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Comparison of percutaneous radiofrequency thermal ablation and surgical resection for small hepatocellular carcinoma

BACKGROUND: The purpose of this investigation was to compare the outcome of percutaneous radiofrequency thermal ablation therapy (PRFA) with surgical resection (SR) in the treatment of single and small hepatocellular carcinoma (HCC). METHODS: We conducted a retrospective cohort study on 231 treatment naive patients with a single HCC ≤ 3 cm who had received either curative PRFA (162 patients) or curative SR (69 patients). All patients were regularly followed up after treatment at our department with blood and radiologic tests. RESULTS: The 1-, 3- and 5-year overall survival rates after PRFA and SR were 95.4%, 79.6% and 63.1%, respectively in the PRFA group and 100%, 81.4% and 74.6%, respectively in the SR group. The corresponding recurrence free survival rates at 1, 3 and 5 years after PRFA and SR were 82.0%, 38.3% and 18.0%, respectively in the PRFA group and 86.0%, 47.2% and 26.0%, respectively in the SR group. In terms of overall survival and recurrence free survival, there were no significant differences between these two groups. In comparison of PRFA group patients with liver cirrhosis (LC) (n = 127) and SR group patients with LC (n = 50) and in comparison of PRFA group patients without LC (n = 35) and SR group patients without LC (n = 19), there were also no significant differences between two groups in terms of overall survival and recurrence free survival. In the multivariate analysis of the risk factors contributing to overall survival, serum albumin level was the sole significant factor. In the multivariate analysis of the risk factors contributing to recurrence free survival, presence of LC was the sole significant factor. The rate of serious adverse events in the SR group was significantly higher than that in the PRFA group (P = 0.023). Hospitalization length in the SR group was significantly longer than in the PRFA group (P = 0.013). CONCLUSIONS: PRFA is as effective as SR in the treatment of single and small HCC, and is less invasive than SR. Therefore, PRFA could be a first choice for the treatment of single and small HCC.

Making sense of perceptions of risk of diseases and vaccinations: a qualitative study combining models of health beliefs, decision-making and risk perception

BACKGROUND: Maintaining high levels of childhood vaccinations is important for public health. Success requires better understanding of parents' perceptions of diseases and consequent decisions about vaccinations, however few studies have considered this from the theoretical perspectives of risk perception and decision-making under uncertainty. The aim of this study was to examine the utility of subjective risk perception and decision-making theories to provide a better understanding of the differences between immunisers' and non-immunisers' health beliefs and behaviours. METHODS: In a qualitative study we conducted semi-structured in-depth interviews with 45 Australian parents exploring their experiences and perceptions of disease severity and susceptibility. Using scenarios about 'a new strain of flu' we explored how risk information was interpreted. RESULTS: We found that concepts of dread, unfamiliarity, and uncontrollability from the subjective perception of risk and ambiguity, optimistic control and omission bias from explanatory theories of decision-making under uncertainty were useful in understanding why immunisers, incomplete immunisers and non-immunisers interpreted severity and susceptibility to diseases and vaccine risk differently. Immunisers dreaded unfamiliar diseases whilst non-immunisers dreaded unknown, long term side effects of vaccines. Participants believed that the risks of diseases and complications from diseases are not equally spread throughout the community, therefore, when listening to reports of epidemics, it is not the number of people who are affected but the familiarity or unfamiliarity of the disease and the characteristics of those who have had the disease that prompts them to take preventive action. Almost all believed they themselves would not be at serious risk of the 'new strain of flu' but were less willing to take risks with their children's health. CONCLUSION: This study has found that health messages about the risks of disease which are communicated as though there is equality of risk in the population may be unproductive as these messages are perceived as unbelievable or irrelevant. The findings from this study have implications beyond the issue of childhood vaccinations as we grapple with communicating risks of new epidemics, and indeed may usefully contribute to the current debate especially in the UK of how these theories of risk and decision-making can be used to 'nudge' other health behaviours.

Development and Applications of VSV Vectors Based on Cell Tropism

Viral vectors have been available in various fields such as medical and biological research or gene therapy applications. Targeting vectors pseudotyped with distinct viral envelope proteins that influence cell tropism and transfection efficiency are useful tools not only for examining entry mechanisms or cell tropisms but also for vaccine vector development. Vesicular stomatitis virus (VSV) is an excellent candidate for development as a pseudotype vector. A recombinant VSV lacking its own envelope (G) gene has been used to produce a pseudotype or recombinant VSV possessing the envelope proteins of heterologous viruses. These viruses possess a reporter gene instead of a VSV G gene in their genome, and therefore it is easy to evaluate their infectivity in the study of viral entry, including identification of viral receptors. Furthermore, advantage can be taken of a property of the pseudotype VSV, which is competence for single-round infection, in handling many different viruses that are either difficult to amplify in cultured cells or animals or that require specialized containment facilities. Here we describe procedures for producing pseudotype or recombinant VSVs and a few of the more prominent examples from envelope viruses, such as hepatitis C virus, Japanese encephalitis virus, baculovirus, and hemorrhagic fever viruses.

Distinct Regulation of Host Responses by ERK and JNK MAP Kinases in Swine Macrophages Infected with Pandemic (H1N1) 2009 Influenza Virus

Swine influenza is an acute respiratory disease in pigs caused by swine influenza virus (SIV). Highly virulent SIV strains cause mortality of up to 10%. Importantly, pigs have long been considered “mixing vessels” that generate novel influenza viruses with pandemic potential, a constant threat to public health. Since its emergence in 2009 and subsequent pandemic spread, the pandemic (H1N1) 2009 (H1N1pdm) has been detected in pig farms, creating the risk of generating new reassortants and their possible infection of humans. Pathogenesis in SIV or H1N1pdm-infected pigs remains poorly characterized. Proinflammatory and antiviral cytokine responses are considered correlated with the intensity of clinical signs, and swine macrophages are found to be indispensible in effective clearance of SIV from pig lungs. In this study, we report a unique pattern of cytokine responses in swine macrophages infected with H1N1pdm. The roles of mitogen-activated protein (MAP) kinases in the regulation of the host responses were examined. We found that proinflammatory cytokines IL-6, IL-8, IL-10, and TNF-α were significantly induced and their induction was ERK1/2-dependent. IFN-β and IFN-inducible antiviral Mx and 2′5′-OAS were sharply induced, but the inductions were effectively abolished when ERK1/2 was inhibited. Induction of CCL5 (RANTES) was completely inhibited by inhibitors of ERK1/2 and JNK1/2, which appeared also to regulate FasL and TNF-α, critical for apoptosis in pig macrophages. We found that NFκB was activated in H1N1pdm-infected cells, but the activation was suppressed when ERK1/2 was inhibited, indicating there is cross-talk between MAP kinase and NFκB responses in pig macrophages. Our data suggest that MAP kinase may activate NFκB through the induction of RIG-1, which leads to the induction of IFN-β in swine macrophages. Understanding host responses and their underlying mechanisms may help identify venues for effective control of SIV and assist in prevention of future influenza pandemics.

