated pulp for 5 min and then pulp capping was carried out using a calcium hydroxide paste mixed with 5% glutaraldehyde. In the other group, the calcium hydroxide paste was mixed with water and no additional chemical was used on the pulp wound before that. This trial had a recall rate of 100% after one year and reported pulp survival rates of 100% for both groups. However, it was

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carried out on a relatively small sample of only 10 teeth per group. Discussion This search revealed two studies that specifically assessed the effect of chemical agents used for pulp wound lavage on the pulp survival after VPT of carious teeth. Unfortunately, both studies were underpowered and no conclusions can be drawn based on this data. Despite a lack of evidence, there appears to be a trend towards using NaOCl to clean pulp wounds in the VPT of teeth with carious exposures. The topic under investigation has not been given the attention it probably deserves. Since it is not possible to precisely identify the level of pulp infection in carious exposures by means of the presently available diagnostic tools, wound disinfection and debridement could be a core topic. Obviously, it is not. A recent review suggested that mechanically removing more pulp tissue in these situations can improve the rate of pulp survival, i.e., pulpotomy seems to yield better results than mere pulp capping [49]. From a histological point of view this makes sense, as the infection advances from the crown towards the root [50]. However, this statement needs to be weighed against the studies by Bjørndal [5] and Asgari and co-workers [24], which until now are the only trials that randomized these levels of intervention (Table 1). Neither found a difference between direct pulp capping and partial or full pulpotomy. Moreover, if more pulp tissue is removed mechanically, it will become harder to check the tooth clinically. Despite the absence of disease, such a tooth may

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no longer respond to simple clinical monitoring tests, such as the cold test [51]. Therefore, chemical wound debridement via lavage of the pulp wound using an antiseptic irrigant in conjunction with minimally invasive mechanical intervention could be the better option. Sodium hypochlorite solutions appear to be ideal for that purpose because of their unique effect on necrotic soft tissue [52] as well as biofilm [53]. Introduced more than 100 years ago, NaOCl solutions remain a standard of care in the disinfection of external soft tissue wounds [54]. Irrigants containing one or multiple antibiotics have also been discussed, as they selectively kill the invading microorganisms and obviously cause less collateral damage than non-specific biocides such as NaOCl [55]. There is also a history of glutaraldehyde usage in antiseptic solutions [56] and despite the fact that it is still contained in some dentine bonding agents [57], the high toxicity and mutagenic potential of this chemical would preclude it from the application proposed by Waly (1995) [47]. Both trials on the effects of wound lavage on the outcome of pulpotomy, identified by this search, were under-powered. The first had four groups and N = 20 [41], the second had two groups and N = 10 [47]. Nowadays, to be accepted by any internationally renowned journal, trials should be registered and a sample size analysis needs to be provided. One of the authors (M.Z.) is part of a team of researchers that planned and are executing a clinical trial on the topic: wound lavage in direct pulp capping of carious

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exposures in adult teeth using 2.5% NaOCl versus 0.9% saline solution. This trial has been registered (CTRI/2019/01/017167). The power analysis revealed that, assuming a 25% difference in pulp survival after one year between the two treatments and a 25% dropout rate, 48 patients per group are necessary to detect a significant difference with a chance (power) of 80% at a type I error of 0.05. As shown in this review, the heterogeneity of the underlying studies is such that comparisons between individual treatment factors and outcomes in any form of meta-analyses would be spurious (Table 1). Success rates in the trials included in this review differed largely for similar interventions e.g., partial pulpotomy using saline as an irrigant and calcium hydroxide as a capping material. The nested clinical trial by Bjørndal and co-workers [5] has been criticized by some clinicians for its reported low success rates after carious exposure of the pulp. It has been suggested that this could be because of the use of an "outdated" pulp capping agent, a calcium hydroxide liner. However, the difference between calcium silicate and calcium hydroxide based capping materials seems to be minor (Table 1). Therefore, other factors that may have had an impact on the outcome must also be considered, such as clinical skills of the operators, inclusion of painful teeth in the trial sample, and/or the lack of disinfection of the pulp wound with NaOCl before capping. In spite or maybe also because of these possible procedural shortcomings, the Bjørndal trial set a new standard in terms

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of its high methodological quality and may have spurred more research. More recent trials showed better outcomes even when calcium hydroxide was used as the pulp capping material (Table 1) but they also used NaOCl for pulp lavage. Whether the use of NaOCl directly influenced the outcome of these trials, remains unclear and cannot be elucidated based on the presently available literature. Although a direct comparison of the selected studies and assessing the influence of individual factors on the outcome of VPT was not possible, correlations between the use of NaOCl and other study parameters could still be identified. Our findings suggest that there is an increasing trend towards using NaOCl solutions for pulp wound lavage in the VPT of carious exposures despite the lack of scientific evidence for it. NaOCl solutions are a popular choice among endodontists [58] and many of the more recent trials identified by this search involved endodontists. This may also be why the recent consensus statement in a prominent endodontology journal recommends the use of NaOCl for pulp wound lavage, as most of the authors of this publication specialize in the field of endodontology [3]. However, practitioners of other specialties e.g., restorative dentistry, who also perform VPT clinically, may not necessarily be inclined to follow this protocol in the absence of available scientific evidence. Lastly, it is important to remember that it is not only the state of pulp tissue at the site of capping, but also the bacterial seal provided by subsequent restoration of the tooth that will determine the

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long-term success of VPT [59]. In view of the lack of scientific evidence in this context, future studies should assess different wound lavage concepts used in other fields of medicine and evaluate these regarding their effectiveness in VPT. Conclusions This systematic review identified a clear trend towards using sodium hypochlorite for pulp wound lavage during vital pulp therapy in recent trials and in combination with the use of calcium silicate cements as pulp capping materials. However, the effect of sodium hypochlorite for this purpose still needs to be substantiated by well-designed clinical trials. Conflicts of Interest: The authors deny any conflicts of interest.

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High Sensitivity of Aged Mice to Deoxynivalenol (Vomitoxin)-Induced Anorexia Corresponds to Elevated Proinflammatory Cytokine and Satiety Hormone Responses Deoxynivalenol (DON), a trichothecene mycotoxin that commonly contaminates cereal grains, is a public health concern because of its adverse effects on the gastrointestinal and immune systems. The objective of this study was to compare effects of DON on anorectic responses in aged (22 mos) and adult (3 mos) mice. Aged mice showed increased feed refusal with both acute i.p. (1 mg/kg and 5 mg/kg) and dietary (1, 2.5, 10 ppm) DON exposure in comparison to adult mice. In addition to greater suppression of food intake from dietary DON exposure, aged mice also exhibited greater but transient body weight suppression. When aged mice were acutely exposed to 1 mg/kg bw DON i.p., aged mice displayed elevated DON and DON3GlcA tissue levels and delayed clearance in comparison with adult mice. Acute DON exposure also elicited higher proinflammatory cytokine and satiety hormone responses in the plasma of the aged group compared with the adult group. Increased susceptibility to DON-induced anorexia in aged mice relative to adult mice suggests that advanced life stage could be a critical component in accurate human risk assessments for DON and other trichothecenes. Introduction Deoxynivalenol (DON, vomitoxin), a trichothecene mycotoxin produced by the fungus Fusarium graminearum, is a common cereal grain contaminant that is highly resistant to heat processing, leading to contamination of human and animal food [1]. Adverse effects of acute exposure to DON include anorexia, diarrhea, and vomiting in experimental animals [2]. In addition, chronic

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DON exposure can lead to immunotoxic effects and growth retardation. Mice, commonly used in experimental studies for DON risk assessment, are incapable of vomiting but exhibit feed refusal and body weight suppression following exposure to the toxin [3]. Previous studies have investigated sex differences and the susceptibility of young animals to DON-induced anorexia [4][5][6][7][8]; however, the effects of this toxin on aged animals are largely unaddressed. Studying the adverse effects of DON in advanced life stage animals is important because approximately 25% and 30% of elderly men and women, respectively, are reported to be in an anorectic state [9][10][11]. This phenomenon of unintentional weight loss in advanced life stage has been termed the "anorexia of aging" and is a high predictor of morbidity and mortality in the elderly [12,13]. The etiology of anorexia is complex and thought to be caused by many factors including depression, increased susceptibility to illness, diet, and the burden of multiple medications [10,14,15]. In a study of elderly Finnish men with depressive symptoms (65-84 years of age; n = 688), many reported gastrointestinal complaints over a two-week period including diarrhea (20%), stomach pains (37%), nausea (29%), vomiting (9%), and loss of appetite (21%) [16]. As these symptoms are also present with DON intoxication, it is possible that elderly individuals who are already predisposed to gastrointestinal ailments might be more sensitive to the negative effects of DON. One potential cause of DON-induced anorexia is the induction of proinflammatory cytokines, including IL-1β, IL-6, and TNF-α, which have been previously shown to cause sickness behavior

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in humans and experimental animals [17][18][19]. Our lab has reported the induction of these cytokines in female mice, as well as an increased plasma IL-6 response in male mice in comparison with female mice [5,20,21]. Prior investigations of the effect of lipopolysaccharide (LPS) in aged and adult mice have shown that the former exhibit a greater cytokine response to LPS than adults [22,23]. Huang et al. (2008) found that plasma IL-6 levels of aged mice were 2-and 6.6-fold higher at 2 and 8 h post intracerebroventricular (i.c.v.) injection of 10 ng of LPS than adult mice [22]. Another study found that plasma levels of IL-1β and IL-6 were significantly increased in aged mice in comparison with adults at 6 h post exposure to 5 μg/g bw LPS administered by intraperitoneal (i.p.) injection [23]. Additionally, our lab discovered that the gut satiety hormones peptide YY (PYY) and cholecystokinin (CCK) are induced in mice upon i.p. and oral DON exposure and both contribute to feed refusal caused by this mycotoxin [24,25]. When Stanström et al. (1998) compared the number of enteroendocrine cells responsible for PYY secretion in the intestinal epithelium of mice that were 3 and 24 months of age, they found that aged mice had significantly more of these cells [26]. Another study in rats revealed that PYY-containing cells per colonic crypt increased with age [27]. In a study comparing the effects of CCK in adult and aged mice, researchers found that aged mice showed increased sensitivity to CCK and that the action of CCK was prolonged

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in aged mice [28]. As PYY and CCK are induced by DON and tend to increase with age, exploring the combined effects of DON exposure and aging is of importance. The aims of this study were to compare feed refusal responses between adult and aged mice after acute and dietary DON exposure. Study 1 compared feed refusal differences by age following acute i.p. DON exposure. In Study 2, we compared DON tissue concentrations, proinflammatory plasma cytokine responses, and satiety hormone responses in adult and aged animals following acute i.p. exposure to the toxin. Finally, Study 3 compared differences in food intake and body weight suppression upon dietary DON exposure in adult and aged mice. The results presented herein indicate that in aged mice the food refusal response is greater than in adult mice following both acute i.p. and dietary DON exposure and that acute exposure is accompanied by delayed DON tissue clearance as well as increased proinflammatory cytokine and gut satiety hormone responses in aged mice. DON-Induced Feed Refusal Is Greater in Aged Mice than in Adult Mice The effects of acute i.p. exposure to 1 mg/kg and 5 mg/kg bw DON were compared in aged and adult mice over 36 h (Figure 1). Aged mice treated with 1 mg/kg DON consumed less food than control aged mice from 1 h to 36 h post-injection (PI) (Figure 2). In comparison, adult mice treated with the same dose of DON consumed less food than adult controls at 1 to 4 h PI and then began to show

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recovery in food intake. By 6 h PI, aged mice exposed to 1 mg/kg DON ingested significantly less food than adult mice. At this dose, aged mice consumed 82% less food than aged control mice, while adult mice at this dose had consumed 41% less food than adult controls at this time point. Figure 1. Study 1 experimental design. On day 1, mice were fasted from 10:00 to 18:00 h, then exposed to DON treatments or vehicle control. Food was immediately replaced following exposure and food measurements were recorded hourly from 1 to 7 h PI, and at 12, 20, 24, and 36 h PI as indicated by arrows. Following exposure to 5 mg/kg bw DON, total food consumption was reduced from 1 to 20 h PI and 1 to 36 h PI for adult and aged mice, respectively. At 7 h PI, 5 mg/kg bw DON-treated adult mice consumed 32% feed of adult vehicle controls, whereas DON-treated aged mice ate only 2% of that of aged vehicle controls. At 36 h PI, aged mice continued to exhibit a substantial depression in food intake compared to aged control mice, with food consumption accounting for 43% and 22% of that of control aged mice at 1 mg/kg and 5 mg/kg bw DON, respectively. In comparison, food intake in adult mice treated with 1 mg/kg and 5 mg/kg bw DON at 36 h PI was 94% and 84%, respectively, of adult control food intake, suggesting that this group has nearly fully recovered from the toxin. DON Tissue Concentrations

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Are Higher in Aged Mice Compared with Adults after Acute Exposure When DON tissue concentrations in the kidney, liver, plasma, spleen, heart, and brain were assessed over 12 h following i.p. exposure to DON at 1 mg/kg bw, toxin levels were consistently higher in aged mice than in adult mice ( Figure 3). The initial rank order of DON concentrations in both aged and adult mice was: kidney > liver > plasma > spleen > heart > brain. At 1 h PI, aged mice had higher DON concentrations in the kidney (1.8-fold increase) and plasma (1.5-fold increase). At 2 h PI, DON concentrations were higher in all tissues of aged mice compared with adults, ranging from 6-fold higher in the kidney to 1.5-fold higher in the brain. Interestingly, DON concentrations in kidneys and brains of aged animals were higher at 2 h PI than at 1 h PI. At 4 h PI, DON concentrations were significantly higher in all organs of aged mice except the brain, with the largest differences existing in the kidney (20-fold higher) and plasma (5-fold higher). Aged mice continued to exhibit higher DON levels in the kidney, spleen, heart, and brain at 12 h PI, with concentrations ranging from 4.3 to 2-fold above the levels of adult mice. Plasma IL-6 and IL-1β Are Higher in Aged Mice Compared with Adults after Acute DON Exposure When the effects of acute exposure to 1 mg/kg bw DON on plasma proinflammatory cytokines were assessed, aged mice exhibited higher plasma levels of IL-6 and IL-1β than