RNA-Seq Based Transcriptional Map of Bovine Respiratory Disease Pathogen “Histophilus somni 2336”

Genome structural annotation, i.e., identification and demarcation of the boundaries for all the functional elements in a genome (e.g., genes, non-coding RNAs, proteins and regulatory elements), is a prerequisite for systems level analysis. Current genome annotation programs do not identify all of the functional elements of the genome, especially small non-coding RNAs (sRNAs). Whole genome transcriptome analysis is a complementary method to identify “novel” genes, small RNAs, regulatory regions, and operon structures, thus improving the structural annotation in bacteria. In particular, the identification of non-coding RNAs has revealed their widespread occurrence and functional importance in gene regulation, stress and virulence. However, very little is known about non-coding transcripts in Histophilus somni, one of the causative agents of Bovine Respiratory Disease (BRD) as well as bovine infertility, abortion, septicemia, arthritis, myocarditis, and thrombotic meningoencephalitis. In this study, we report a single nucleotide resolution transcriptome map of H. somni strain 2336 using RNA-Seq method. The RNA-Seq based transcriptome map identified 94 sRNAs in the H. somni genome of which 82 sRNAs were never predicted or reported in earlier studies. We also identified 38 novel potential protein coding open reading frames that were absent in the current genome annotation. The transcriptome map allowed the identification of 278 operon (total 730 genes) structures in the genome. When compared with the genome sequence of a non-virulent strain 129Pt, a disproportionate number of sRNAs (∼30%) were located in genomic region unique to strain 2336 (∼18% of the total genome). This observation suggests that a number of the newly identified sRNAs in strain 2336 may be involved in strain-specific adaptations.

A review on chemical and biological properties of Cayratia trifolia Linn. (Vitaceae)

Cayratia trifolia Linn. Domin Syn. Vitis trifolia (Family: Vitaceae) is commonly known as Fox grape in English; Amlabel, Ramchana in Hindi and Amlavetash in Sanskrit. It is native to India, Asia and Australia. It is a perennial climber having trifoliated leaves with 2-3 cm long petioles and ovate to oblong-ovate leaflets. Flowers are small greenish white and brown in color. Fruits are fleshy, juicy, dark purple or black, nearly spherical, about 1 cm in diameter. It is found throughout the hills in India. This perennial climber is also found in the hotter part of India from Jammu and Rajasthan to Assam extending into the peninusular India upto 600 m height. Whole plant of Cayratia trifolia has been reported to contain yellow waxy oil, steroids/terpenoids, flavonoids, tannins upon preliminary phytochemical screening. Leaves contain stilbenes (piceid, reveratrol, viniferin, ampelopsin). Stem, leaves, roots are reported to possess hydrocyanic acid, delphinidin and several flavonoids such as cyanidin is reported in the leaves. This plant also contains kaempferol, myricetin, quercetin, triterpenes and epifriedelanol. Infusion of seeds along with extract of tubers is traditionally given orally to diabetic patients to check sugar level of blood. Paste of tuberous is applied on the affected part in the treatment of snake bite. Whole plant is used as diuretic, in tumors, neuralgia and splenopathy. Its climbers wrapped around the neck of frantic bullock and poultice of leaves are used to yoke sores of bullock. The bark extract shows the antiviral, antibacterial, antiprotozoal, hypoglycemic, anticancer and diuretic activity. This article focuses on the upgraded review on chemical and biological properties of Cayratia trifolia Linn. and triggers further investigation on this plant.

Molecular mechanism for 3:1 subunit stoichiometry of rod cyclic nucleotide-gated ion channels

Molecular determinants of ion channel tetramerization are well characterized, but those involved in heteromeric channel assembly are less clearly understood. The heteromeric composition of native channels is often precisely controlled. Cyclic nucleotide-gated (CNG) channels from rod photoreceptors exhibit a 3:1 stoichiometry of CNGA1 and CNGB1 subunits that tunes the channels for their specialized role in phototransduction. Here we show, using electrophysiology, fluorescence, biochemistry, and X-ray crystallography, that the mechanism for this controlled assembly is the formation of a parallel 3-helix coiled-coil domain of the carboxy-terminal leucine zipper region of CNGA1 subunits, constraining the channel to contain three CNGA1 subunits, followed by preferential incorporation of a single CNGB1 subunit. Deletion of the carboxy-terminal leucine zipper domain relaxed the constraint and permitted multiple CNGB1 subunits in the channel. The X-ray crystal structures of the parallel 3-helix coiled-coil domains of CNGA1 and CNGA3 subunits were similar, suggesting that a similar mechanism controls the stoichiometry of cone CNG channels. SUPPLEMENTARY INFORMATION: The online version of this article (doi:10.1038/ncomms1466) contains supplementary material, which is available to authorized users.

The 2011 Retrovirology Prize winner Masao Matsuoka: forward looking and antisense

Masao Matsuoka wins the 2011 Retrovirology Prize.

Effect modification of environmental factors on influenza-associated mortality: a time-series study in two Chinese cities

BACKGROUND: Environmental factors have been associated with transmission and survival of influenza viruses but no studies have ever explored the role of environmental factors on severity of influenza infection. METHODS: We applied a Poisson regression model to the mortality data of two Chinese metropolitan cities located within the subtropical zone, to calculate the influenza associated excess mortality risks during the periods with different levels of temperature and humidity. RESULTS: The results showed that high absolute humidity (measured by vapor pressure) was significantly (p < 0.05) associated with increased risks of all-cause and cardiorespiratory deaths, but not with increased risks of pneumonia and influenza deaths. The association between absolute humidity and mortality risks was found consistent among the two cities. An increasing pattern of influenza associated mortality risks was also found across the strata of low to high relative humidity, but the results were less consistent for temperature. CONCLUSIONS: These findings highlight the need for people with chronic cardiovascular and respiratory diseases to take extra caution against influenza during hot and humid days in the subtropics and tropics.

Influenza A/H1N1 septic shock in a patient with systemic lupus erythematosus. A case report

BACKGROUND: Immunocompromised patients, such as systemic lupus erythematosus (SLE) sufferers have an increased risk of mortality, following influenza infection. In the recent pandemic, influenza A H1NI virus caused 18449 deaths, mainly because of adult respiratory distress syndrome or bacterial co-infections. CASE PRESENTATION: In this case report, an SLE patient with viral-induced septic shock, without overt pulmonary involvement, is discussed. The patient was administered oseltamivir and supportive treatment, including wide-spectrum antibiotics, vasopressors and steroids, according to the guidelines proposed for bacterial sepsis and septic shock. She finally survived and experienced a lupus flare soon after intensive care unit (ICU) discharge. CONCLUSIONS: To our knowledge, this is the first case to report severe septic shock from influenza A/H1N1 virus, without overt pulmonary involvement.

Timeliness of contact tracing among flight passengers for influenza A/H1N1 2009

BACKGROUND: During the initial containment phase of influenza A/H1N1 2009, close contacts of cases were traced to provide antiviral prophylaxis within 48 h after exposure and to alert them on signs of disease for early diagnosis and treatment. Passengers seated on the same row, two rows in front or behind a patient infectious for influenza, during a flight of ≥ 4 h were considered close contacts. This study evaluates the timeliness of flight-contact tracing (CT) as performed following national and international CT requests addressed to the Center of Infectious Disease Control (CIb/RIVM), and implemented by the Municipal Health Services of Schiphol Airport. METHODS: Elapsed days between date of flight arrival and the date passenger lists became available (contact details identified - CI) was used as proxy for timeliness of CT. In a retrospective study, dates of flight arrival, onset of illness, laboratory diagnosis, CT request and identification of contacts details through passenger lists, following CT requests to the RIVM for flights landed at Schiphol Airport were collected and analyzed. RESULTS: 24 requests for CT were identified. Three of these were declined as over 4 days had elapsed since flight arrival. In 17 out of 21 requests, contact details were obtained within 7 days after arrival (81%). The average delay between arrival and CI was 3,9 days (range 2-7), mainly caused by delay in diagnosis of the index patient after arrival (2,6 days). In four flights (19%), contacts were not identified or only after > 7 days. CI involving Dutch airlines was faster than non-Dutch airlines (P < 0,05). Passenger locator cards did not improve timeliness of CI. In only three flights contact details were identified within 2 days after arrival. CONCLUSION: CT for influenza A/H1N1 2009 among flight passengers was not successful for timely provision of prophylaxis. CT had little additional value for alerting passengers for disease symptoms, as this information already was provided during and after the flight. Public health authorities should take into account patient delays in seeking medical advise and laboratory confirmation in relation to maximum time to provide postexposure prophylaxis when deciding to install contact tracing measures. International standardization of CT guidelines is recommended.

Validation of Self-swab for Virologic Confirmation of Influenza Virus Infections in a Community Setting

Few studies have investigated the validity of self-collected nose and throat swabs for influenza confirmation in community settings. We followed outpatients with confirmed influenza with sequential measurement of viral loads and applied log-linear regression models to the viral shedding patterns. Among 176 outpatients with confirmed influenza, the detection of virus and quantitative viral loads obtained from self-swabs was consistent with statistical predictions based on earlier and later measurements, suggesting that self-collected nose and throat swabs can be a valid alternative for virologic confirmation of influenza A or B infection in a community setting.

Molecular Mimicry as a Mechanism of Autoimmune Disease

A variety of mechanisms have been suggested as the means by which infections can initiate and/or exacerbate autoimmune diseases. One mechanism is molecular mimicry, where a foreign antigen shares sequence or structural similarities with self-antigens. Molecular mimicry has typically been characterized on an antibody or T cell level. However, structural relatedness between pathogen and self does not account for T cell activation in a number of autoimmune diseases. A proposed mechanism that could have been misinterpreted for molecular mimicry is the expression of dual T cell receptors (TCR) on a single T cell. These T cells have dual reactivity to both foreign and self-antigens leaving the host vulnerable to foreign insults capable of triggering an autoimmune response. In this review, we briefly discuss what is known about molecular mimicry followed by a discussion of the current understanding of dual TCRs. Finally, we discuss three mechanisms, including molecular mimicry, dual TCRs, and chimeric TCRs, by which dual reactivity of the T cell may play a role in autoimmune diseases.

Low usage of government healthcare facilities for acute respiratory infections in guatemala: implications for influenza surveillance

BACKGROUND: Sentinel surveillance for severe acute respiratory infections in hospitals and influenza-like illness in ambulatory clinics is recommended to assist in global pandemic influenza preparedness. Healthcare utilization patterns will affect the generalizability of data from sentinel sites and the potential to use them to estimate burden of disease. The objective of this study was to measure healthcare utilization patterns in Guatemala to inform the establishment of a sentinel surveillance system for influenza and other respiratory infections, and allow estimation of disease burden. METHODS: We used a stratified, two-stage cluster survey sample to select 1200 households from the Department of Santa Rosa. Trained interviewers screened household residents for self-reported pneumonia in the last year and influenza-like illness (ILI) in the last month and asked about healthcare utilization for each illness episode. RESULTS: We surveyed 1131 (94%) households and 5449 residents between October and December 2006 and identified 323 (6%) cases of pneumonia and 628 (13%) cases of ILI. Treatment for pneumonia outside the home was sought by 92% of the children <5 years old and 73% of the persons aged five years and older. For both children <5 years old (53%) and persons aged five years and older (31%) who reported pneumonia, private clinics were the most frequently reported source of care. For ILI, treatment was sought outside the home by 81% of children <5 years old and 65% of persons aged five years and older. Government ambulatory clinics were the most frequently sought source of care for ILI both for children <5 years old (41%) and persons aged five years and older (36%). CONCLUSIONS: Sentinel surveillance for influenza and other respiratory infections based in government health facilities in Guatemala will significantly underestimate the burden of disease. Adjustment for healthcare utilization practices will permit more accurate estimation of the incidence of influenza and other respiratory pathogens in the community.

Understanding the clinical spectrum of complicated Plasmodium vivax malaria: a systematic review on the contributions of the Brazilian literature

The resurgence of the malaria eradication agenda and the increasing number of severe manifestation reports has contributed to a renewed interested in the Plasmodium vivax infection. It is the most geographically widespread parasite causing human malaria, with around 2.85 billion people living under risk of infection. The Brazilian Amazon region reports more than 50% of the malaria cases in Latin America and since 1990 there is a marked predominance of this species, responsible for 85% of cases in 2009. However, only a few complicated cases of P. vivax have been reported from this region. A systematic review of the Brazilian indexed and non-indexed literature on complicated cases of vivax malaria was performed including published articles, masters' dissertations, doctoral theses and national congresses' abstracts. The following information was retrieved: patient characteristics (demographic, presence of co-morbidities and, whenever possible, associated genetic disorders); description of each major clinical manifestation. As a result, 27 articles, 28 abstracts from scientific events' annals and 13 theses/dissertations were found, only after 1987. Most of the reported information was described in small case series and case reports of patients from all the Amazonian states, and also in travellers from Brazilian non-endemic areas. The more relevant clinical complications were anaemia, thrombocytopaenia, jaundice and acute respiratory distress syndrome, present in all age groups, in addition to other more rare clinical pictures. Complications in pregnant women were also reported. Acute and chronic co-morbidities were frequent, however death was occasional. Clinical atypical cases of malaria are more frequent than published in the indexed literature, probably due to a publication bias. In the Brazilian Amazon (considered to be a low to moderate intensity area of transmission), clinical data are in accordance with the recent findings of severity described in diverse P. vivax endemic areas (especially anaemia in Southeast Asia), however in this region both children and adults are affected. Finally, gaps of knowledge and areas for future research are opportunely pointed out.

Protective Role of the ACE2/Ang-(1–9) Axis in Cardiovascular Remodeling

Despite reduction in cardiovascular (CV) events and end-organ damage with the current pharmacologic strategies, CV disease remains the primary cause of death in the world. Pharmacological therapies based on the renin angiotensin system (RAS) blockade are used extensively for the treatment of hypertension, heart failure, and CV remodeling but in spite of their success the prevalence of end-organ damage and residual risk remain still high. Novel approaches must be discovered for a more effective treatment of residual CV remodeling and risk. The ACE2/Ang-(1–9) axis is a new and important target to counterbalance the vasoconstrictive/proliferative RAS axis. Ang-(1–9) is hydrolyzed slower than Ang-(1–7) and is able to bind the Ang II type 2 receptor. We review here the current experimental evidence suggesting that activation of the ACE2/Ang-(1–9) axis protects the heart and vessels (and possibly the kidney) from adverse cardiovascular remodeling in hypertension as well as in heart failure.