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adult animals ( Figure 4). Beginning at 2 h PI, aged mice treated with DON had a 2.2-fold increase in both plasma IL-6 and IL-1β when compared with adult mice at 2 h PI. At 4 h PI, aged mice had a 120-fold difference of IL-6 in comparison with adult animals, which had nearly returned to adult control levels at this time point. Aged animals also had a 4.4-fold increase in IL-1β at 4 h PI when compared with adult mice. Plasma IL-6 levels remained significantly elevated in aged mice when compared with control aged mice and DON-treated adult mice at 12 h PI, while IL-1β levels were not statistically significant in any group at this time point. Vehicle-treated aged mice had higher plasma IL-6 at 2 and 4 h post exposure than treated adults. However, IL-6 levels were very low compared to DON-treated mice at these time points. Adult mice had higher levels of IL-1β at 2 and 4 h PI. TNF-α was not detectable in the plasma of either aged or adult mice at any time point. This latter result is in agreement with a previous study in our lab comparing proinflammatory cytokines in male and female mice treated with 1 mg/kg bw DON [21]. DON Induction of PYY and CCK Is Greater in Aged Animals than in Adults with DON Exposure When the effects of acute exposure to 1 mg/kg bw DON on plasma levels of gut satiety hormones were measured, DON elicited greater increases in PYY and CCK in aged mice than

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in adult mice ( Figure 5). DON-treated aged mice exhibited a trend toward greater PYY than similarly treated adult mice at 1 and 2 h PI. At 4 h PI, aged mice treated with DON exhibited a robust increase in plasma PYY, having levels that were 51% higher than control aged mice. In contrast, adult mice treated with DON at this time point were not different from control adults. Plasma PYY had returned to baseline levels by 12 h PI in both DON-treated groups. In mice exposed to the vehicle only, PYY plasma levels trended higher in aged mice than adults and PYY was significantly higher in control aged mice at 2 and 4 h PI than in control adults. DON-treated aged mice exhibited a trend toward greater CCK than similarly treated adult mice at 1 and 4 h PI. At 1 h PI, aged mice exposed to DON had a 99% increase in plasma CCK in comparison to control aged animals and were significantly higher than control aged mice and DON-treated adults. In comparison, while adult mice exhibited a 59% increase in CCK over adult controls at 1 h PI, they were not significantly higher than control adults. DON-treated animals continued to show trends toward increased plasma levels of CCK at 2 and 4 h PI, although these were not statistically significant. At 12 h PI, CCK concentrations were no longer impacted by DON in either age group. CCK plasma levels were higher in vehicle-treated aged mice than in adults, though not significantly. Study 3

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2.3.1. Reduction in Food Intake Is Greater in Aged Mice than in Adults with Dietary DON Exposure The impact of dietary exposure to DON at 0, 1, 2.5, and 10 ppm on food intake was compared between aged and adult mice. Aged mice showed a greater reduction in food intake upon dietary DON exposure than adult mice (Table 1). After 1 day of dietary DON exposure, both aged and adult animals exhibited a statistically significant negative correlation between decreasing food intake with increasing DON concentration in the diet. Aged mice fed DON diets containing 2.5 and 10 ppm consumed 9% and 62% less food, respectively, than aged control mice over the first day, while adult mice fed the same concentration diets consumed 2% and 58% less food than adult controls, respectively, at this time point. After 2 days, aged mice continued to exhibit decreasing food intake with increasing DON concentration, while adult mice no longer exhibited this negative correlation. Aged mice fed diets containing 2.5 and 10 ppm DON consumed 13% and 54% less food, respectively, than aged control mice at day 2, while adult mice fed the same concentration diets consumed 3% and 10% less food than adult controls at this time point. Dietary DON Transiently Suppresses Body Weights to a Greater Extent in Aged Mice Effects of consuming feed containing 1, 2.5, and 10 ppm DON on body weight suppression were further compared by age. Aged mice were more sensitive to body weight suppression than adults ( Figure 6). Aged mice fed a diet

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containing 2.5 ppm DON weighed less than group control mice and adult mice fed the same diet beginning on day 6 of the exposure period, weighing 5% less than aged control mice, while adult treated mice weighed 0.4% less than adult control mice. At the study termination (day 14), aged mice fed 2.5 ppm DON weighed 6% less than aged control mice. Adult mice fed the same diet weighed 5% less than adult controls at day 14 and the significance between the differences in weight loss by age no longer existed. Aged mice fed a diet containing 10 ppm initially exhibited a rapid depression of weight gain in comparison with controls and on day 4 weighed 11% less than aged control mice. In comparison, adult mice fed 10 ppm DON diet weighed 6% less than adult controls at day 4. After 14 days of exposure to a diet containing 10 ppm DON, aged and adult mice continued to show significant weight suppression in comparison to the respective group controls; however, differences between adult and aged mice were no longer evident upon termination of the study, suggesting that these effects were transient. Figure S1). No significant differences in DON equivalent tissue concentrations were observed between aged and adult mice; however, adult mice fed diets containing 10 ppm DON had higher DON equivalent concentrations in the spleen than aged animals fed the same diet. As animals were allowed ad libitum access to food prior to euthanasia, the amount of toxin consumed and time of consumption are unknown. The

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observation that aged mice tended to have lower DON equivalent levels than adult mice fed the same diet could indicate that aged animals were consuming less food, and thus less toxin, than adults. Proinflammatory Plasma Cytokines Are not Detectable after 14 Days of Dietary DON Exposure Plasma levels of the proinflammatory cytokines IL-6, IL-1β, and TNF-α were not detectable in aged or adult mice after 14 days of dietary DON exposure at any treatment level. These findings are consistent with previous research in our lab also reporting no change in the plasma levels of IL-6, IL-1β, or TNF-α after 17 days of exposure to diets containing 1, 2.5, and 10 ppm DON in either male or female mice [21]. A study examining mice exposed to 2 mg/mL DON in drinking water for 36 days could not detect these proinflammatory cytokines in the plasma [29]. Discussion Prior investigations have addressed increased susceptibility of young mice, poultry, and swine to DON, but the negative effects experienced by older animals has been largely unstudied [5][6][7]. This study is the first to report increased anorectic responses in aged mice compared with young adults following DON exposure. The discovery that anorectic responses to DON are greater in old animals is critical because advanced age in humans leads to a phenomenon known as the "anorexia of aging" in which elderly individuals show unintended weight loss [9,12,13]. There is a possibility that DON consumption could exacerbate this condition. The finding that aged mice have higher tissue concentrations of DON following acute exposure over

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a prolonged period compared with adults suggests that aged mice might differ in their ability to absorb, metabolize, or excrete the toxin. The contention that aged animals have a lower capacity to metabolize and thus excrete DON is supported by the previous observation of lower mRNA levels of UDP-glucuronosyltransferases, the family of enzymes responsible for DON glucuronidation, in aged mice when compared with their adult counterparts [30]. Increased levels of DON in the kidneys of aged mice relative to adults are consistent with the notion that aged animals have a reduced capacity to excrete the toxin. As kidney DON concentrations in aged mice were higher at 2 h PI than at 1 h PI, it appears that the toxin accumulated in the kidney. This result could be due to changes in metabolic abilities or to reduced kidney functioning, including reduced glomerular filtration rate and renal blood flow, known pharmacokinetic changes observed with aging [15,31]. Analyzing differences in the DON metabolite profile in different tissues and the integrity of kidney functioning with aging will therefore be an important objective in future studies. DON concentrations in the brains of aged mice were also elevated at 2 h PI compared with 1 h PI, as seen in the kidney, while the levels of this toxin in the brains of young adults decreased from 1 h PI to 2 h PI. Importantly, DON concentrations were still higher in aged mice at 12 h PI. These findings are notable because DON is known to directly affect the brain, including the induction

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of c-Fos, a strong indicator of neuronal activation [19,32,33]. Further exploration of the possible differential effects of DON on the brains of aged mice could provide new insight into why animals are more sensitive than adults to the anorectic effects of DON. Increased proinflammatory plasma cytokine responses in aged mice compared with adults might be a critical factor behind the heightened anorectic responses observed in aged mice [34]. Previous investigations of LPS-induced cytokine responses also reported an increase in proinflammatory cytokine responses in aged animals compared with adults [22,23]. Here, we observed that peak elevation of plasma IL-1β and IL-6 at 2 h PI in aged mice coincides with the highest kidney concentration of DON. Together these observations suggest that a decreased ability to excrete DON is linked to increased proinflammatory cytokine responses. Elevated tissue IL-1β might be more important to DON-induced anorexia than the observed IL-6 increase for several reasons. First, a recent study by Wu and Zhang [35] reported that type 1 IL-1 receptor (IL-1R1) antagonist IL-1RA dose-dependently attenuated both IL-1 beta-and acute DON-induced anorexia. Second, we have previously observed that IL-6 deficient mice are recalcitrant to food refusal caused by dietary DON exposure [36]. Third, while both peripheral and central injection of IL-1β into rodents elicits anorexia in rodents, only central injection of IL-6 suppresses food intake [34]. Thus, the contributions of DON-induced IL-6 to food refusal are unclear at this time and require further study, particularly with regard to the effects of this trichothecene on the expression of IL-6 in the brain.

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Some studies have reported increased baseline levels of proinflammatory cytokines in aged mice, a process known as inflammaging [37,38]. Plasma IL-6 was slightly elevated in aged control mice (0.02-0.3 ng/mL) in our studies of acute i.p. exposure in comparison with adults (0.01-0.02 ng/mL). The baseline levels of plasma IL-6 are likely a response to the i.p. injection. As no elevation in plasma cytokine levels was observed at 12 h PI in control aged mice, elevated plasma cytokine levels in DON-treated aged mice are a result of toxin exposure and not a proinflammatory phenotype prior to exposure, and highly consistent with their potential to contribute to the increased anorectic response in aged mice. Elevated PYY and CCK responses in aged mice relative to adults could also elicit increased food refusal in aged mice. These hormones are known to cause satiety and have been previously shown by our lab to increase with exposure to DON [24,25,39]. Again, increased PYY and CCK could be caused by slower excretion of DON. It is also possible that DON evokes a greater induction of these gut satiety hormones innately, as previous research in humans has shown greater induction of PYY and CCK with food consumption in aged subjects compared with younger individuals [40][41][42]. Our findings also support that PYY, and to a lesser extent CCK, are indeed basally elevated in aged animals and exposure to DON elicited a further elevation of these satiety hormones. One limitation of the acute studies is that food intake and hormones were measured upon acute i.p. exposure

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to DON to minimize stress to aged mice and microflora effects. Future research should also address whether these satiety hormones are similarly elevated in aged mice with oral exposure to DON. The greater suppression of food intake in aged mice than in adults upon dietary DON exposure is consistent with the observation of greater feed refusal in aged animals vs. adults in the acute i.p. exposure to DON. Aged mice consuming less food than adult animals also indicates that they are more sensitive to the adverse effects of DON. Because aged animals ingest less food, they also consume less DON, suggesting that aged mice have a lower threshold for the amount of toxin that they can tolerate. At the termination of Study 3, weight loss in aged and adult mice appeared to plateau. It might be speculated that this results from induction by DON of UGT enzymes and/or bacterial ability to convert DON to DOM-1, resulting in a greater capacity to metabolize DON. In a two-year study assessing chronic exposure of mice to diets containing 1, 5, and 10 ppm DON, body weight gain appeared to be unaffected by DON exposure for the first 100 days of treatment diets [43]. After this time point, weight gain in mice on diets containing 5 and 10 ppm begins to decrease while the body weight gain of mice on diets containing diets 0 and 1 ppm DON continued to increase until approximately 500 days of treatment diets. The results presented in the two-year chronic feeding study, along with our

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findings in Study 3, indicate that while some adaptation to DON does occur, chronic exposure to DON over a lifetime will still result in weight reduction. Animals The Institutional Animal Care and Use Committee at Michigan State University approved all animal experiments. Adult male (3 mos) and aged male C57BL6 mice (22 mos) were purchased from the National Institute on Aging colony (Charles River Laboratories, Wilmington, MA, USA). Mice were housed singly in polycarbonate cages with sifted aspen bedding on a 12 h light/dark cycle, with constant temperature (21-24°C) and humidity (40%-55%). In all experiments mice were acclimated to a high fat pellet diet (45% kcal from fat; Research Diets, Inc., New Brunswick, NJ, USA) one week prior to DON exposure. The high fat diet has previously been determined to provide the most efficient food recovery in acute DON exposure studies [44]. Study 1 Effects of age on food intake after acute i.p. DON exposure were assessed as summarized in Figure 1 using a protocol previously described by our laboratory [44]. After acclimation, adult and aged mice (n = 5/group) were fasted from 10:00 to 18:00 h, and injected i.p. with either 0 (vehicle), 1 or 5 mg/kg bw DON. Food consumption was measured hourly 1 to 7 h post exposure, and at 12, 20, 24, and 36 h post injection (PI). Measurements were conducted under red light conditions during dark cycle. We choose to use i.p. exposure for this and the following study to (1) minimize stress [45] and (2) bypass microbial metabolism to DOM-1,

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a non-toxic metabolite of DON, because rodent gastrointestinal tract microflora are capable of forming this metabolite, while human microflora are typically unable of this type of metabolism [46]. Study 2 Effects of age on DON tissue levels and plasma concentration of proinflammatory cytokines and satiety hormones were measured after acute i.p toxin injection. Adult and aged mice (n = 9-10/group) were fasted as in Study 1 and exposed to 1 mg/kg bw DON in PBS or PBS vehicle via i.p. injection. Mice were euthanized with CO2 at 1, 2, 4, and 12 h PI without food replacement. Blood was collected via cardiac puncture and then kidney, liver, spleen, heart, and brain samples were collected. Plasma was isolated from blood by centrifugation at 3500× g for 10 min at 4 °C. Plasma and organs were stored at −80 °C until analysis. Study 3 Effects of age on food intake and body weight changes elicited by dietary DON exposure were assessed. After acclimation, adult and aged mice were randomized into equal weight groups by age group (n = 5-6/group) and placed on high fat diets containing 0, 1, 2.5, and 10 ppm DON. Mice were given two weighed pellets (approximately 7 g) to allow for ad libitum food consumption. Body weights were measured daily at 10:00 AM for 14 days. Food intake was also measured each day at this time for 2 days. After 2 days, mice on diets containing DON progressively began to shred the pellets into fine particles, preventing accurate food recovery. After 14 days of