Viral Proteins Acquired from a Host Converge to Simplified Domain Architectures

The infection cycle of viruses creates many opportunities for the exchange of genetic material with the host. Many viruses integrate their sequences into the genome of their host for replication. These processes may lead to the virus acquisition of host sequences. Such sequences are prone to accumulation of mutations and deletions. However, in rare instances, sequences acquired from a host become beneficial for the virus. We searched for unexpected sequence similarity among the 900,000 viral proteins and all proteins from cellular organisms. Here, we focus on viruses that infect metazoa. The high-conservation analysis yielded 187 instances of highly similar viral-host sequences. Only a small number of them represent viruses that hijacked host sequences. The low-conservation sequence analysis utilizes the Pfam family collection. About 5% of the 12,000 statistical models archived in Pfam are composed of viral-metazoan proteins. In about half of Pfam families, we provide indirect support for the directionality from the host to the virus. The other families are either wrongly annotated or reflect an extensive sequence exchange between the viruses and their hosts. In about 75% of cross-taxa Pfam families, the viral proteins are significantly shorter than their metazoan counterparts. The tendency for shorter viral proteins relative to their related host proteins accounts for the acquisition of only a fragment of the host gene, the elimination of an internal domain and shortening of the linkers between domains. We conclude that, along viral evolution, the host-originated sequences accommodate simplified domain compositions. We postulate that the trimmed proteins act by interfering with the fundamental function of the host including intracellular signaling, post-translational modification, protein-protein interaction networks and cellular trafficking. We compiled a collection of hijacked protein sequences. These sequences are attractive targets for manipulation of viral infection.

Treatment of Neuroterrorism

Bioterrorism is defined as the intentional use of biological, chemical, nuclear, or radiological agents to cause disease, death, or environmental damage. Early recognition of a bioterrorist attack is of utmost importance to minimize casualties and initiate appropriate therapy. The range of agents that could potentially be used as weapons is wide, however, only a few of these agents have all the characteristics making them ideal for that purpose. Many of the chemical and biological weapons can cause neurological symptoms and damage the nervous system in varying degrees. Therefore, preparedness among neurologists is important. The main challenge is to be cognizant of the clinical syndromes and to be able to differentiate diseases caused by bioterrorism from naturally occurring disorders. This review provides an overview of the biological and chemical warfare agents, with a focus on neurological manifestation and an approach to treatment from a perspective of neurological critical care. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13311-011-0097-2) contains supplementary material, which is available to authorized users.

New Cardiovascular and Pulmonary Therapeutic Strategies Based on the Angiotensin-Converting Enzyme 2/Angiotensin-(1–7)/Mas Receptor Axis

Angiotensin (Ang)-(1–7) is now recognized as a biologically active component of the renin-angiotensin system (RAS). The discovery of the angiotensin-converting enzyme homologue ACE2 revealed important metabolic pathways involved in the Ang-(1–7) synthesis. This enzyme can form Ang-(1–7) from Ang II or less efficiently through hydrolysis of Ang I to Ang-(1–9) with subsequent Ang-(1–7) formation. Additionally, it is well established that the G protein-coupled receptor Mas is a functional ligand site for Ang-(1–7). The axis formed by ACE2/Ang-(1–7)/Mas represents an endogenous counter regulatory pathway within the RAS whose actions are opposite to the vasoconstrictor/proliferative arm of the RAS constituted by ACE/Ang II/AT(1) receptor. In this review we will discuss recent findings concerning the biological role of the ACE2/Ang-(1–7)/Mas arm in the cardiovascular and pulmonary system. Also, we will highlight the initiatives to develop potential therapeutic strategies based on this axis.

Unraveling the structural complexity in a single-stranded RNA tail: implications for efficient ligand binding in the prequeuosine riboswitch

Single-stranded RNAs (ssRNAs) are ubiquitous RNA elements that serve diverse functional roles. Much of our understanding of ssRNA conformational behavior is limited to structures in which ssRNA directly engages in tertiary interactions or is recognized by proteins. Little is known about the structural and dynamic behavior of free ssRNAs at atomic resolution. Here, we report the collaborative application of nuclear magnetic resonance (NMR) and replica exchange molecular dynamics (REMD) simulations to characterize the 12 nt ssRNA tail derived from the prequeuosine riboswitch. NMR carbon spin relaxation data and residual dipolar coupling measurements reveal a flexible yet stacked core adopting an A-form-like conformation, with the level of order decreasing toward the terminal ends. An A-to-C mutation within the polyadenine tract alters the observed dynamics consistent with the introduction of a dynamic kink. Pre-ordering of the tail may increase the efficacy of ligand binding above that achieved by a random-coil ssRNA. The REMD simulations recapitulate important trends in the NMR data, but suggest more internal motions than inferred from the NMR analysis. Our study unmasks a previously unappreciated level of complexity in ssRNA, which we believe will also serve as an excellent model system for testing and developing computational force fields.

Soluble RAGE as a severity marker in community acquired pneumonia associated sepsis

BACKGROUND: Community-acquired pneumonia (CAP) is considered the most important cause of death from infectious disease in developed countries. Severity assessment scores partially address the difficulties in identifying high-risk patients. A lack of specific and valid pathophysiologic severity markers affect early and effective sepsis therapy. HMGB-1, sRAGE and RAGE have been involved in sepsis and their potential as severity markers has been proposed. The aim of this study was to evaluate HMGB-1, RAGE and sRAGE levels in patients with CAP-associated sepsis and determine their possible association with clinical outcome. METHOD: We evaluated 33 patients with CAP-associated sepsis admitted to the emergency room and followed in the medical wards. Severity assessment scores (CURB-65, PSI, APACHE II, SOFA) and serologic markers (HMGB-1, RAGE, sRAGE) were evaluated on admission. RESULTS: Thirty patients with a diagnosis of CAP-associated sepsis were enrolled in the study within 24 hours after admission. Fourteen (46.6%) had pandemic (H1N1) influenza A virus, 2 (6.6%) had seasonal influenza A and 14 other diagnoses. Of the patients in the study group, 16 (53.3%) had a fatal outcome. ARDS was observed in 17 (56.6%) and a total of 22 patients had severe sepsis on admission (73%). The SOFA score showed the greatest difference between surviving and non-surviving groups (P = .003) with similar results in ARDS patients (P = .005). sRAGE levels tended to be higher in non-surviving (P = .058) and ARDS patients (P = .058). Logistic regression modeling demonstrated that SOFA (P = .013) and sRAGE (P = .05) were the only variables that modified the probability of a fatal outcome. CONCLUSION: The association of elevated sRAGE with a fatal outcome suggests that it may have an independent causal effect in CAP. SOFA scores were the only clinical factor with the ability to identify surviving and ARDS patients.

Virus Identification in Unknown Tropical Febrile Illness Cases Using Deep Sequencing

Dengue virus is an emerging infectious agent that infects an estimated 50–100 million people annually worldwide, yet current diagnostic practices cannot detect an etiologic pathogen in ∼40% of dengue-like illnesses. Metagenomic approaches to pathogen detection, such as viral microarrays and deep sequencing, are promising tools to address emerging and non-diagnosable disease challenges. In this study, we used the Virochip microarray and deep sequencing to characterize the spectrum of viruses present in human sera from 123 Nicaraguan patients presenting with dengue-like symptoms but testing negative for dengue virus. We utilized a barcoding strategy to simultaneously deep sequence multiple serum specimens, generating on average over 1 million reads per sample. We then implemented a stepwise bioinformatic filtering pipeline to remove the majority of human and low-quality sequences to improve the speed and accuracy of subsequent unbiased database searches. By deep sequencing, we were able to detect virus sequence in 37% (45/123) of previously negative cases. These included 13 cases with Human Herpesvirus 6 sequences. Other samples contained sequences with similarity to sequences from viruses in the Herpesviridae, Flaviviridae, Circoviridae, Anelloviridae, Asfarviridae, and Parvoviridae families. In some cases, the putative viral sequences were virtually identical to known viruses, and in others they diverged, suggesting that they may derive from novel viruses. These results demonstrate the utility of unbiased metagenomic approaches in the detection of known and divergent viruses in the study of tropical febrile illness.