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DON exposure, mice were euthanized under CO2 at 8:00 AM. Food access was ad libitum prior to euthanasia. Blood was collected via cardiac puncture and then kidney, liver, spleen, heart, and brain samples were collected. Plasma was isolated from the blood. Plasma and organs were stored at −80 °C until analysis. DON Analysis DON quantification in plasma and organs in Study 2 were analyzed using the Veratox high sensitivity (HS) ELISA (Neogen, Lansing, MI, USA) as described by Pestka et al. (2008), with slight modifications. Briefly, organs were homogenized 1:1 in PBS (except the heart, which was homogenized 1:2). Tissue homogenates were heated at 100 °C for 5 min and then centrifuged at 14,000× g for 10 min at 4 °C. The resulting supernatant was analyzed by ELISA. Chromogenic end product was measured using an F3 ELISA plate reader at 650 nm and Softmax software (Molecular Devices, Menlo Park, CA, USA). As previously described by our lab [21], the ELISA was found to be 100% cross reactive with DON3GlcA, a major metabolite of the toxin produced in rodents. Cross reactivity with other potential glucuronides was not determined. Since we did not measure ratio of active vs. conjugated DON, results are reported as DON equivalents. Proinflammatory Cytokine Analyses Plasma levels of the proinflammatory cytokines IL-6, IL-1β, and TNF-α were determined in Study 2 and 3 using Duoset ELISAs from R & D Systems (Minneapolis, MN, USA). Due to limited plasma quantities, equal volumes of mouse plasma samples from Study 2 were pooled within groups and ran in

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technical replicates (n = 4 rep). Statistical Analysis Statistical analysis was conducted by SigmaPlot version 11.0 (Jandel Scientific; San Rafael, CA, USA). Statistical comparisons between ages were made at each time point using a Student's t-test, unless normality failed. If normality was not met, a Mann-Whitney Rank Sum test was performed. Statistical comparisons between age and dose were made at each time point using a one-way analysis of variance (ANOVA), unless normality failed. A Kruskal-Wallis one-way analysis of variance by ranks was performed if normality was not met. Student-Neuman-Keuls was used in all post hoc analysis for parametric and non-parametric animal groups of equal numbers. Dunn's test was used in post hoc analysis of non-parametric analysis of unequal animal group numbers. Pearson product moment correlations were performed to determine the statistical significance between food consumption and treatment levels of DON diets by age and day in Study 3. Differences were considered significant when p < 0.05. Conclusions The results presented herein indicate that aged mice are more susceptible than their young adult counterparts to DON-induced anorexia following either acute i.p. or dietary DON exposure. In addition to reduced food intake in dietary exposure studies, a greater suppression of body weight gain was seen in aged mice compared with adult mice. Aged mice exhibited slower tissue clearance of DON and increased proinflammatory plasma cytokine and gut satiety hormone responses compared with adult mice after acute i.p. exposure to DON. Collectively, these findings suggest that advanced life stage should be considered when formulating risk assessments for DON and

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other trichothecene mycotoxins.

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Influence of Spatial and Chromatic Noise on Luminance Discrimination Pseudoisochromatic figures are designed to base discrimination of a chromatic target from a background solely on the chromatic differences. This is accomplished by the introduction of luminance and spatial noise thereby eliminating these two dimensions as cues. The inverse rationale could also be applied to luminance discrimination, if spatial and chromatic noise are used to mask those cues. In this current study estimate of luminance contrast thresholds were conducted using a novel stimulus, based on the use of chromatic and spatial noise to mask the use of these cues in a luminance discrimination task. This was accomplished by presenting stimuli composed of a mosaic of circles colored randomly. A Landolt-C target differed from the background only by the luminance. The luminance contrast thresholds were estimated for different chromatic noise saturation conditions and compared to luminance contrast thresholds estimated using the same target in a non-mosaic stimulus. Moreover, the influence of the chromatic content in the noise on the luminance contrast threshold was also investigated. Luminance contrast threshold was dependent on the chromaticity noise strength. It was 10-fold higher than thresholds estimated from non-mosaic stimulus, but they were independent of colour space location in which the noise was modulated. The present study introduces a new method to investigate luminance vision intended for both basic science and clinical applications. Pseudoisochromatic stimuli are composed of a combination of luminance and colour contrast 30 . The design of the stimuli is a mosaic that has a spatial noise added to a

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luminance noise within its patches. The target highlights from the mosaic field by the chromaticity difference 31 . Pseudoisochromatic stimulus is widely used to investigate colour discrimination. In this particular type of test, the chromatic contrast is embedded in luminance and spatial noise, and the subject performs a colour discrimination task 28,29,[31][32][33][34][35][36] . Other stimulus design have been used to investigate the colour discrimination 37,38 . Our research group has described that different luminance and colour interactions within the pseudoisochromatic stimuli can have an influence on colour discrimination tasks 28 . We investigated how the number of luminance values in the luminance noise influenced the colour perception 28 . We observed that when the stimuli have two or four luminance values in the luminance noise the color discrimination was worse than when the stimuli had six or more luminance values in the luminance noise. We also observed that the colour discrimination thresholds and reaction times were dependent on the relationship between the mean luminance of the mosaic and the range of the luminance noise 29 . In the present investigation, a novel stimulus was introduced that could be used to investigate the influence of the luminance and color interactions within the luminance discrimination tasks. This new stimulus, similar to the pseudoisochromatic stimulus, is also composed of a mosaic with spatial noise, but it has chromatic noise randomly distributed within the mosaic. The chromatic noise, along with spatial noise, mask the presence of a target that differs from the mosaic field by luminance contrast. We investigated the

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influence of this chromatic noise on the luminance discrimination thresholds as well as the influence of the mosaic design in the measurement. Results Experiment #1. Luminance contrast threshold as a function of the chromatic noise length. The luminance contrast thresholds varied as a function of the vector length of the chromatic noise values. The presence of the chromatic noise was associated with a reduction in the luminance discrimination threshold. The higher chromatic noise length, the higher luminance contrast threshold. Figure 1 shows the mean contrast threshold (n = 40, 2 trials) as a function of the chromatic noise (Fig. 1). The mean luminance thresholds for the different chromatic noise conditions (in u'v' units) were: 0.06: 38.9% ± 6.8; 0.03: 35.6% ± 5.7; 0.015: 30.2% ± 4.6; 0.0075: 26.6% ± 4.6; and 0: 24.2% ± 5. For a subgroup of observers, we compared the luminance contrast thresholds in three consecutive sessions. Figure 2 shows the comparison of three individual results ( Fig. 2A-C) and group results of the sessions 1, 2, and 3. We observed that all the sessions showed a inhibitory influence of the chromatic vector length in the luminance contrast thresholds. We found statistical difference between the luminance contrast threshold obtained with chromatic noise of 0.06 and 0.03 u'v' units. The contrast thresholds estimated for these stimuli in the first session were significantly higher than those obtained for the same conditions during the second and third session (Fig. 2D, p < 0.05). All the other intersession comparisons for the same chromatic noise length had no

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significant differences. Experiment #2. Comparison of luminance contrast threshold estimated using mosaic and non-mosaic stimulus. Figure 3 illustrates the comparison of luminance contrast thresholds estimated using mosaic stimulus at the different chromatic noise conditions (the same from the Experiment #1) and those estimated using non-mosaic stimulus. All contrast thresholds obtained with the mosaic stimulus were significantly higher than that estimated with the non-mosaic stimulus (p < 0.01). The frequency distribution of the log10 of the ratio between the thresholds estimated with mosaics and the threshold estimated using non-mosaic stimulus are shown in Fig. 3B. The data had a normal distribution and the median of the log10 of the ratio of the distribution was 1.19, the first quartile was 1.08, and the third quartile was 1.28. Experiment #3. Comparison of the luminance contrast thresholds estimated using stimuli with different chromatic noise content. Seven observers carried out the experiment #3. No difference was observed in the contrast thresholds estimated from a same chromatic noise length around the five reference chromaticities (p > 0.05). Figure 4 shows the multiple comparisons of contrast threshold obtained from five different chromatic noises. Comparison of the luminance contrast thresholds estimated using liquid crystal display (LCD) and cathode ray tube (CRT). We compared the results obtained from the same observers (n = 7) tested using both screens and the chromatic noise generated from the C2 reference chromaticity (Fig. 5). There was no influence of the system used to stimulate the psychophysical experiment. For the same chromatic noise length, the luminance contrast thresholds estimated using

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LCD and CRT had no significant difference. We also compared the results from these observers (n = 7) tested using CRT and LCD and subgroups of the sample tested using only LCD. To compose these subgroups tested using LCD, we randomly select 7 observers from 33 observers. The results of the multiple comparisons between CRT observers and LCD observers is shown in the Table 1. We observed that after 10 comparisons between the CRT observer group and the LCD observer subgroups, 18% of the comparisons had statistical significance and it was quite constrained to the higher chromatic noise lengths. Discussion A new mosaic stimulus was used to evaluate luminance discrimination in this current study. The luminance contrast was masked by chromatic and spatial noise randomly distributed in the mosaic. The most important observation was that the presence of chromatic noise impaired luminance discrimination. This was similar to what was observed using pseudoisochromatic stimulus 28,29 ; therefore, this new mosaic stimulus can be used to investigate colour and luminance interactions on visual perception. Most of the psychophysical studies that have combined colour and luminance in the stimulus have shown that colour influenced the luminance perception and vice-versa. This influence of chromatic information within the luminance discrimination is controversial. Some investigators have shown that interaction between colour and luminance increased the luminance contrast sensitivity 25,39 . These investigators observed that when color and luminance contrasts were combined, the contrast sensitivity was enhanced for several spatial frequencies, thus reflecting a facilitatory interaction between the chromatic and luminance systems 25,39

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. An additional group investigated the effect of luminance masking in the chromatic discrimination and the effect of chromatic masking in the luminance discrimination 26 . It was reported that luminance masking improved the chromatic discrimination at all ranges of luminance contrast of the masking, while chromatic contrast masking in the luminance contrast stimulus had a double effect in the luminance discrimination 26 . Low chromatic contrast masking had no influence in the luminance discrimination, intermediate chromatic contrast masking impaired the luminance discrimination, and high chromatic contrast masking increased the luminance discrimination 26 . The results reported in the Figure 3. Comparison of the luminance contrast thresholds obtained using mosaic (gray bars) and non-mosaic (white bar) stimuli (A). The thresholds estimated using mosaic stimulus were significantly higher than those estimated using non mosaic stimulus for all subjects at all conditions. (B) Normal frequency distribution of the log10 ratio between the thresholds estimated using mosaic stimulus and non-mosaic stimulus. The median of the distribution was 1.18 log units. Error bars represents the standard deviation (n = 40). Error bars represent the standard deviation of the mean. . There was no significant difference between the thresholds estimated using each one of the adapting chromatic state for the same chromatic noise length. Error bars represent the standard deviation of the mean. present investigation as well as previous studies 28,29 confirmed previous results concerning masking experiments on visual perception. In that similar mechanisms govern the detection of luminance contrast in the presence of colour contrast, and detection of colour contrast

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in the presence of luminance contrast 40 . No significant subthreshold cross modal effect has been observed, but there was facilitation for the detection of one attribute in the presence of the other in a broad range of suprathreshold contrast. At high contrast, both colour and luminance inhibited the detection of the other contrast modality 40 . Several models of how luminance and colour channels interact have been proposed. The existence of one pathway that shares the transduction of luminance and colour has not been supported by published data from several investigators [40][41][42][43] . It is well known that P, M, and K ganglion cells have responses for colour and luminance, but with different gains 5,[11][12][13]15 , supporting the existence of multiples channels of luminance and colour from the retina to V1. After V1, distinct pathways for colour and luminance information could then combine the M, P, and K inputs with different weights in higher order neurons 44 . The results from Experiment #1 showed an inhibitory effect of the presence of chromatic information on the luminance contrast discrimination. The responsible mechanism for the task in Experiment #1 had low contrast sensitivity. This was even after the exclusion of all chromatic noise from the stimulus, and all the circles of the mosaic field had the same chromaticity. In this experimental procedure, the luminance thresholds were quite high compared to the peak of the contrast sensitivity function normally observed in a healthy subject. Another clue to the potential mechanism involved in the luminance contrast discrimination masked by

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chromatic noise can be inferred from the results in Experiment #2. This protocol sought to determine if the presence of the mosaic would potentially explain the results of high contrast thresholds. When the same task was used to identify the C-gap orientation, but in a stimulus without the mosaic array, the response results returned lower contrast thresholds. These results corresponded to data reported previously under similar testing conditions 45,46 . The comparison of chromatic discrimination estimated using pseudoisochromatic stimulus with that used an array of four homogeneous squares, one of which had a different chromaticity showed impaired discrimination in the tasks using mosaic stimulus 47 . These investigators suggested that the observer needed different strategies to identify the chromatic differences in both kinds of stimuli 47 . In the display array of squares, a simple search strategy was used, while a scan strategy was used as the choice in order to determine the C gap orientation in pseudoisochromatic design. The mosaic structure would contribute to a loss of information within the target. In the current investigation it was found that the ratio between the contrast thresholds estimated using non-mosaic and mosaic stimulus was around 1 log unit. Many authors have reported 1 log unit as the ratio of the luminance contrast sensitivity between M and P pathways activity at the retina 48,49 , LGN [50][51][52] , and psychophysics 53 . The presence of the mosaic caused the main effect on the luminance contrast thresholds. It impaired the contrast detection compared to the Figure 5. Comparison of

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the luminance contrast thresholds (n = 7) estimated from stimulus presented in the LCD (white bars) and CRT (black bars) and chromatic noise generated around C2. There was no significant difference between the thresholds estimated from the same chromatic noise length. Error bars represent the standard deviation of the mean. Table 1. p-value of the analysis of variance comparing the luminance contrast threshold obtained using stimuli with different chromatic noise length presented in LCD (10 randomly chosen subgroups with 7 observers) and CRT (7 observers). ns: no statistical significance. Condition (u'v' units) non-mosaic condition. The mosaic design favored the activity of a low contrast discrimination mechanism, such as determined by the P pathway. During our experiments, we used 16 different spatial noises during the experiments, while Cambridge Colour Test 2.3 version keeps the spatial noise constant during the test. There was no systematic investigation to evaluate the specific effects of the random or constant spatial noise in the luminance or colour discrimination. Future investigations can be done focused this question. Previous study investigated the chromatic discrimination thresholds with random spatial noise with and without luminance temporal noise and they found no differences on the thresholds obtained for normal trichromats 54 . Added to this mosaic effect, we also found the influence of the chromatic noise on the luminance thresholds. Even in the chromatic noise condition of 0.0075 u'v' units, we found impairment of the luminance contrast thresholds. The vector size of 0.0075 u'v' units is just above to the colour discrimination thresholds observed for the subjects

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between 20 and 30 years 35 . This would indicate the existence of a mechanism with high chromatic discrimination that also was working to detect the luminance contrast thresholds or the interaction between a mechanism with low luminance contrast sensitivity and a mechanism with high chromatic discrimination. The observation of low luminance contrast sensitivity and the high chromatic discrimination within the mechanisms that underly the contrast discrimination (using our novel stimulus), suggests that P pathway (and probably K pathway) potentially has a fundamental role in these mechanisms. The P pathway could be the common trunk for new streams that encode exclusively colour information and that process colour and lumiance combined information in the visual cortex 22,23 . It is probable that other visual pathways also contribute to the inputs of the colour and luminance combined pathways 23 . Using the novel stimulus, a set of chromaticities was used to introduce chromatic noise. The influence of the spectral content of the chromatic noise over the luminance discrimination was examined in Experiment 3. Five sets of chromaticity noise were used located in different places on the CIE1976 colour space: C1 (yellowish chromaticities), C2 (no colour predominance), C3 (bluish chromaticities), C4 (greenish chromaticities), and C5 (reddish chromaticities). These reference chromaticities were the same used in Regan et al. (1994) to estimate colour ellipses discrimination around them. Similar luminance discrimination was observed under these different chromatic adaptation states. This indicated that the mechanism was dependent on the masking effect of the chromatic noise over the luminance contrast discrimination. The chromatic

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content of the noise seemed to have no effect on this phenomenon. All the results presented are comparative, as such the use of the "C" as the stimuli is constant across the different sessions. It is not clear the impact on the results if another stimulus was used. Several studies have discussed about misreading in the Ishihara test 55-58 . The normal trichromat observer can read incorrectly figures that looks like other figures, for example the numbers 3 and 8. The use of other symbols could have led to slightly changes in the task we did in the present study, but the real impact of their use in the threshold values needs for additional investigations. Our results were consistent using two test platforms with different colour depth (MacBook + LCD, 10 bits per channel; ViSaGe system + CRT, 14 bits per channel). We found no systematic difference for results of the observers tested using both systems, and it enables the use of the new stimulus for applications programmed in low-cost system. We also observed that there was good replicability of the results and some learning effect after three consecutive sessions. For the higher values of the chromatic noise (0.06 and 0.03 u'v' units), there was a significant decreasing of the contrast thresholds after the first session. The novel stimulus that uses mosaic design and chromatic noise to mask luminance discrimination opens up an alternative way to investigate the colour and luminance interactions. These data, increase the comparability of the results obtained from colour discrimination using pseudoisochromatic

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stimulus and results of luminance contrast discrimination. New investigations with this novel stimulus are needed to further elucidate the mechanism that underlies the impairment of luminance discrimination in the presence of chromatic noise. Methods The influence of the chromatic and spatial noises on the luminance discrimination, was investigated using three different experimental conditions: #1, #2, and #3. Experiment #1 was designed to determine the luminance contrast threshold as a function of the chromatic noise length; Experiment #2 aimed to compare the results estimated from mosaic and non-mosaic stimuli; and finally experiment #3 compared the estimated luminance contrast threshold using different chromatic noises. All subjects exhibited normal visual acuity or corrected at 20/20. Each participant had their colour vision evaluated by three assessments; Ishihara test, Cambridge Colour Test, and Color Assessment and Diagnosis test. Additionally, the subject's general health history was also noted; no subject reported to suffer of diabetes, high blood pressure, neurological diseases, exposure to organic solvents or any other disease that could impair the visual function. All participants gave written informed consent for both study participation, and publication of identifying information/images. The Ethics Committee on Human Research of the Tropical Medicine Nucleus of the Federal University of Pará approved all procedures reported in the present study (report ##570.434). All methods were performed in accordance with Declaration of Helsinki. Experiment #1. Forty normal trichromat subjects (mean age of 27.3 ± 3.91 years old) participated in this portion of the investigation. The stimulus was presented within a software programmed in MATLAB language environment (MATLAB 2012b, Mathworks,

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Natick, MA, USA) on a MacBook PRO platform (Apple Inc., Palo Alto, USA) the liquid crystal display (LCD) had 1680 × 1050 pixels of spatial resolution. The 17" MacBook Pro drove a NVIDIA GeForce 8600 M GT graphics processor with 512 MB of GDDR3 SDRAM and dual-link DVI and 10 bits of colour resolution per channel. We used a chromameter (CS-100A, Konica Minolta, Osaka, Japan) to calibrate the display. All chromatic calculations of the present study assumed a two degrees observer angle and a D65 illuminant. Target and background were formed by a mosaic of about 428 circles. We used 16 different mosaics. The spatial dimension of the circles ranged between 0.1226 and 0.4876 degrees. We do not consider that the spatial dimensions of the mosaic elements could insert some cue in the luminance discrimination task. The chromatic noise was composed by 8 different chromaticities angled in 0, 45, 90, 135, 180, 225, 270 and 315 degrees from a reference chromaticity in the CIE1976 colour space (u = 0.219; v' = 0.48). The chromatic noise was quantified by the vector length from the reference chromaticity to each one of the 8 chromaticities. We used vector lengths of 0.06, 0.03, 0.015, 0.0075 u'v' units, and one condition without the chromatic noise, i.e. all the circles had the reference chromaticity. The Landolt C target was subgroup of circles which differed in luminance from the background. Figure 6A-E shows the stimuli in five chromatic noise conditions. The background covered 5°of visual angle, the outer and inner diameters of the

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target were 4.4° and 2.2° of visual angle, respectively, and the C gap had 1° of visual angle (Fig. 7). The psychophysical experiment consisted of estimating the luminance threshold using a four alternatives forced-choice procedure. The task to perform was to identify the orientation of the Landolt C gap (up, down, left, and right). An adaptive stochastic approximation staircase method controlled the luminance of the target, which started with 2 cd/m 2 . Throughout the duration of the experiment, the background luminance was kept at 40 cd/m 2 . The staircase rule was that after two correct responses the target luminance increased, and after one wrong response the target luminance decreased. The step size of target luminance increase/decrease followed the equation (1). The target luminance (targetlum) of the trial (t) was summed (after two correct responses) or decreased (after one wrong response) by the difference between the background luminance (bglum) and target luminance of the previous trial times a factor (f), which value was 0.5 for increase luminance step and 1.5 for decrease luminance step. After 12 reversals the staircase stopped, and the Weber contrast between the target and background was recorded. The contrast threshold was calculated averaging the last seven reversals. The forty participants performed the experimental procedure for twice. We also did the experimental procedure in a subgroup of thirteen participants for three consecutive trials to investigate the learning curve for the test. Figure 8 shows the stimulus in different luminance Weber contrast. Experiment #2. The Experiment #2 was the comparison between the Experiment

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#1 results with the luminance thresholds estimated using a non-mosaic stimulus. During the first session of Experiment 1, we also estimated the contrast threshold in a non-mosaic condition. The non-mosaic condition was randomly shown among the first session mosaic conditions. Using the same system, a non-mosaic stimulus was programmed (Fig. 6F). A Landolt-C target with the same dimensions as in Experiment 1 was centered on an isoluminant and isochromatic field. The psychophysical method to estimate the luminance threshold was the same as used in Experiment 1. We calculated the log 10 of the ratio between the thresholds obtained with mosaics and the threshold obtained using non-mosaic stimulus in order to estimate their difference. Experiment #3. Seven normal trichromats participated in this procedure (#3). We used ViSaGe system (Cambridge Research System, CRS, Rochester, UK) to present the stimulus on a CRT monitor with high spatial and temporal resolution (1680 × 1050 pixels, 75 Hz, 14 bits of colour resolution per channel). CRS toolbox for MATLAB was used to program the stimulus in MATLAB language environment (MATLAB 2012b, Mathworks, Natick, MA, USA) and to drive the ViSaGe graphic card (CRS). We used the same design of the mosaic stimulus described in Experiment #1, but the chromatic noise was modulated around by five reference chromaticities: (Fig. 9). The same chromatic noise lengths used in experimental procedure #1 were also used in the third protocol (Experiment #3). The luminance contrast threshold was estimated similarly to that of Experiment #1. Data analysis. One-way ANOVA followed by a Tukey posthoc test was

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used to compare the contrast thresholds estimated in the different chromatic noise length in the Experiments #1 and #2. Two-Way ANOVA followed by a Bonferroni posthoc test was used to determine contrast thresholds estimated for the five chromatic noise lengths and three stimuli conditions of the Experiment #3. We considered the significance level of 5%. Limitations The sample size of the Experiment #3 is different of the sample sizes of the Experiments #1 and #2. Conclusion The present study introduces a new method to investigate luminance vision for basic science and clinical applications. A combination of spatial and chromatic noise impaired the luminance contrast threshold detection. The presence of one or more colour-and luminance-sensitive visual pathways could be physiological substrate of the mechanism that underlies the luminance contrast perception of this novel stimulus.

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Isolation and Characterisation of Human-Derived blaKPC-3-Producing Salmonella enterica Serovar Rissen in 2018 In this study, we describe a Salmonella enterica serovar (S.) Rissen strain with a reduced susceptibility to meropenem, isolated from a urinary infection in an 89-year-old woman in 2018 during activity surveillance in Italy (Enter-Net Italia). The genomic characteristics, pathogenicity, and antimicrobial resistance mechanisms were investigated via a genomic approach. Antimicrobial susceptibility testing revealed a “susceptible, increased exposure” phenotype to meropenem in the S. Rissen strain (4_29_19). Whole-genome sequencing (WGS) was performed using both the NovaSeq 6000 S4 PE150 XP platform (Illumina, San Diego, CA, USA) and MinION (Oxford Nanopore). The S. Rissen 4_29_19 strain harboured two plasmids: a pKpQIL-like plasmid carrying the blaKPC-3 resistance gene in a Tn4401a transposon (pKPC_4_29_19), and a ColE-like plasmid (p4_4_29_19) without resistance genes, highly prevalent among Enterobacterales. Comparative analysis revealed that the pKPC_4_29_19 plasmid was highly related to the pKpQIL reference plasmid (GU595196), with 57% coverage and 99.96% identity, but lacking a region of about 30 kb, involving the FIIK2 replicon region and the entire transfer locus, causing the loss of its ability to conjugate. To our knowledge, this is the first time that a pKpQIL-like plasmid, carrying blaKPC-3, highly diffused in Klebsiella pneumoniae strains, has been identified in a Salmonella strain in our country. The acquisition of blaKPC genes by Salmonella spp. is extremely rare, and is reported only sporadically. In zoonotic bacteria isolated from humans, the presence of a carbapenem resistance gene carried by mobile genetic elements, usually described in healthcare-associated infection bacteria, represents an important

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concern for public health. Introduction The rapid spread of carbapenem-resistant Enterobacterales is emerging as a growing worldwide public health threat, mainly due to the production of carbapenemase enzymes causing limited therapeutic options to treat such infections [1].Carbapenem resistance is often conveyed by carbapenem-resistant beta-lactamase (bla) genes found on mobile genetic elements (MGEs), such as integrons, insertion sequences, transposons, and plasmids.These MGEs can spread across bacterial cells of the same or different species.Carbapenem resistance emerged worldwide from the 2000s, mainly in Klebsiella pneumoniae, due to the diffusion of the bla KPC gene, encoding the class A KPC enzymes, carried by high-risk clones of clonal group 258 (CG258, including sequence types ST258 and ST512) and ST307 [2][3][4].The bla KPC genes are generally located on Tn4401, a Tn3-like transposon [1,5,6].It has been detected in the plasmids of several incompatibility groups, including IncFIIK plasmids related to pKpQIL [7], IncN, ColE [8,9], IncI2, and IncX3 plasmids [10,11].This enzyme was initially described in K. pneumoniae, and is now readily detected in many members of the Enterobacterales family, but very rarely in Salmonella enterica (S.) [5][6][7]12,13].The bla KPC-3 gene has been mainly described in pKpQIL plasmids in K. pneumoniae CG258 in Italy, and more recently, also in other K. pneumoniae lineages, including ST307.pKpQILlike plasmids usually carry one or both the IncFIIk and FIBk replicons, and a cluster of approximately 35 kb encoding the conjugation machinery of pKpQIL [8,10,14,15].The mating pair stabilisation mediated by TraN drives the high-efficiency transfer of IncF plasmids, supports the role of shaping the plasmid host range, and plays a

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prominent role in pKpQIL dissemination [16].The dissemination of the pKpQIL plasmids may evolve in the rearrangement of the plasmid scaffolds with the loss of the tra region, the IncFIIk replicon, or resistance genes [17]. The minimum inhibitory concentration (MIC) to carbapenems may exhibit significant variation, depending on the membrane permeability status, the rate of carbapenem hydrolysis by the associated enzyme, and the gene expression level [18,19]. Salmonella enterica is one of the most important zoonotic foodborne pathogens causing human gastroenteritis.It comprises more than 2600 antigenically different serovars [20].In the last decade, S. Rissen, an uncommonly reported serotype in humans, has been reported to play a significant role in the outbreak of foodborne diseases in Asian countries [21].The role of this serotype has also been highlighted in Europe, and it is frequently reported in the carcasses of food-producing animals, contributing to the spread of resistance in Salmonella spp.On the other hand, cases of S. Rissen in humans are not abundant [22]. This study describes the phenotypic and genotypic analysis of a bla KPC -producing S. Rissen strain (4_29_19) isolated in Italy in 2018, during activity surveillance (Enter-Net Italia) from a woman's urine sample.This strain showing a reduced susceptibility or a susceptible, increased exposure to meropenem.A susceptible, increased exposure has been defined by EUCAST when there is a high likelihood of therapeutic success, because exposure to the agent is increased through adjustment of the dosing regimen, or by its concentration at the site of infection (https://www.eucast.org/newsiandr,accessed on 10 December 2022).Salmonella urinary tract infections are uncommon in routine hospital

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practice, and are infrequently documented in the scientific literature, although they may cause serious morbidity in immunocompromised, and even in immunocompetent, patients [23].The presence of certain pathogenesis islands (SPIs) or single virulence genes could represent an advantage for bacteria to establish the infection.The whole genome sequencing (WGS) approach elucidates the antimicrobial resistance (AMR) mechanisms, plasmid evolution, and chromosomal characteristics of this isolate. The FullForcePlasmidAssembler (FFPA.py),performing an Illumina and Nanopore WGS hybrid assembly, generated a circularised chromosome and two circularised plasmid sequences.The chromosome was 4,919,121 bp in size, and the GC content was 52.1%, containing 4538 coding sequences (CDSs), 22 rRNAs, and 86 tRNAs.The multilocus sequence typing (MLST) obtained through the chromosomal sequence resulted in ST469.The in silico analysis for the presence of antimicrobial resistance genes in the chromosome identified tet(A) and aac(6 )-Iaa genes; the last one is intrinsic to the Salmonella genus, and it is considered a cryptic gene that does not contribute to aminoglycoside resistance [24].According to SPIFinder, 4_29_19 strain contained the following pathogenicity SPIs: SPI-1 to SPI-5, SPI-8, SPI-9, and C63PI.The numerous gene clusters located in SPIs are implicated in the pathogenesis of Salmonella spp.The five SPI-1-5 are common to all serotypes of Salmonella spp.SPI-1, SPI-2, SP4, SP8, and SPA9 modulate bacterial invasion into intestinal epithelial cells; SPI-3 is involved in the survival of Salmonella spp. in macrophages (intracellular survival and replication).SPI-5 encodes five genes (pipA, pipB, pipC, pipD, and sopB) involved in intestinal mucosal fluid secretion and inflammatory responses, and C63PI encodes an iron transport system [25]. The Salmonella 4_29_19 strain presented