Effects of a Non-Conservative Sequence on the Properties of β-glucuronidase from Aspergillus terreus Li-20

We cloned the β-glucuronidase gene (AtGUS) from Aspergillus terreus Li-20 encoding 657 amino acids (aa), which can transform glycyrrhizin into glycyrrhetinic acid monoglucuronide (GAMG) and glycyrrhetinic acid (GA). Based on sequence alignment, the C-terminal non-conservative sequence showed low identity with those of other species; thus, the partial sequence AtGUS(-3t) (1–592 aa) was amplified to determine the effects of the non-conservative sequence on the enzymatic properties. AtGUS and AtGUS(-3t) were expressed in E. coli BL21, producing AtGUS-E and AtGUS(-3t)-E, respectively. At the similar optimum temperature (55°C) and pH (AtGUS-E, 6.6; AtGUS(-3t)-E, 7.0) conditions, the thermal stability of AtGUS(-3t)-E was enhanced at 65°C, and the metal ions Co(2+), Ca(2+) and Ni(2+) showed opposite effects on AtGUS-E and AtGUS(-3t)-E, respectively. Furthermore, Km of AtGUS(-3t)-E (1.95 mM) was just nearly one-seventh that of AtGUS-E (12.9 mM), whereas the catalytic efficiency of AtGUS(-3t)-E was 3.2 fold higher than that of AtGUS-E (7.16 vs. 2.24 mM s(−1)), revealing that the truncation of non-conservative sequence can significantly improve the catalytic efficiency of AtGUS. Conformational analysis illustrated significant difference in the secondary structure between AtGUS-E and AtGUS(-3t)-E by circular dichroism (CD). The results showed that the truncation of the non-conservative sequence could preferably alter and influence the stability and catalytic efficiency of enzyme.

Protein sequence analysis based on hydropathy profile of amino acids

Biology sequence comparison is a fundamental task in computational biology. According to the hydropathy profile of amino acids, a protein sequence is taken as a string with three letters. Three curves of the new protein sequence were defined to describe the protein sequence. A new method to analyze the similarity/dissimilarity of protein sequence was proposed based on the conditional probability of the protein sequence. Finally, the protein sequences of ND6 (NADH dehydrogenase subunit 6) protein of eight species were taken as an example to illustrate the new approach. The results demonstrated that the method is convenient and efficient.

Filovirus Tropism: Cellular Molecules for Viral Entry

In human and non-human primates, filoviruses (Ebola and Marburg viruses) cause severe hemorrhagic fever. Recently, other animals such as pigs and some species of fruit bats have also been shown to be susceptible to these viruses. While having a preference for some cell types such as hepatocytes, endothelial cells, dendritic cells, monocytes, and macrophages, filoviruses are known to be pantropic in infection of primates. The envelope glycoprotein (GP) is responsible for both receptor binding and fusion of the virus envelope with the host cell membrane. It has been demonstrated that filovirus GP interacts with multiple molecules for entry into host cells, whereas none of the cellular molecules so far identified as a receptor/co-receptor fully explains filovirus tissue tropism and host range. Available data suggest that the mucin-like region (MLR) on GP plays an important role in attachment to the preferred target cells, whose infection is likely involved in filovirus pathogenesis, whereas the MLR is not essential for the fundamental function of the GP in viral entry into cells in vitro. Further studies elucidating the mechanisms of cellular entry of filoviruses may shed light on the development of strategies for prophylaxis and treatment of Ebola and Marburg hemorrhagic fevers.

Replication-Competent Recombinant Porcine Reproductive and Respiratory Syndrome (PRRS) Viruses Expressing Indicator Proteins and Antiviral Cytokines

Porcine reproductive and respiratory syndrome virus (PRRSV) can subvert early innate immunity, which leads to ineffective antimicrobial responses. Overcoming immune subversion is critical for developing vaccines and other measures to control this devastating swine virus. The overall goal of this work was to enhance innate and adaptive immunity following vaccination through the expression of interferon (IFN) genes by the PRRSV genome. We have constructed a series of recombinant PRRS viruses using an infectious PRRSV cDNA clone (pCMV-P129). Coding regions of exogenous genes, which included Renilla luciferase (Rluc), green and red fluorescent proteins (GFP and DsRed, respectively) and several interferons (IFNs), were constructed and expressed through a unique subgenomic mRNA placed between ORF1b and ORF2 of the PRRSV infectious clone. The constructs, which expressed Rluc, GFP, DsRed, efficiently produced progeny viruses and mimicked the parental virus in both MARC-145 cells and porcine macrophages. In contrast, replication of IFN-expressing viruses was attenuated, similar to the level of replication observed after the addition of exogenous IFN. Furthermore, the IFN expressing viruses inhibited the replication of a second PRRS virus co-transfected or co-infected. Inhibition by the different IFN subtypes corresponded to their anti-PRRSV activity, i.e., IFNω5 ° IFNα1 > IFN-β > IFNδ3. In summary, the indicator-expressing viruses provided an efficient means for real-time monitoring of viral replication thus allowing high‑throughput elucidation of the role of host factors in PRRSV infection. This was shown when they were used to clearly demonstrate the involvement of tumor susceptibility gene 101 (TSG101) in the early stage of PRRSV infection. In addition, replication‑competent IFN-expressing viruses may be good candidates for development of modified live virus (MLV) vaccines, which are capable of reversing subverted innate immune responses and may induce more effective adaptive immunity against PRRSV infection.

A Co-Opted DEAD-Box RNA Helicase Enhances Tombusvirus Plus-Strand Synthesis

Replication of plus-strand RNA viruses depends on recruited host factors that aid several critical steps during replication. In this paper, we show that an essential translation factor, Ded1p DEAD-box RNA helicase of yeast, directly affects replication of Tomato bushy stunt virus (TBSV). To separate the role of Ded1p in viral protein translation from its putative replication function, we utilized a cell-free TBSV replication assay and recombinant Ded1p. The in vitro data show that Ded1p plays a role in enhancing plus-strand synthesis by the viral replicase. We also find that Ded1p is a component of the tombusvirus replicase complex and Ded1p binds to the 3′-end of the viral minus-stranded RNA. The data obtained with wt and ATPase deficient Ded1p mutants support the model that Ded1p unwinds local structures at the 3′-end of the TBSV (−)RNA, rendering the RNA compatible for initiation of (+)-strand synthesis. Interestingly, we find that Ded1p and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is another host factor for TBSV, play non-overlapping functions to enhance (+)-strand synthesis. Altogether, the two host factors enhance TBSV replication synergistically by interacting with the viral (−)RNA and the replication proteins. In addition, we have developed an in vitro assay for Flock house virus (FHV), a small RNA virus of insects, that also demonstrated positive effect on FHV replicase activity by the added Ded1p helicase. Thus, two small RNA viruses, which do not code for their own helicases, seems to recruit a host RNA helicase to aid their replication in infected cells.