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several virulence genes, on the chromosome, linked with the pathogenesis of Salmonella spp.[26], involving adhesion systems, iron uptake, magnesium uptake and manganese uptake, motility, and type III secretion systems (T3SS) (Table 2).The Salmonella 4_29_19 strain harboured two plasmids: an IncFIB (pKpQIL)-like plasmid of 70,085 bp (pKPC_4_29_19), carrying various resistance genes, and a ColE-like plasmid of 4657 bp (p4_4_29_19), without antibiotic resistance genes, highly prevalent among Enterobacterales.The pKPC_4_29_19 plasmid harboured bla TEM-1 and a truncated bla OXA-9 gene between two IS26 elements; a bla KPC-3 gene located in Tn4401a, a Tn3-like element; an aac(6')-Ib gene positioned 15,260 bp downstream of the bla KPC-3 gene and flanked by a Tn3 transposon and an IS26; and a mer operon, conferring resistance to mercuric ions (Figure 1).The pKPC_4_29_19 plasmid carried the toxin/antitoxin vapB/vapC systems and parB/parM partitioning genes encoding proteins involved in plasmid stability.Comparative analysis of the pKPC_4_29_19 plasmid was performed using the reference pKpQIL plasmid (GU595196), the bla KPC-3 -carrying plasmid harboured by the carbapenemresistant K. pneumoniae clone ST258 isolated in Israel [14], and the p120Kb (CP095782) plasmid, showing the best match using BLASTN, isolated in Italy from a K. pneumoniae strain co-producing OXA-181 and KPC-121, a KPC variant conferring resistance to ceftazidime/avibactam (Figure 1) [27].The plasmid comparison revealed that the reference pKpQIL plasmid (GU595196) [14] showed 57% coverage, and 99.96% nucleotide identity, with pKPC_4_29_19.Similarly, the p120Kb (CP095782) plasmid [27] displays 58% coverage and 99.99% identity with the pKPC_4_29_19 plasmid.With respect to the reference pKpQIL plasmid (GU595196), the pKPC_4_29_19 plasmid showed a deletion of 49,520 bp, including the FIIK

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2 replicon and the entire transfer locus regions.The deletion of the transfer locus caused the loss of self-conjugation of pKPC_4_29_19 (Figure 1).Conjugation experiments on the pKPC_4_29_19 plasmid were not successful.This deletion was probably mediated by the MGE regions.It is plausible that significant rearrangements of the plasmid scaffold could be caused by the presence of six copies of IS facilitating homologous recombination through the exchange of sequences between identical or related segments. Interestingly, the pKPC_4_29_19 plasmid presented a region of 5969 bp, encoding the Tn1331 transposon, aac(6')-Ib, and IS26, which has been very rarely reported in pKpQILlike plasmids (identified in p120Kb and pKPN39428, CP056026) (Figure 1).The Tn1331 was identified to be associated with the bla KPC-3 gene in K. pneumoniae strains isolated in Jerusalem from 2005 to 2009, but it was located on a different plasmid type, and not on pKpQIL [28]. To investigate the genomic characteristics of the S. Rissen strain in a global context, the phylogenetic analysis relationship between S. Rissen 4_29_19 and 359 S. Rissen isolates belonging to ST469 in Europe (retrieved from EnteroBase in February 2023) was performed. The core genome Multilocus Sequence typing (cgMLST) analysis performed via the chewB-BACA (BSR-Based Allele Calling Algorithm) revealed that the S. Rissen isolates were widely distributed in Europe, differing by 1 allele distance (AD) from 81 AD.Italian isolates were poorly represented (three isolates), and scattered along the tree, making it impossible to identify a geographical cluster (Figure 2).The closest relatives of S. Rissen 4_29_19 were the isolates PT_SE0020 (ERR3317801) and PT_SE0012 (ERR3317780) recovered in 2015 in

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humans and pigs in Portugal, differing by 40AD and 34AD, respectively, and the 191,884 (SRR7426878) strain, isolated in the UK in humans, differing by 34 AD.PlasmidFinder analysis revealed that these isolates did not carry plasmids.To investigate the genomic characteristics of the S. Rissen strain in a global context, the phylogenetic analysis relationship between S. Rissen 4_29_19 and 359 S. Rissen isolates belonging to ST469 in Europe (retrieved from EnteroBase in February 2023) was performed.The core genome Multilocus Sequence typing (cgMLST) analysis performed via the chew-BBACA (BSR-Based Allele Calling Algorithm) revealed that the S. Rissen isolates were widely distributed in Europe, differing by 1 allele distance (AD) from 81 AD.Italian isolates were poorly represented (three isolates), and scattered along the tree, making it impossible to identify a geographical cluster (Figure 2).The closest relatives of S. Rissen 4_29_19 were the isolates PT_SE0020 (ERR3317801) and PT_SE0012 (ERR3317780) recovered in 2015 in humans and pigs in Portugal, differing by 40AD and 34AD, respectively, and the 191,884 (SRR7426878) strain, isolated in the UK in humans, differing by 34 AD.PlasmidFinder analysis revealed that these isolates did not carry plasmids. Discussion S. Rissen in the European Union (EU) is among the top 20 serovars recovered from humans and animals (pigs and bovines) in 2021 [29].Different virulence genes are involved in the pathogenicity of Salmonella spp., which contributes to their invasion and proliferation in complex environments.The virulence components, including adhesins, invasins, and toxins, frequently cluster in specific chromosomal regions, known as SPIs.S. Rissen is characterised by a variety of genetic patterns accounting for its

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virulence and antibiotic resistance [30].The S. Rissen 4_29_19 strain contains important SPIs and many virulenceassociated genes, which highlight its pathogenesis.In recent years, an increase in multidrug resistant (MDR) S. Rissen isolates has been described.Most of the S. Rissen individuals isolated from pigs were MDR (93%).The most diffused resistance pattern is ampicillin, chloramphenicol, sulfamethoxazole, tetracycline, and trimethoprim, mainly carried on integrons [31,32].In some parts of Asia, S. Rissen is also a common serovar in pigs, chickens, pork, and humans [33].It has been demonstrated that S. Rissen isolates from Thai pig farms were frequently MDR to most of the antimicrobials listed above.Considering the risk associated with Salmonella spp.contamination, humans can acquire bacterial infections containing antimicrobial-resistant genes through the consuming or handling of contaminated food [29,34,35].No meropenem-resistant human isolates were reported in the EU from 2020 to 2021, while one human isolate was identified in 2019 (OXA-48 producing S. Typhimurium var.Copenhagen, isolated from a domestically acquired infection in Spain) [36,37].In 2018 and 2021, none of the Salmonella spp.isolates recovered from animals or carcasses were reported as being resistant to meropenem [34].However, it should be noted that, in some European reporting countries, the meropenem results were interpreted using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoint, which is four dilutions higher than the ECOFF, leading to a sub-detection of isolates with carbapenem resistance genes, not conferring full resistance.EUCAST suggests, for carbapenemase, a meropenem screening cut-off of >0.125 mg/L (zone diameter < 28 mm) (http://www.eucast.org,accessed on 10 December 2022). We reported the description of an S. Rissen isolate with

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resistance to imipenem and ertapenem, and a reduced susceptibility to meropenem, that produced the KPC-3 enzyme.In Italy, the most frequent carbapenemase is KPC-3.In the last decade, KPC-3-producing K. pneumoniae, belonging to CG258, has been responsible for a high incidence of invasive, nosocomial-acquired infections in our country [15,38].The resistance to carbapenems is mainly based on the horizontal gene transfer of plasmids carrying multiple resistance genes [1], and the most frequent plasmid to be associated with the bla KPC-3 gene is the pKpQIL-like plasmid.Several reports have already described the spread of this plasmid type among different bacterial isolates and patients during hospital outbreaks [39].The description of the plasmid provides interesting confirmatory insights into the plasticity of pKpQIL-like plasmids, and their role in the ability to adapt to different bacterial species and genera by facilitating the spread of bla KPC-3 .These plasmids were isolated in clinically relevant Enterobacterales across the globe [11,40], but pKpQIL complete sequences are present in the NCBI database only in isolates of Klebsiella spp., E. coli, Enterobacter cloacae, and Raoultella ornithinolytica.The presence of bla KPC-3 in a pKpQIL-like plasmid is described in Salmonella spp.for the first time in this study.The acquisition of a KPC-3 positive pKpQIL plasmid by S. Rissen could be explained by the clinical history of the patient from whom the S. Rissen strain 4_29_19 was isolated.That patient, indeed, during previous hospitalisations, presented an acute urinary infection caused by ESBL-producing Klebsiella spp., which could be responsible for the diffusion of the pKpQIL-like plasmid to the S. Rissen strain. As previously described, our results

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indicated that pKpQIL-like plasmids are in constant genetic flux, creating new pKpQIL plasmid variants [7,9].The reported excision of the replicon region FIIK 2 and the entire transfer locus in pKPC_4_29_19 is a non-rare event.The plasticity of pKpQIL plasmids due to genetic rearrangements has been highlighted several times.In the pKpQIL-SC29 plasmid (JX442977), a reversion of the strain to carbapenem susceptibility was probably caused by IS26-mediated looping out, resulting in the deletion of the entire Tn4401-bla KPC-3 transposon and transfer locus in the plasmid [17].The expression of bla KPC-3 in S. Rissen conferred resistance to imipenem and ertapenem carbapenems, and increased the MICs for meropenem (from MIC < 0.12 mg/L in a control strain to MIC = 4.0 mg/L in S. Rissen strain 4_29_19), a phenotype compatible with an efficient membrane permeability, as described in other species [13].The acquisition of bla KPC genes by Salmonella spp. is highly unusual.It has been reported only sporadically in the S. Javiana strain from Brazil, where bla KPC-2 was carried by a small IncQ1 plasmid, or in S. Schwarzengrund in Argentina, where bla KPC-2 was present in an IncL/M plasmid, or in S. Typhimurium in China, where bla KPC-2 was located in an IncX4 plasmid [12,13,41].Other authors reported the presence of bla KPC-2 in the serovars Cubana, Javiana, and Typhimurium, from the United States, Paraguay, and Colombia, respectively, but lacked, however, genotyping studies [12,13,[42][43][44].Interestingly, the serovars Schwarzengrund and Javiana displayed a similar low-level resistance to meropenem to that of the S. Rissen 4_29_19 isolate [12,13]. The rare conjugative acquisition of pKpQIL by

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Salmonella spp.isolates is probably due to the lack of a wild-type OmpK36 porin in this species.It has been demonstrated that the mating pair stabilisation during the conjugation process requires the interaction between the TraN factor of pKpQIL and OmpK36 present in K. pneumoniae, but absent in Salmonella spp.[16].It could be hypothesised that the acquisition of the pKpQIL plasmid by the S. Rissen strain 4_29_19 occurred due to an event of conjugation supported in trans by a co-resident plasmid. The MIC of antibiotics tested was determined using the MicroScan WalkAway system (Beckman Coulter, Inc., Brea, CA, USA). The conjugation experiments of the pKPC_4_29_19 plasmid were performed using rifampicin-resistant E. coli DH5-α and Luria-Bertani agar plates (A, 10 mg/L and rifampicin 50 mg/L) at 37 • C. Conclusions The identification, in zoonotic bacteria isolated from humans, of carbapenem resistance genes carried by MGE, usually described in healthcare-associated infections, is an important concern for public health.Our findings reinforce the importance of maintaining active AMR surveillance in Salmonella spp.responsible for community infections.Integrated AMR surveillance in nosocomial and veterinary infections must be promoted and consolidated. Author Contributions: D.F. and L.V. contributed to the study's conception and planning design, genomic analysis, data analysis, data evaluation and manuscript writing.A.G.-F.contributed to the MinION sequencing, genomic analysis, data analysis, data evaluation, and manuscript writing. Informed Consent Statement: As the data in this study were analysed retrospectively, the study did not infringe upon the rights or welfare of the patient, and did not require her consent. Data Availability Statement: The genomic and plasmidic sequences have been

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deposited in GenBank under BioProject accession number PRJNA972423.The strains have been stored under the BioSample accession number SAMN34994532.The chromosome sequences were released under accession number CP126310.The manually curated plasmid sequences were released under the accession numbers OR126577 (pKPC_4_29_19) and OR126578 (p4_4_29_19). Antibiotics 2023 , 11 Figure 1 . Figure 1.Comparison of the plasmid sequences of pKPC_4_29_19 (OR126577), pKpQIL (GU595196), and p120Kb (CP095782), using the Easyfig program.Yellow arrows indicate the resistance genes; blue arrows indicate the replication genes; red arrows indicate the mobile genetic elements; green arrows indicate the tra locus; and white arrows indicate the genes involved in plasmid duplication and stability, and hypothetical proteins. Figure 1 . Figure 1.Comparison of the plasmid sequences of pKPC_4_29_19 (OR126577), pKpQIL (GU595196), and p120Kb (CP095782), using the Easyfig program.Yellow arrows indicate the resistance genes; blue arrows indicate the replication genes; red arrows indicate the mobile genetic elements; green arrows indicate the tra locus; and white arrows indicate the genes involved in plasmid duplication and stability, and hypothetical proteins. Figure 2 . Figure 2. The phylogenetic relationship between the S. Rissen strain 4_29_19 and other S. Rissen strains belonging to ST469 available in the EnteroBase database in February 2023 (strains with differences of less than 500 AD from cgMLST) via cgMLST analysis.The colours indicate different countries. S.O., A.M.D., S.A., and F.S. performed the microbiological characterisation.M.E.contributed to the Illumina sequencing.A.C. and C.L. contributed to the manuscript revision.R.O. and S.P. provided the Salmonella strain and related epidemiological data.S.R. provided clinical data.All authors have read and agreed to the published version of the

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manuscript.Funding: This research was supported by EU funding within the NextGeneration EU-MUR PNRR Extended Partnership initiative on Emerging Infectious Diseases (Project no.PE00000007, PE13 INF-ACT, Spoke 3). Table 1 . The MIC in mg/L measured for the Salmonella enterica Rissen 4_29_19 strain using the MicroScan WalkAway system (Beckman Coulter, Inc., Brea, CA, USA). Table 2 . Salmonella enterica Rissen 4_29_19 virulence factors present in the chromosome.