Transmission of Infectious Diseases En Route to Habitat Hotspots

BACKGROUND: The spread of infectious diseases in wildlife populations is influenced by patterns of between-host contacts. Habitat “hotspots” - places attracting a large numbers of individuals or social groups - can significantly alter contact patterns and, hence, disease propagation. Research on the importance of habitat hotspots in wildlife epidemiology has primarily focused on how inter-individual contacts occurring at the hotspot itself increase disease transmission. However, in territorial animals, epidemiologically important contacts may primarily occur as animals cross through territories of conspecifics en route to habitat hotspots. So far, the phenomenon has received little attention. Here, we investigate the importance of these contacts in the case where infectious individuals keep visiting the hotspots and in the case where these individuals are not able to travel to the hotspot any more. METHODOLOGY AND PRINCIPAL FINDINGS: We developed a simulation epidemiological model to investigate both cases in a scenario when transmission at the hotspot does not occur. We find that (i) hotspots still exacerbate epidemics, (ii) when infectious individuals do not travel to the hotspot, the most vulnerable individuals are those residing at intermediate distances from the hotspot rather than nearby, and (iii) the epidemiological vulnerability of a population is the highest when the number of hotspots is intermediate. CONCLUSIONS AND SIGNIFICANCE: By altering animal movements in their vicinity, habitat hotspots can thus strongly increase the spread of infectious diseases, even when disease transmission does not occur at the hotspot itself. Interestingly, when animals only visit the nearest hotspot, creating additional artificial hotspots, rather than reducing their number, may be an efficient disease control measure.

The Dispanins: A Novel Gene Family of Ancient Origin That Contains 14 Human Members

The Interferon induced transmembrane proteins (IFITM) are a family of transmembrane proteins that is known to inhibit cell invasion of viruses such as HIV-1 and influenza. We show that the IFITM genes are a subfamily in a larger family of transmembrane (TM) proteins that we call Dispanins, which refers to a common 2TM structure. We mined the Dispanins in 36 eukaryotic species, covering all major eukaryotic groups, and investigated their evolutionary history using Bayesian and maximum likelihood approaches to infer a phylogenetic tree. We identified ten human genes that together with the known IFITM genes form the Dispanin family. We show that the Dispanins first emerged in eukaryotes in a common ancestor of choanoflagellates and metazoa, and that the family later expanded in vertebrates where it forms four subfamilies (A–D). Interestingly, we also find that the family is found in several different phyla of bacteria and propose that it was horizontally transferred to eukaryotes from bacteria in the common ancestor of choanoflagellates and metazoa. The bacterial and eukaryotic sequences have a considerably conserved protein structure. In conclusion, we introduce a novel family, the Dispanins, together with a nomenclature based on the evolutionary origin.

Health System Resource Gaps and Associated Mortality from Pandemic Influenza across Six Asian Territories

BACKGROUND: Southeast Asia has been the focus of considerable investment in pandemic influenza preparedness. Given the wide variation in socio-economic conditions, health system capacity across the region is likely to impact to varying degrees on pandemic mitigation operations. We aimed to estimate and compare the resource gaps, and potential mortalities associated with those gaps, for responding to pandemic influenza within and between six territories in Asia. METHODS AND FINDINGS: We collected health system resource data from Cambodia, Indonesia (Jakarta and Bali), Lao PDR, Taiwan, Thailand and Vietnam. We applied a mathematical transmission model to simulate a “mild-to-moderate” pandemic influenza scenario to estimate resource needs, gaps, and attributable mortalities at province level within each territory. The results show that wide variations exist in resource capacities between and within the six territories, with substantial mortalities predicted as a result of resource gaps (referred to here as “avoidable” mortalities), particularly in poorer areas. Severe nationwide shortages of mechanical ventilators were estimated to be a major cause of avoidable mortalities in all territories except Taiwan. Other resources (oseltamivir, hospital beds and human resources) are inequitably distributed within countries. Estimates of resource gaps and avoidable mortalities were highly sensitive to model parameters defining the transmissibility and clinical severity of the pandemic scenario. However, geographic patterns observed within and across territories remained similar for the range of parameter values explored. CONCLUSIONS: The findings have important implications for where (both geographically and in terms of which resource types) investment is most needed, and the potential impact of resource mobilization for mitigating the disease burden of an influenza pandemic. Effective mobilization of resources across administrative boundaries could go some way towards minimizing avoidable deaths.

First Dating of a Recombination Event in Mammalian Tick-Borne Flaviviruses

The mammalian tick-borne flavivirus group (MTBFG) contains viruses associated with important human and animal diseases such as encephalitis and hemorrhagic fever. In contrast to mosquito-borne flaviviruses where recombination events are frequent, the evolutionary dynamic within the MTBFG was believed to be essentially clonal. This assumption was challenged with the recent report of several homologous recombinations within the Tick-borne encephalitis virus (TBEV). We performed a thorough analysis of publicly available genomes in this group and found no compelling evidence for the previously identified recombinations. However, our results show for the first time that demonstrable recombination (i.e., with large statistical support and strong phylogenetic evidences) has occurred in the MTBFG, more specifically within the Louping ill virus lineage. Putative parents, recombinant strains and breakpoints were further tested for statistical significance using phylogenetic methods. We investigated the time of divergence between the recombinant and parental strains in a Bayesian framework. The recombination was estimated to have occurred during a window of 282 to 76 years before the present. By unravelling the temporal setting of the event, we adduce hypotheses about the ecological conditions that could account for the observed recombination.

Isothermal Amplification Using a Chemical Heating Device for Point-of-Care Detection of HIV-1

BACKGROUND: To date, the use of traditional nucleic acid amplification tests (NAAT) for detection of HIV-1 DNA or RNA has been restricted to laboratory settings due to time, equipment, and technical expertise requirements. The availability of a rapid NAAT with applicability for resource-limited or point-of-care (POC) settings would fill a great need in HIV diagnostics, allowing for timely diagnosis or confirmation of infection status, as well as facilitating the diagnosis of acute infection, screening and evaluation of infants born to HIV-infected mothers. Isothermal amplification methods, such as reverse-transcription, loop-mediated isothermal amplification (RT-LAMP), exhibit characteristics that are ideal for POC settings, since they are typically quicker, easier to perform, and allow for integration into low-tech, portable heating devices. METHODOLOGY/SIGNIFICANT FINDINGS: In this study, we evaluated the HIV-1 RT-LAMP assay using portable, non-instrumented nucleic acid amplification (NINA) heating devices that generate heat from the exothermic reaction of calcium oxide and water. The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients. CONCLUSION: The RT-LAMP isothermal amplification method used in conjunction with a chemical heating device provides a portable, rapid and robust NAAT platform that has the potential to facilitate HIV-1 testing in resource-limited settings and POC.

Design and management of an orthopaedic bone bank in the Netherlands

The design and management of an orthopaedic bone bank is a complex process in which medical organisation and legislation intertwine. Neither in the Netherlands, nor in any other European country, there are official guidelines for the organisation and management of an orthopaedic bone bank. In the Netherlands, the recently modified ‘law of security and quality for using human materials’ (WVKL) dictates requirements for technical and organisational aspects for the use of human tissue and cells. The bone bank procedures include a thorough questionnaire for donor selection, extensive serological, bacteriological and histopathological examination, as well as standard procedures for registration, processing, preservation, storage and distribution of bone allografts. This article describes the organisation of an accredited bone bank and can be used as a proposition for an official guideline or can be useful as an example for other orthopaedic bone banks in Europe.

Dectin-1 and DC-SIGN Polymorphisms Associated with Invasive Pulmonary Aspergillosis Infection

The recognition of pathogen-derived structures by C-type lectins and the chemotactic activity mediated by the CCL2/CCR2 axis are critical steps in determining the host immune response to fungi. The present study was designed to investigate whether the presence of single nucleotide polymorphisms (SNPs) within DC-SIGN, Dectin-1, Dectin-2, CCL2 and CCR2 genes influence the risk of developing Invasive Pulmonary Aspergillosis (IPA). Twenty-seven SNPs were selected using a hybrid functional/tagging approach and genotyped in 182 haematological patients, fifty-seven of them diagnosed with proven or probable IPA according to the 2008 EORTC/MSG criteria. Association analysis revealed that carriers of the Dectin-1 (rs3901533 T/T) and Dectin-1 (rs7309123 G/G) genotypes and DC-SIGN (rs4804800 G), DC-SIGN (rs11465384 T), DC-SIGN (7248637 A) and DC-SIGN (7252229 C) alleles had a significantly increased risk of IPA infection (OR = 5.59 95%CI 1.37–22.77; OR = 4.91 95%CI 1.52–15.89; OR = 2.75 95%CI 1.27–5.95; OR = 2.70 95%CI 1.24–5.90; OR = 2.39 95%CI 1.09–5.22 and OR = 2.05 95%CI 1.00–4.22, respectively). There was also a significantly increased frequency of galactomannan positivity among patients carrying the Dectin-1 (rs3901533_T) allele and Dectin-1 (rs7309123_G/G) genotype. In addition, healthy individuals with this latter genotype showed a significantly decreased level of Dectin-1 mRNA expression compared to C-allele carriers, suggesting a role of the Dectin-1 (rs7309123) polymorphism in determining the levels of Dectin-1 and, consequently, the level of susceptibility to IPA infection. SNP-SNP interaction (epistasis) analysis revealed significant interactions models including SNPs in Dectin-1, Dectin-2, CCL2 and CCR2 genes, with synergistic genetic effects. Although these results need to be further validated in larger cohorts, they suggest that Dectin-1, DC-SIGN, Dectin-2, CCL2 and CCR2 genetic variants influence the risk of IPA infection and might be useful in developing a risk-adapted prophylaxis.

Lethal Nipah Virus Infection Induces Rapid Overexpression of CXCL10

Nipah virus (NiV) is a recently emerged zoonotic Paramyxovirus that causes regular outbreaks in East Asia with mortality rate exceeding 75%. Major cellular targets of NiV infection are endothelial cells and neurons. To better understand virus-host interaction, we analyzed the transcriptome profile of NiV infection in primary human umbilical vein endothelial cells. We further assessed some of the obtained results by in vitro and in vivo methods in a hamster model and in brain samples from NiV-infected patients. We found that NiV infection strongly induces genes involved in interferon response in endothelial cells. Among the top ten upregulated genes, we identified the chemokine CXCL10 (interferon-induced protein 10, IP-10), an important chemoattractant involved in the generation of inflammatory immune response and neurotoxicity. In NiV-infected hamsters, which develop pathology similar to what is seen in humans, expression of CXCL10 mRNA was induced in different organs with kinetics that followed NiV replication. Finally, we showed intense staining for CXCL10 in the brain of patients who succumbed to lethal NiV infection during the outbreak in Malaysia, confirming induction of this chemokine in fatal human infections. This study sheds new light on NiV pathogenesis, indicating the role of CXCL10 during the course of infection and suggests that this chemokine may serve as a potential new marker for lethal NiV encephalitis.

Influenza Virus Respiratory Infection and Transmission Following Ocular Inoculation in Ferrets

While influenza viruses are a common respiratory pathogen, sporadic reports of conjunctivitis following human infection demonstrates the ability of this virus to cause disease outside of the respiratory tract. The ocular surface represents both a potential site of virus replication and a portal of entry for establishment of a respiratory infection. However, the properties which govern ocular tropism of influenza viruses, the mechanisms of virus spread from ocular to respiratory tissue, and the potential differences in respiratory disease initiated from different exposure routes are poorly understood. Here, we established a ferret model of ocular inoculation to explore the development of virus pathogenicity and transmissibility following influenza virus exposure by the ocular route. We found that multiple subtypes of human and avian influenza viruses mounted a productive virus infection in the upper respiratory tract of ferrets following ocular inoculation, and were additionally detected in ocular tissue during the acute phase of infection. H5N1 viruses maintained their ability for systemic spread and lethal infection following inoculation by the ocular route. Replication-independent deposition of virus inoculum from ocular to respiratory tissue was limited to the nares and upper trachea, unlike traditional intranasal inoculation which results in virus deposition in both upper and lower respiratory tract tissues. Despite high titers of replicating transmissible seasonal viruses in the upper respiratory tract of ferrets inoculated by the ocular route, virus transmissibility to naïve contacts by respiratory droplets was reduced following ocular inoculation. These data improve our understanding of the mechanisms of virus spread following ocular exposure and highlight differences in the establishment of respiratory disease and virus transmissibility following use of different inoculation volumes and routes.

Placement of Leucine Zipper Motifs at the Carboxyl Terminus of HIV-1 Protease Significantly Reduces Virion Production

Natural HIV-1 protease (PR) is homodimeric. Some researchers believe that interactions between HIV-1 Gag-Pol molecules trigger the activation of embedded PR (which mediates Gag and Gag-Pol cleavage), and that Gag-Pol assembly domains outside of PR may contribute to PR activation by influencing PR dimer interaction in a Gag-Pol context. To determine if the enhancement of PR dimer interaction facilitates PR activation, we placed single or tandem repeat leucine zippers (LZ) at the PR C-terminus, and looked for a correlation between enhanced Gag processing efficiency and increased Gag-PR-LZ multimerization capacity. We found significant reductions in virus-like particles (VLPs) produced by HIV-1 mutants, with LZ fused to the end of PR as a result of enhanced Gag cleavage efficiency. Since VLP production can be restored to wt levels following PR activity inhibition, this assembly defect is considered PR activity-dependent. We also found a correlation between the LZ enhancement effect on Gag cleavage and enhanced Gag-PR multimerization. The results suggest that PR dimer interactions facilitated by forced Gag-PR multimerization lead to premature Gag cleavage, likely a result of premature PR activation. Our conclusion is that placement of a heterologous dimerization domain downstream of PR enhances PR-mediated Gag cleavage efficiency, implying that structural conformation, rather than the primary sequence outside of PR, is a major determinant of HIV-1 PR activation.