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APPROPRIATE STRATEGIES FOR DESIGNING CONTEMPORARY ART MUSEUMS WITH THE AIM OF ATTRACTING MORE PEOPLE IN SOCIOCULTURAL SPACES OF THE COUNTRY ( CASE STUDY: SARI, MAZANDARAN) Museums that are brimful of precious cultural treasures and indicate the identity of a society reflect human thought and artistic creativity during different generations and can convey concepts to visitors through its public displays. Withthe assumptionthatthe museumscanenrich the culture of a country youngcommunity, this study has triedtotargetitsresearchtoinvestigateways toimprove the design ofthe contemporary art museumin Mazandaran. Therefore,using theSPSS softwaresample size was estimated 384 based on Morgan table, among which55.7% were men and 44.3% were women. According tothesignificance level that wasless than 0.05, the frequencydifferencebetween the two groups of responses turned out to be significantat 99%. So the assumption that qualitative factors such as (aesthetic style designed for the set, easy access to the collections and availability of educational facilities) compared to individual and social factors such as (users' cultural and social conditions, visitors' economic situation, the sense of peace created by the presence of people in the building, etc.) have a greater impact on the category of visiting a museum is accepted. INTRODUCTION As a new phenomenon in the world of art, museums have taken different roles and functions. Exhibition, conservation, education and research can be regarded as museums different functions that have increasingly changed them intoculturalinstitutionsthat are affectingvarious aspects of today's societies (Mirzaie and Nadalian 2009, 93). Museumsshould not be considered asplaces where onlyancientmonumentsare shown, butallart and scientific fairs, galleries, libraries, archivesandmosthistoricalmonuments can be museums. Allobjects being displayed in museums have

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messages to send to their visitors and we canperceivethese messages by contemplationand study them from differentpoints of view (Kasiri, 2012, 70).One-off the most importantfunctions ofa museum is thecommunication it makes betweenvisitors andobjects displayed. In fact, we must striveto transfertheconnection and the feeling existed between thecreatorof a workandthe work itselftothevisitors, and thisis not beyond reach (DabiriNejad 2004, 96). Statistics indicate that in 19 th century only a specific group of people spent their time visiting museums (Dasam, 2008,70-120) and countries with various free admission museums have a small number of visitors, in fact for every 200 people going to movie theaters, only one person is visiting a museum. There is no room for doubt that establishing museums is important for showing human achievements. Additionally, in economicand profitability aspects, the world famousmuseumshave been successfulin attractingdomestic andforeign tourists (Nafisi, 2001, 30-43). Not only are museums an effective factor in cultural and educational fields, but also they affect countries process of gaining international identity. Todays, almost all tourists know museums such as the Louvre Museum in Paris, the British Museum in London, and the Hermitage Museum in Saint Petersburg. These museums are a part of the identity and civilization of the country holding them (Lotfi, 2007, 74-82). It is important for countries like Iran with young, educated and talented population to increase the number of their museums. This way the art status can affect the injection of new ideas through its evolution. Additionally, it should be noted that in today's world the museums have a comprehensive mission to conduct that

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is interpretation of covert concepts existing in artistic works and transferring their values to all groups of people especially ordinary people. This way people can enjoy their free time with their families as they are learning about educational and cultural values of the works. Actually, museums are presented to all people in a society disregarding their age, gender or cultural and social groups and it can be claimed that museums are appropriate for all groups (DabiriNejad 2004, 96). This mission is conducted through cultural interaction such as beliefs, attitudes, preferences and other personality aspects of individuals that have been considered as the most important factors for controlling human behaviors in behavioral science-related theories. In this regard, a public favorablespace with the capability of gathering people together canleadto the development ofcultural interaction (Yazdanfar et al, 2013, 7). Collective life is alsoan opportunityforyouthtoget away from thestressesof everyday life and spend their leisure timeand have cultural interaction and is an opportunity for people of different groups to gather together and enjoy freedom of speech and express their ideas, thusincreasingtoleranceof different groups which is encouraged among them can create more socializationand anactiveandlively space (Behzadfar and Tahmasebi, 2013, 19). Regarding the importance of interaction between people and the fact that it has not been considered seriously in today's design in the country, The present studyis toinvestigatethe factors influencingthe design ofpublicplacessuch as thecontemporary art museumto providestrategies forincreasingculturalinteractionin the community. Statement of the problem The study of evolution, in the midst ofallhuman phenomenais the most charismatic, and perhapsthe historyof cultureand civilization can be

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counted as themost fascinating branch of history. Because culture and civilizationis as expended as human life, all from scientists to readers are eager to delve into the history and the past of their job and thought especially the history of the time by which they can observe and evaluate achievements. Culture is comprised of art, literature, the science of creation, philosophy and religion. Public culture is defined as ethnic solidarity, Coexistence, assistance, cooperation, friendship, love, andfinally, afactor of mutual understanding. Mutual understanding is a two-sided understanding which is a point where culture and art converge (architecture and culture, 1999).In other words, culture is a set of human valuable and spiritual dynamic achievements that are learnt over time and in different places (In the form of non-hereditary) and are transferred from one generation to another and therefore lead to excellence of mind and body and eventually to truth and human perfection. Thus, cultural globalization requires various cultural components and indicators to be considered. Culture in this sense, includes all valuable lessons and creativity of individuals and communities and includes all ideas and great, dynamic traditions, technical talks, cultural and artistic works and various methods of communication (Rezaienabard, 2012). In fact, themuseumwithrareandancientobjects, guardourcultural and artistic heritage. However, today function of museumshas changed considerably. Themuseumshould not be considered as a placewhereonlytheancient and historical monumentsare displayed. Allobjects being displayedin museums have messages to send to their visitors and we canperceivethese messages by contemplation. Oneof the most importantfunctions ofa museum is thecommunication it makes betweenvisitors andobjects displayed. In fact, we must

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striveto transfertheconnection and the feeling existed between thecreatorof a workandthe work itselftothevisitors, and thisis notbeyond reach because a work can make connections between the present and past time and thus people can observe their deep connection with ancestors who created our culture and recover their identity. By seeing historical objects in museums, people can observe the evolution of human thought in creation and innovation of works overtly and then can see the effects of last generation culture. It is because of the fact that human made objects are a reflection of every society culture and need. Therefore we could say that in today's world, museums as a cultural institution are passages where a generation's works are preserved for the next generation ,various cultures are reflected, traditions and customs are crystallized, and cultural heritage of different nations from old times to the present time are shown and visitors' passing from this passage can build their recognition. Due to the fact that Iran is rich in art and culture; moreover, this artistic and cultural originality could be evident, thus by organizing it through various ways such as establishing contemporary art museum we can achieveglory, pride and identityincontemporaryart in today's youngsociety. The aim ofthisstudy with a cultural interaction approach is to identifyappropriate waysfor designingcontemporary art museums;moreover, in this studywe aretoanswer the following questions:  Is the designandconstruction of a contemporary art museumwith the cultural interaction approach necessary in Sari -provincial capital city of Mazandaran, Iran?  To what extent the citizens' personal characteristics (age, sex, education, etc.) can influence

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the design of a contemporary artmuseumwiththe cultural interaction approach?  To what extent aresocial and culturalfeatures effectivein the design ofacontemporary artmuseumwith cultural interaction approach?  Are the regional climatic conditionseffective in the design of contemporary artmuseumwith thecultural interaction approach?  Is the quality ofthe designedspace effective inthe prosperity of contemporary artmuseumwiththe cultural interaction approach? Table 1: A summary of conducted studies in the field of assessment of strategies for improving design of contemporary art museum Subject researcher The success of urban spaces is dependent on the use of that space and human presence in it. In fact, the architecture must increase Theoretical foundations Museum definition The wordmuseumin Persian is the pronunciation of the French word"Mouse" which has a Greekroot"Mouse" and is the name of all nineGreekgoddess ofart, poetryandmusic inAncientGreece. In English, Italian, etc.the word museumhas been used to mean "gazing, contemplating, and thinking" (Falahi, Ali 1966). Residents ofthe West defines this term as a placewhere theancient worksare kept and is considered to be a source of precious and delicate objects…. It can be said thatmuseum isthe scale of wisdom, degree of perception and reflection of orders. There, unsolvable problems are solved and historical knowledge of visitors is increased (Shirazi, 1992, 2) The rootof thisword is taken from Greek word "Mousine" meaningthe domicile ofMouse,the Goddess of art and imageryinancient Greek mythology. This Greek term is pronounced "Museum" in English and "Mousee" in French. Around 1873,the word "Mousee" entered Persian from French. International Council of MuseumsICOMsays "The museumisapermanentandnonfinancial institutionwhose doors areopen to everyoneandserve thecommunity. The aim ofestablishing museums

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is to provide conditionsfor conducting researchon evidence and works inherited fromhuman generations and environments, and to collect, conserve, produce, and create a relationship between these works and specifically, display them in order to exploit them spiritually (Rahimi and Hosseini, 2009, 79). TO put it simply, themuseumcan be definedas a permanentnon-profitorganizationthatserves thecommunity and isopen to the public. This organizationcollects and protects and displays materialevidencerelatedtohumansand their environmentso that it can bestudied andtaught, and can be a source of enjoyment (Zahedi et al. 2008, 13). Classification of Museums Museums can be classified the best way by the collections they hold. Collection or the way museums' objects are considered is the basis of this collection. It is because of the fact the nature of theworksandideas is associated with itand determine thegoals and activities ofthe museum. Thus, in general, museums can be placed in three groups: a) History(museum ofarcheology, anthropology) b) Artistic ( museum of contemporary art) c) Scientific (Museum ofgeology, botany, natural science and science and technology) (Nafisi, 2001, 34) In modern world of today, the concept of objects and the way their values are displayed and transferredtovisitors have changed.Museumsarecommunicative tools of objects, andthey are ineffective unless they are in connection with humans. Transfer of interest, information and values are the basis of educationandmuseums enjoy a special place incultural, educational, and researchpracticesincommunity level (Yavari, 1999, 66). Cultural interaction in a public space (The museum of contemporary art) Culturalinteraction is a relationship betweentwo or more peoplewhich leads toa reactionbetweenthemandthistype ofreactionis knowntoboth sides. "Actually,there are otherdefinitionsfor example, cultural and socialinteractionand communication, can

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be aphysicalissue, a look, a conversationor communication between people, which requiresthe definition ofappropriateevents and activitiesandthe roleofpeoplein a spaceandtheir participation inculturalgroupsandsocialnetworks. " (Daneshpur and Charkian, 2007, 22). What weare witnessingin today's society is a reduction in people's communicationwitheach other, although these spaces are considerably effective in the formation of meetings, chats and doing sociocultural activities (Yazdani and Teymuri, 2013, 84). Today, One of the approaches that has been considered for reviving the society, is attention to its public spaces, and it is believed to have significant impact on determination of the identity of the city and eventually, promotion of citizens' culture (Rafeyan, 2013, 16), because these places are convenient for people. Hence, they can be places forlocalcitizens to visit constantly.Differentspacesin apublic placesuch as(museum of contemporary art with the cultural interactions approach) make it possible for people tovisit different generations (Behzadfar and Tahmasebi, 2013, 18). Research methodology According to the nature, subject and objective predicted for this research, we could say it is a descriptive-analytical research and can be categorized as applied research studies. Since questionnaire and interview were used for collecting required data, this research can be survey research. Required data were collected through both libraries and surveys. Statistical population in this research is all citizens living in Sari-provincial capital city of Mazandaran. Systematic random sampling was conducted according to Morgan table and questions were given to 384 respondents. After collecting survey data through completed questionnaires, and by using SPSS software, Kolmogorov-Smirnovtest and binominal test, variables and their relationships were studied. Hypothesis and discussion Results are the

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most important part of research that lead to the development of hypotheses and add new information to past knowledge with the help of research theories (Hafeznia, 2003). The normality of data distribution (Kolmogorov-Smirnovtest) Most statistical tests including parametric tests are based upon the normality of data distribution and they are applied with this presumption that data distribution in a community or in samples selected from the community follows a normal distribution. Thus, before conducting any statistical analyses on variables, analyzers need to know variables type of distribution. Applying Kolmogorov-Smirnov test, we can achieve this objective. In Kolmogorov-Smirnov test, null hypothesis is that data follow a normal distribution; on the other hand, the alternative hypothesis is that data don't follow a normal distribution. According to the table presented below, as it can be seen in this test, the probability level and P value is more than error level in all variables (0.05). Given the P value, Null hypothesis is not rejected and so data distribution is considered to follow a normal distribution. Consequently, parametric tests have been used for testing research hypotheses. Evaluation of the research main hypothesis H0: Seemingly, in comparison with personal and social factors ( personal features such as religious beliefs, culture etc.) qualitative factors (the quality of spaces designed for museums) are more effective for contemporary art museum to be welcomed. H1: In comparison with personal and social factors (personal features such as religious beliefs, culture etc.) qualitative factors (the quality of spaces designed for museums) don't seem to be more effective for

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contemporary art museum to be welcomed. Results of table 2 show that the frequency of responses more than average was 384(52%) and the frequency of responses less than average was 48%. Given the fact that P value is less than 0.05, the distribution difference in 2 groups turned out to be significant at 99%. Therefore, H0 that stated in comparison with personal and social factors ( personal features such as religious beliefs, culture etc.) qualitative factors (the quality of spaces designed for museums) are more effective for contemporary art museum to be welcomed, is accepted. 1. Attention to exquisite design that is commensurate with the museum applicability: The results fromquestionnairesshow thatappearance plays an important role and can attract more visitors. Due to existing needs in Sari as the provincial capitalof Mazandaran, exquisite design and the use of structural systems can attract more people, even from other cities. The availability ofspaceand familiarity with that: Accordingtothedailytrafficin the cityandmoreuseofprivate vehicles, theavailabilityandlocationofthebuildingin an areawithfacilitiesforparkingandspacesecurity, and not imposing more traffic tothe cityare amongthe most importantfactorsin designing. The resultsachieved from the samplesemphasize thisissue. Designing a space that induces a sense of relaxation to visitors: Creating a sense ofrelaxationandalleviatingeveryday stressand tensionin peopleare among the most important factors thatshouldbe considered. Usingspeciallighting, selecting the correcttonesin interior designandexteriorbody of the buildingas well as designing greenspaces accordingtothemethodsof environmental psychology can help achieve this factor. Considering an appropriate place for inviting adept instructors: Cities in Iran are suffering from the paucity of cultural and social facilities. The lack of an appropriate place for inviting well-known instructors and taking

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advantage of their experience has always been artists' concern in this city. By designing such places (contemporary art museum) not only it is possible to remove this need but also we can change this exhibition to an applicable space.

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Combining FEM and Neural Networking in the Design of Optimization of Traditional Montenegrin chair The paper presents the results of tests and analysis of strength of traditional Montenegrin chair using FEM (The Finite Element Method) according to the procedure described in the relevant European standards. Stresses in the critical points of the structure were obtained and compared with values of permissible stresses for appropriate material. The influence of design parameters of chair on distribution and intensity of the stresses was considered, so it was selected the appropriate combination of parameters that presents the optimal solution from the aspect of the mentioned criterion. The obtained solution is compared with a volume equivalent model of usual chair with four legs. Based on the results we trained neural network in order to make it enable selection of design parameters of the traditional Montenegrin chairs that provide a satisfactory solution INTRODUCTION The traditional Montenegrin chair ("Stolovaca") is a traditional piece of furniture in Montenegro.The name, according to tradition, originates from the verb "sit"govern, manage and, due to the fact that patriarchy is expressed in the Montenegrin regions, in the households it was a part of the furniture on which usually only the head of the household was sitting. Recommendations for the choice of materials for its production are the result of experience.The seating panel (seat) is usually made of spruce, while the rest is mostly made of beech wood.Spruce is taken because it is lighter and less prone to distortion (bending) compared to deciduous trees, which is very important considering

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the fact that the sizes of the panel are large.Its advantage in comparison to other conifer is reflected in the fact that there is usually no resin bag (as opposed to pine) and that it is lighter (for example, than fir tree).For making the other parts of Montenegrin chair, the following can be used along with beech: maple, ash, oak, walnut, pear (suitable for carving), or other timber of higher load capacity, although beech is often chosen due to the fact that it is widespread in this region. Mechanical properties of the same types of wood [1,2], vary from region to region and some of them are presented in Table 1. Montenegrin chair compared to conventional chairs with four legs shows the following advantages: • considering that the plane is defined by three non collinear points, the reliance is made through three and not four legs, thus saving materials; • saving of materials is also reflected in the fact that in a chair with four legs for supporting the seat plate and connecting the legs in order to increase the stability of the chair additional slats are needed (Figure 1), which are not present in Montenegrin chair; • in order not to come to the wobbling of the chair, the base on which the four-legs chair is put needs to be ideally flat, while the chair with three legs will not wobble on uneven surfaces; • although the layout is a matter of taste, it can be concluded that from the aesthetic, but also the ergonomic

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viewpoint the Montenegrin chair is in advantage compared to traditional chair with four legs, among other things because of its unusualness.As the fiddle represents a traditional Montenegrin instrument which Montenegrins have been proud of trzing to decorate it with a variety of motifs, so the owners devoted time to finesses of Montenegrin chair trying to carve and shape it, leaving in some way their own mark.Thus, they all introduced some novelty, either large or small, and over time its design changed and the experience crystallized the most appropriate one (Figure 2).The backrest is shaped so that space for supporting the back is higher and thinner, becomes reduced and the spread as it follows from the back to "apples" that fall into the palm (Figure 3).This form of backrest enables proper reliance of both arms and back.The trestles have a slightly rounded shape and slantwise position to increase seat capacity and stability. OBJECTIVES AND HYPOTHESIS OF THE PAPER Testing the strength, durability and safety of furniture is done according to the procedure explained in the standards MEST EN 12520 [3] and MEST EN 1728 [4].In static testing of chairs strength a standard default value of external load is set through an appropriate tool (Figure 4.) by menas of which the load is transferred to the seating pad and is considered that the observed chair meets the criterion of strength if it doesn't come to its breaking or permanent damage after testing in accordance with MEST EN 12520.One of the aims is the optimization of the structural parameters

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of Montenegrin chair from the aspect of static strength using FEM and neural networks.Thereat, the following parameters were taken into account: a) the thickness of the seating panel; b) the thickness of the legs; c) the front legs distance; d) the tilting angle of the legs (the angle that the axis forms with the corresponding gravity line of the triangle formed by the legs).The hypothesis of this paper is: using FEM and neural networks can optimize relevant parameters of the Montenegrin chair. EXPERIMENTAL RESEARCH USING THE FINITE ELEMENT METHOD To determine the moment in which there will come to the chair fracture, the equivalent von-Mises's stresses were measured at critical points of the structure, i.e. the points of junction of legs with the seat.The assumption was introduced that fracture occurs at the moment when the stress value exceeds the lowest of the values set out in Table 1.For the purpose of testing, the ANSYS program was used based on the finite element method. The model of the traditional Montenegrin chair was drawn with a variation of the above mentioned parameters.Additionally, the tool is drawn in accordance with the dimensions given in clause 5.4 of standard MEST EN 1728.Among the tools and the seat plate foam is placed, and the load of 1.3 kN is given by means of tools.The standard MEST EN 1728 provides that during the static test of chair strength with four legs in the testing machine the last two legs of the chair are fixed to prevent it from slipping during the test.Analogously,

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a solid connection (bounded) of the front legs of the Montenegrin chair and the base is defined in the software, while between the third (rear) leg and the base the type of contact no separation is defined (the connection which does not separate the body, but it provides a very little slippage (without friction) of one body over another). In order to obtain more accurate data, at critical points, i.e. the point of junction of legs with the seat, spheres of influence were formed in which the finer finite element mesh was defined compared to the rest of the structure. For For each combination of the previously reported values the maximum stress was determined, giving a total of 81 analyses.The obtained results are shown in table 2. Table 2 shows that from the stated input values the minimum stress (5 MPa) is provided by the combination of the following parameters: panel thickness 30 mm, thickness of legs 35 mm, legs distance 380 mm and the angle of the legs 80°.It was completely expected that the smallest stresses occurred for the thickest plate and the thickest legs.In the arrangement with stresses lower than the allowable, the stresses variation is quite small with a changing range and legs tilt.However, with the increase of the stresses, the variation is greater in their values with the change of the parameters mentioned above.Minimum stress is much lower than all limit stresses set out in Table 1, which means that this chair can withstand much higher loads than those of the standard

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MEST EN 12520 (110 kg).The solution with minimal von-Misses stresses was compared with a volume equivalent constructive solution for the usual chair with four legs.Their volumes are at 10,57•10-3 m3.Distribution of stresses obtained according to the previously described procedure is shown in Figure 5.The network was selected with a maximum size of elements of 20 mm, and in the contact of legs with a seat plate spheres of influence were formed of the radius of 50 mm with a maximum size of elements of 5 mm.Such a network in the Montenegrin chair consisted of 38 969 elements and 122 442 nodes, and in a chair with four legs 31 622 elements and 101 104 nodes. Legend: a) the panel thickness in (mm) b) the thickness of the legs in (mm) c) the distance between the front legs in (mm) d) the legs tilt (°) Notes: The stresses in Table are in (MPa) The permitted stress is 44 MPa The gray background is for values that are not satisfactory On the basis of the previous figure it is observed that the maximum stresses in both cases are approximately equal. APPLICATION OF NEURAL NETWORKS IN OPTIMIZATION FUNCTION Artificial neural networks are useful for a great number of inputs over a short period of time and when the connectivity of the data is not known [5].Artificial neural networks are evolutionary optimization-based algorithms developed by [6] and [7].A neural network is defined by the neurons and their connections.All neurons are organized into layers; the sequence of layers defines the order

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in which the activations are computed [8].Back propagation is the best known and widely used learning algorithm in training multi-layers, such as the Feed Forward Neural Networks [9].The architecture of the Feed Forward Back-propagation Neural Network is presented in [10].Back-propagation provides a computationally efficient method for changing the weights in the Feed Forward Neural Network.In [11] proposes that the prediction accuracy increases when the Back-propagation Neural Networks are applied and as the number of hidden neurons increases.For these reasons, the current research uses a Feed Forward Backpropagation Neural Network with a higher number of hidden neurons to obtain faster convergence. For the purposes of this paper, work with neural networks is implemented in the software package Matlab.The rapid rise of this software package is accomplished by the development of the possibility of upgrading the modular type, i.e. the development of additional modules, the so called, Toolboxes that complement MATLAB "by the functions of interest for a certain mathematical and engineering disciplines" [12].One of the modules and Neural Network Toolbox includes functions for the design and simulation of neural networks. Neural Network Toolbox provides complete engineering of neural network in MATLAB environment, starting from design through training to simulation of a variety of neural network algorithms [13].The main application of this tool is reflected in [14, 15 and16]: • approximation and modeling of the functions; • signal processing; • forecasting, classification and grouping of data. To build the Neural Network (Results presented in Back propagation Neural Network), the data must inputted into the software package MATLAB.The training

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set for the neural network represents a set of data given in the earlier introduced Table 2. For this set of input data Feed Forward Back propagation Neural Network is created which is shown in Figure 6.The best results on the training sample has made network with five layers of neurons, each of which has per 25 neurons and the last as a rule 1.Thus the selected network, trained at the set of selected input data has performances of progress, as in Figure 7. Testing of such network training was conducted at three examples.The first example is extracted from the training sample as follows: (the plate thickness: 10; the thickness of the legs: 15; the legs distance: 380; the legs tilt: 75).The test results in this case show a very good agreement (Figure 9). CONCLUSIONS This paper is based on the application of FEM and neural networks on a traditional Montenegrin chair ("stolovaca").Bearing in mind that the majority of producers in Montenegro returns to the original shape of furniture, the traditional Montenegrin chair appears as a demanded product. For this particular shape (chair with three pillars) the appropriate standards for testing do not exist.This lack of standards has caused that on the basis of the same density values of the chair with four legs and the traditional Montenegrin chair optimizing of relevant parameters of the Montenegrin chair is performed (thickness of the seat plate, the thickness of the legs, the distance between legs and the angle of the legs). Modelling by using FEM [17] was implemented

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with the assumption that fracture occurs at the moment when the stress value exceeds the strength of the material.In this way, the data (data 81) were provided which served for the neural network training and testing.The trained neural network enables that for the variation of parameters the optimal solution is reached (e.g.minimum size), which confirms the starting hypothesis.But this paper also confirms the advantages related to the use of neural networks as a support for designers. The next fundamental step in this research deals with the desire to investigate in the future the applicability of the neural networks when multiple conditions have to be simultaneously optimized.In that case, characterized by a larger complexity, the procedure cannot benefit of a single formula to be minimized or maximized, anymore.On the contrary, an N-dimensional space for optimization emerges with the necessity to define optimization laws, acting at an upper level, before any final decision.It happens, for instance, when the chair design has to be optimized not only on the basis of a stress-strain relation (mainly related to the product's safety), but also considering additional aspects as costs, lead time, etc... Miscellaneous parameters related to the wood manufacturing, as machine tools, tools, productivity and many others have to be taken in count [18,19,20,21].Also, in this case neural networks can represent a valid approach, but instead of implementing complex networks and elaborated training procedures, it is sometimes preferable to couple them with alternative fuzzy code (as genetic algorithms).All these aspects will be investigated soon. Figure 1 .Figure 2 . Figure 1.Additional

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slats for increased stability of ordinary chair Figure 3 . Figure 3.The backrest and handrail of Montenegrin chair Figure 4 . Figure 4. Montenegrin chair and tool for testing static strength. Figure 5 ( Figure 5 (a).The arrangement of von-Mises's stresses at the Montenegrin chair Figure 8 ( a) shows the regression model of trained network where a very good convergence is observed as evidenced by the output and the value of errors in Figure 8 (b). Figure 6 . Figure 6.A Model of Feed forward Back-propagation Neural Network. Figure 7 . Figure 7. Training progress of Feed forward Backpropagation Neural Network. Figure 8 .Figure 9 . Figure 8. Results of training of Feed forward Backpropagation Neural Network.a) Figure 10 . Figure 10.The results of the tests on 2 test samples.The test results shown in Figure 10 indicate logical results in relation to the Table 2.The result lower than 44, which relates to a state of stress indicates that the chair is satisfactory as is the case in Figure 10 (a), i.e. to a unsatisfactory result as in the case of the Figure 10 (b). Table 2 . Dependence of maximum stresses on design parameters of Montenegrin chair. Fragassa C.: Improvements in wood thermoplastic composite materials properties by physical and chemical treatments, International Journal of Quality Research, Vol. 10, No. 1: pp 205-218, 2016.[21] Savoia M., Stefanovic M. and Fragassa C.: Merging Technical Competences and Human Resources with the Aim at Contributing to Transform the Adriatic Area in a Stable Hub for a Sustainable Technological Development,

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International Journal of Quality Research, Vol.10, No.1, pp.1-16, 2016.

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Diagnostic evaluation of food-related allergic diseases Food allergy is a serious and potentially life-threatening problem for an estimated 6% of children and 3.7% of adults. This review examines the diagnostic process that begins with a patient's history and physical examination. If the suspicion of IgE-mediated food allergy is compelling based on the history, skin and serology tests are routinely performed to provide confirmation for the presence of food-specific IgE antibody. In selected cases, a provocation challenge may be required as a definitive or gold standard reference test for confirmation of IgE mediated reactions to food. Variables that influence the accuracy of each of the diagnostic algorithm phases are discussed. The clinical significance of food allergen-specific IgE antibody cross-reactivity and IgE antibody epitope mapping of food allergens is overviewed. The advantages and limitations of the various diagnostic procedures are examined with an emphasis on future trends in technology and reagents. Introduction Approximately 6% of children and 3.7% of adults experience IgE-mediated allergic symptoms following the ingestion of food [1]. This contrasts with approximately 20% of the population that alters their diet for a perceived adverse reaction to food [2]. The allergist has the challenge of accurately identifying immunologically and non-immunologically-mediated reactions in the setting of this perception using information provided by the patient's history, skin and serology testing for food-specific IgE and food challenges. A number of general issues must be considered when reviewing studies on the diagnosis of food allergy. These considerations include the characteristics of the patient population in individual studies, the instrumentation and interpretation

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of allergen-specific IgE skin and serology testing and variations in food challenge protocols [3]. This review examines the diagnostic process that begins with a patient's history and physical examination. We will overview considerations involved in skin testing and then focus on specific IgE testing, which has become of paramount importance in both diagnosing and following the natural history of food allergy. We highlight potential problems with the "gold standard" of food allergy diagnosis, the double-blinded, placebo-controlled food challenge. We then review the importance of considering cross-reactivity in the interpretation of skin testing and specific-IgE testing while discussing new technologies that may help decipher the degree of cross-reactivity. Finally, we mention the experimental studies of food-allergen epitope mapping in predicting the natural history of milk and egg allergy. Clinical history The patient's history and physical examination are the foundation for the diagnosis of food allergy. The first goal is to distinguish whether the patient's reaction has an immunologic or a non-immunologic basis. Immunologic reactions include immediate-type, IgE-mediated reactions that involve the skin (pruritus, urticaria, angioedema, flushing), GI tract (oral pruritus, nausea, vomiting, diarrhea), nasal/respiratory tract (nasal congestion, rhinnorhea, ocular pruritus, sneezing, nasal pruritus, laryngeal edema, wheezing, shortness of breath) and/or the cardiovascular system (light-headedness, syncope, hypotension). These reactions can lead to death [4,5]. These symptoms typically begin within an hour of ingestion of the culprit food. The foods most commonly involved in food allergy are cow's milk, hen's egg, peanuts, tree nuts, seeds, soy, wheat, fish and crustaceans [6]. "Oral allergy syndrome" is an IgE-mediated reaction to

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fresh fruit, and less frequently nuts and vegetables, due to cross-reactivity to aeroallergens such as birch tree pollen or ragweed that cause oral pruritus, tingling and/or angioedema of the lips, palate, tongue or oropharynx [7]. Other food-mediated immunological or non-immunological reactions have different history and physical examination features from immediate-type hypersensitivity reactions. Conditions with both non-IgE and IgE based mechanisms include eosinophilic gastrointestinal disorders and atopic dermatitis. Types of cell-mediated food hypersensitivity include food-induced enterocolitis, foodinduced pulmonary hemosiderosis (Heiner's syndrone), celiac disease, contact dermatitis and dermatitis herpetiformis. Non-immunologic reactions include lactose intolerance or other problems with food digestion. This review will focus on the diagnosis of immediate-type, IgE-mediated food allergy. Skin prick testing In conjunction with the history and physical exam, diagnostic skin testing is a cornerstone in the evaluation of food allergy. It offers an in-office, rapid, and sensitive assessment of allergen sensitization. General considerations of skin testing should be discussed first before exploring the specific details of food allergen skin testing. Extensive variability exists in skin prick test devices, skin testing techniques used, and the grading and interpretation of results [8][9][10]. Each variable needs to be carefully considered before extrapolating the conclusions from a published study to one's own clinical practice [10]. Inter-physician variation in scoring and interpretation of skin tests is of particular concern in tests that are not strongly positive or definitively negative [8]. Extending this discussion to food allergy, none of the food extracts used in diagnostic skin testing have been standardized, and therefore, significant heterogeneity in allergenic protein content and

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variability in the ultimate biological potency of these extracts often occurs between lots. Fruits and vegetables produce extracts that contain particularly labile allergens, and thus the use of fresh produce may offer increased sensitivity using the prick-prick method [11]. Intradermal skin testing can also be associated with systemic reactions and it is generally not recommended for the diagnosis of food allergy [12]. In one study, no patient with a positive intradermal skin test and a negative SPT to food had a positive double-blind placebo controlled food challenge (DBPCFC) [12]. Age must also be taken into account when assessing skin test reactivity. Children younger than 2 years of age may have less skin reactivity and thus smaller wheals than older children. Children less than 1 year of age may have IgE-mediated allergic disease related to a particular food in the absence of skin test reactivity [13] While the diagnostic sensitivity of negative puncture skin test results is >95% in ruling out food allergy [2], its diagnostic specificity is limited. A larger puncture skin test wheal size in conjunction with a positive clinical history has been correlated with an increased likelihood of a positive open food challenge [14][15][16][17]. Specifically, using a lancet puncture technique and an open food challenge for confirmation, Sporik et al. demonstrated no negative challenges if puncture skin test wheal sizes were ≥ 8 mm for cow's milk or peanut and ≥ 7 mm for hen's egg [16]. Other studies have reported similar findings specifically for hen's egg [18] and tree nut allergy [19]. Specific

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IgE testing To date, the ImmunoCAP (Phadia, Uppsala, Sweden) has been the only clinically used IgE antibody immunoassay that has been systematically evaluated for its predictive value in food allergy studies. Recent studies have shown that the Immulite (Siemens Healthcare Diagnostics, Los Angeles, CA, USA) may overestimate specific IgE measurements in comparison to ImmunoCAP results [20,21]. Moreover, the Turbo RAST (currently HYTECH-288, Hycor Biomedical-Agilent, Garden Grove, CA, USA) reportedly overestimated egg-specific IgE but underestimated IgE antibody levels to birch and D. farinae in another study in comparison to the ImmunoCAP [20]. These data emphasize that the different clinically-used IgE antibody autoanalyzers detect different populations of IgE antibody. While the majority of research performed to date [1,2,[21][22][23][24][25][26] on the predictive power of quantitative food-specific IgE antibody levels has been performed using the Immu-noCAP System, it is likely that this is not the only assay method that possesses the ability to predict individuals who will experience positive food challenges. The research to investigate the predictive power of other specific IgE assays has simply not yet been performed. A 2008 Clinical Laboratory Standards Institute consensus guideline on quantitative IgE antibody methods [27] emphasizes that each of the principal serological IgE antibody assays used in clinical laboratories worldwide measures a different population of IgE antibody for any given allergen specificity. Thus, IgE antibody results generated with one method should thus not be used to make predictive clinical judgments with data in the literature generated using another assay method [20,27]. The different quantitative IgE antibody results among assays is most likely

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not the result of an inherent assay design issue or their total IgE calibration systems that are standardized to the same World Health Organization 75/502 IgE reference preparation. Rather, these different IgE antibody results are more likely to be a result of differential expression on allergenic molecules/epitopes on the allergen-containing reagent in each of these assays. A new chip-based IgE antibody technology has emerged to enhance the food allergen-specific IgE antibody data that are available to both the clinician and the patient. The microarray chip technology [28,29] has been commercialized in the form of the ImmunoCAP-ISAC or Immuno Solid phase Allergen Chip (VBC Genomics-Vienna, Austria; Phadia, Uppsala, Sweden). It currently has 103 native/recombinant component allergens from 43 allergen sources that are dotted in triplicate onto glass slides. Twenty microliters of serum are pipetted onto the chip and antibodies specific for the allergens attached to the chip surface bind during a 2 hour incubation period. Following a buffer wash, bound IgE is detected with a fluorescently-labeled anti-IgE. The chip is read in a fluorometer and fluorescent signal units are interpolated into ISU or ISAC units as semi-quantitative estimates of specific IgE antibody in the original serum. The analytical sensitivity of the ISAC varies as a function of the particular allergen specificity and is generally viewed as less than the Immu-noCAP system when the same allergens are coupled to sponge allergosorbent. This device has been providing clinical data to clinicians in Europe for several years, but is not yet cleared by North American regulatory agencies for clinical use.

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Historically, specific IgE testing has been considered by the allergist to be less sensitive than skin testing in the diagnosis of food allergy [30]. However, during the 21 st century, serological measurements of food-specific IgE antibody have become vital to the evaluation of food allergy, especially in children. Serological IgE antibody assays have the advantage of providing quantitative values that can aid in predicting with high certainty the presence of clinically significant food allergy, and thereby decreasing the need for food challenges. While this has been clearly demonstrated by Sampson and Ho [26] with the ImmunoCAP, future work needs to be done to evaluate the predictive cutpoints of the other IgE antibody assay methods. Food-specific IgE measurements on retrospectively evaluated sera were used to develop 95% positive predictive values for food allergy to milk, egg, peanut and fish in a group of children with atopic dermatitis [26]. These cutoff values were then confirmed in a prospective study of a similar patient population to achieve 90% diagnostic specificity threshold values that can be used to avoid the need for food challenge [25]. Predictive values for walnut have also been developed [23]. Importantly, different predictive values have emerged beyond the initial studies which represent differences in diet, demographics (especially age), disease states (e.g. presence or absence of atopic dermatitis) of the study populations, and the challenge protocols (see Table 1). Therefore, it is critical to consider these factors when extrapolating the clinical relevance of the quantitative measures of IgE antibody. Specific IgE values have also been used to

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determine the appropriateness of a food challenge. For instance, a specific IgE level of 2 kUa/L in a group of children with a high prevalence of atopic dermatitis represented an approximately 50% likelihood of passing a food challenge to milk, egg or peanut [31]. This 50% likelihood is considered an acceptable risk/benefit level for a food challenge [31]. In general, the magnitude of a food-specific IgE level cannot predict the severity of the clinical reaction [32]. However, there was one recent report demonstrating a significant correlation between the magnitude of specific IgE and severity of clinical reaction in egg allergic children. But there were a number of important exceptions to this association [33]. An inverse relationship was reported between the ratio of total peanut-specific IgE and challenge score to peanut allergy (r = -0.561) [34]. A study also found that the food specific to total IgE ratio was no more helpful than the specific IgE value in predicting the outcome of a food challenge [35]. The allergen-specific IgE antibody level can also aid in predicting the natural history of allergies to peanut [36], tree nuts [37], cow's milk [38] and hen's egg [39]. The rate of decline of hen egg and cow's milk-specific IgE level can help predict the resolution of the allergy [40]. Food challenge The DBPCFC has been long considered the "gold standard" for the diagnosis of food allergy and as a benchmark test from which to judge the diagnostic performance characteristics of the clinical history, skin test and IgE antibody serology. Open challenges

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may have false positive results ranging from 20.5-71% [41][42][43]. However, positive placebo reactions, that occur during the DBPCFC may be as high as 35% [44,45]. False-negative open challenges occur 1-3% of the time [2]. Some authors argue that performing several placebo and active oral provocations may be necessary to increase the specificity of DBPCFC to ~95% [3]. The same authors and others [46,47] point out that the general lack of standardized methods for the oral challenges is a primary limitation of the DBPCFC. Given a reported placebo reaction rate of 27% in adults undergoing oral drug challenge [48], oral food challenges in adults may have similar limitations. In summary, these limitations should be considered when estimating the overall diagnostic performance of SPT and specific IgE antibody testing. Cross-reactivity IgE antibody (immunological) cross-reactivity between different foods or between food and aeroallergens such as trees and grasses occurs much more readily than clinically evident cross-reactivity. These immunological cross-reactions, which are seen with both skin testing and serological measures of IgE antibody, are generally reproducible and effectively inhibited by soluble allergen. However, they can often fail to translate into a clinical response following allergen exposure. Thus, a positive IgE antibody response that is associated with a cross-reaction may be considered a false positive result in relation to the sub- Techniques to better understand the intricacies of cross reactivity remain one of the great challenges in the accurate laboratory diagnosis of food allergy. ISAC [28,29] is one IgE antibody assay that is specifically designed to aid the clinician in identifying

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the presence and quantifying the degree cross-reactive IgE antibody among the different food and pollen allergen groups that are known to share extensive homology. Bet-v 1 from Birch tree pollen, for instance, has structural homology in the PR10 family with allergenic proteins from alder tree pollen (Aln-g 1), hazelnut pollen (Cor-a 1), apple (Mal-d 1), peach (Pru-p 1), soybean (Gly-m 4), peanut (Ara-h 8), celery (Apr-g 1), carrot (Dau-c 1) and kiwi (Act-d 8). A primary sensitivity to Bet-v 1 may result in oral allergy symptoms after exposure to any of these other structurally similar (cross-reactive) allergenic molecules. The ISAC chip also can aid in identifying cross-reactivity among other allergen families such as the profilins (e.g., Bet-v 2-Birch, Ole-e 2-Olive, Hev-b 8-Latex, Phi-p 12-timothy grass), the lipid transfer proteins (e.g., Cor-a 9-hazelnut, Pru-p 3-peach, Art-v 3-mugwort and Par-j 2-Wall pellitory), the calcium binding proteins (e.g., Bet-v 4-birch, Phl-p 7-timothy grass), the tropomyosins (e.g, Pen-a 1-shrimp, Der-p 10-house dust mite, Blag 7-cockroach, Ani s 3-Anisakis), and the serum albumin family (e.g, Bos-d 6-bovine, Fel-d 2-cat, Can-f 3-dog, Equc 3-Horse and Gal-d 5-chicken). Knowledge of the extent of IgE cross-reactivity among these structurally similar proteins provides unique information to the allergist as support to the clinical history in diagnosis and management of the food allergic patient [53]. Combined with personal computer-based intelligent software algorithms that aid the practicing allergy specialist in digesting and interpreting the vast amount of IgE antibody data from the chip-based microarray assay, the issue of food crossreactivity should become more manageable. One high profile

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serological issue involving PR-10/Bet v 1 homologue cross-reactivity is the recent supplementation of the hazelnut Phadia ImmunoCAP allergosorbent with recombinant hazelnut Cor a 1 that is known to cross-react with Birch Bet v 1 [54]. Following this supplementation, serum from birch pollen allergic individuals containing IgE anti-Bet v 1 produced high IgE anti-hazelnut levels in the ImmunoCAP due to cross-reactivity. The clinical significance of these levels has been questioned and some clinicians have returned to evaluating their patients for hazelnut sensitivity using Cor a 1 unsupplemented hazelnut allergosorbents. Epitope mapping in food allergy Recent scientific advances have allowed for the identification and cloning of specific food epitopes [55]. The identification of specific IgE epitopes with immunoblot analyses may theoretically be used to better define the likelihood of clinical reactivity and/or natural history of food allergy than traditional allergen specific IgE measurements as described above in the section on "specific IgE testing" [55]. Special attention has been given to the quantitative detection of linear versus conformational food epitopes. One hypothesis is that conformational epitopes on food allergens may degrade in the GI tract, while linear epitopes retain their immunogenicity and allergenicity even in the enzyme rich, acidic gut environment [55]. Thus, children who have IgE antibodies specific for linear epitopes to alpha-s-1 and beta-casein, for instance, may be more likely to have persistent milk allergy [56]. Caseins comprise 80% of milk proteins and are composed of 4 protein fractions: α s1 -, α s2 -, β-, and κcaseins. Whey proteins comprise the remainder of milk protein. The

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relative allergenicity of each cow's milk protein is unclear, although caseins seem to be the major allergen [57] Likewise, children with persistent hen egg allergy develop IgE antibodies against more sequential and conformational epitopes of ovomucoid, the dominant and most allergenic egg allergen, and ovalbumin [58]. However, epitope mapping of peanut allergens has not offered substantial clinical benefit over specific IgE measurements for the assessment of peanut allergy [59,60]. Summary Diagnosis of IgE-mediated food allergy has progressed over the last ten years. Threshold values for allergen-specific IgE have provided allergy specialists with a new diagnostic tool to define the need for a food challenge and allowed greater insight into the natural history of allergic reactions to selected foods. These IgE antibody threshold values should be carefully used, however, while taking into consideration the potential variability resulting from differences in the study populations and the methods used in provocation testing. Better definition of the IgE cross-reactivity among foods and between foods and pollens needs to be factored into the diagnostic process to more accurately predict clinical reactivity. Furthermore, use of recombinant and native purified allergenic molecules in the micro-array chip-based ISAC assay for specific IgE antibody should help clarify some common crossreactivity seen among foods. Finally, exploration of food allergen epitope diversity and IgE avidity and specific activity (specific to total IgE ratio) may allow for improved diagnostic specificity.

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