Using Support Vector Machine and Evolutionary Profiles to Predict Antifreeze Protein Sequences

Antifreeze proteins (AFPs) are ice-binding proteins. Accurate identification of new AFPs is important in understanding ice-protein interactions and creating novel ice-binding domains in other proteins. In this paper, an accurate method, called AFP_PSSM, has been developed for predicting antifreeze proteins using a support vector machine (SVM) and position specific scoring matrix (PSSM) profiles. This is the first study in which evolutionary information in the form of PSSM profiles has been successfully used for predicting antifreeze proteins. Tested by 10-fold cross validation and independent test, the accuracy of the proposed method reaches 82.67% for the training dataset and 93.01% for the testing dataset, respectively. These results indicate that our predictor is a useful tool for predicting antifreeze proteins. A web server (AFP_PSSM) that implements the proposed predictor is freely available.

Reliability and External Validity of AMSTAR in Assessing Quality of TCM Systematic Reviews

Objective. The aim of this study is to measure the reliability and external validity of AMSTAR by applying it to a sample of TCM systematic reviews. Study Design and Methods. We tested the agreement, reliability, construct validity, and feasibility of AMSTAR through comparisons with OQAQ. Statistical analyses were performed by using SPSS 13.0. Results. A random of sample with 41 TCM systematic reviews was selected from a database. The interrater agreement of the individual items of AMSTAR was moderate with a mean kappa of 0.50 (95% CI: 0.26, 0.73). The ICC for AMSTAR against OQAQ (total score of 9 items, excluding item 10) was 0.87 (95% CI: 0.76, 0.93). Conclusions. Although there is room for improvement on few items, the new tool is reliable, valid, and easy to use for methodological quality assessment of systematic reviews on TCM.

Innate Immune Response of Human Alveolar Macrophages during Influenza A Infection

Alveolar macrophages (AM) are one of the key cell types for initiating inflammatory and immune responses to influenza virus in the lung. However, the genome-wide changes in response to influenza infection in AM have not been defined. We performed gene profiling of human AM in response to H1N1 influenza A virus PR/8 using Affymetrix HG-U133 Plus 2.0 chips and verified the changes at both mRNA and protein levels by real-time RT-PCR and ELISA. We confirmed the response with a contemporary H3N2 influenza virus A/New York/238/2005 (NY/238). To understand the local cellular response, we also evaluated the impact of paracrine factors on virus-induced chemokine and cytokine secretion. In addition, we investigated the changes in the expression of macrophage receptors and uptake of pathogens after PR/8 infection. Although macrophages fail to release a large amount of infectious virus, we observed a robust induction of type I and type III interferons and several cytokines and chemokines following influenza infection. CXCL9, 10, and 11 were the most highly induced chemokines by influenza infection. UV-inactivation abolished virus-induced cytokine and chemokine response, with the exception of CXCL10. The contemporary influenza virus NY/238 infection of AM induced a similar response as PR/8. Inhibition of TNF and/or IL-1β activity significantly decreased the secretion of the proinflammatory chemokines CCL5 and CXCL8 by over 50%. PR/8 infection also significantly decreased mRNA levels of macrophage receptors including C-type lectin domain family 7 member A (CLEC7A), macrophage scavenger receptor 1 (MSR1), and CD36, and reduced uptake of zymosan. In conclusion, influenza infection induced an extensive proinflammatory response in human AM. Targeting local components of innate immune response might provide a strategy for controlling influenza A infection-induced proinflammatory response in vivo.

Successful treatment of Chlamydophila pneumoniae acute respiratory distress syndrome with extracorporeal membrane oxygenator: a case report and diagnostic review

INTRODUCTION: Chlamydophila pneumoniae is a respiratory pathogen known to infect the upper and lower respiratory tracts. Infection severity can range from sub-clinical pulmonary infection to acute respiratory distress syndrome. CASE PRESENTATION: A previously healthy 62-year-old Caucasian man was admitted to our hospital for acute respiratory failure. Serum samples obtained every week starting from the day of admission showed clear-cut seroconversion for C. pneumoniae antibodies. All other cultures obtained during the first days of hospitalization were negative. Despite maximal ventilatory support (high positive end expiratory pressure, fraction of inspired oxygen of 1.0, nitric oxide inhalation, neuromuscular blocking agents and prone positioning), our patient remained severely hypoxemic, which led us to initiate an extracorporeal membrane oxygenation treatment. Extracorporeal membrane oxygenation and hemodiafiltration were withdrawn on day 12. Our patient was extubated on day 18 and discharged from our Intensive Care Unit on day 20. He went home a month later. CONCLUSION: We describe the first published case of acute respiratory distress syndrome due to C. pneumoniae infection successfully treated by extracorporeal membrane oxygenation, a very useful tool in this syndrome. A quick and specific method for the definite diagnosis of Chlamydophila infection should be developed.

Environmental Sampling for Avian Influenza Virus A (H5N1) in Live-Bird Markets, Indonesia

To identify environmental sites commonly contaminated by avian influenza virus A (H5N1) in live-bird markets in Indonesia, we investigated 83 markets in 3 provinces in Indonesia. At each market, samples were collected from up to 27 poultry-related sites to assess the extent of contamination. Samples were tested by using real-time reverse transcription–PCR and virus isolation. A questionnaire was used to ascertain types of birds in the market, general infrastructure, and work practices. Thirty-nine (47%) markets showed contamination with avian influenza virus in >1 of the sites sampled. Risk factors were slaughtering birds in the market and being located in West Java province. Protective factors included daily removal of waste and zoning that segregated poultry-related work flow areas. These results can aid in the design of evidence-based programs concerning environmental sanitation, food safety, and surveillance to reduce the risk for avian influenza virus A (H5N1) transmission in live-bird markets.

Influenza in Refugees on the Thailand–Myanmar Border, May–October 2009

We describe the epidemiology of influenza virus infections in refugees in a camp in rural Southeast Asia during May–October 2009, the first 6 months after identification of pandemic (H1N1) 2009 in Thailand. Influenza A viruses were detected in 20% of patients who had influenza-like illness and in 23% of those who had clinical pneumonia. Seasonal influenza A (H1N1) was the predominant virus circulating during weeks 26–33 (June 25–August 29) and was subsequently replaced by the pandemic strain. A review of passive surveillance for acute respiratory infection did not show an increase in acute respiratory tract infection incidence associated with the arrival of pandemic (H1N1) 2009 in the camp.

Early Clinical Features of Dengue Virus Infection in Nicaraguan Children: A Longitudinal Analysis

BACKGROUND: Tens of millions of dengue cases and approximately 500,000 life-threatening complications occur annually. New tools are needed to distinguish dengue from other febrile illnesses. In addition, the natural history of pediatric dengue early in illness in a community-based setting has not been well-defined. METHODS: Data from the multi-year, ongoing Pediatric Dengue Cohort Study of approximately 3,800 children aged 2–14 years in Managua, Nicaragua, were used to examine the frequency of clinical signs and symptoms by day of illness and to generate models for the association of signs and symptoms during the early phase of illness and over the entire course of illness with testing dengue-positive. Odds ratios (ORs) and 95% confidence intervals were calculated using generalized estimating equations (GEE) for repeated measures, adjusting for age and gender. RESULTS: One-fourth of children who tested dengue-positive did not meet the WHO case definition for suspected dengue. The frequency of signs and symptoms varied by day of illness, dengue status, and disease severity. Multivariable GEE models showed increased odds of testing dengue-positive associated with fever, headache, retro-orbital pain, myalgia, arthralgia, rash, petechiae, positive tourniquet test, vomiting, leukopenia, platelets ≤150,000 cells/mL, poor capillary refill, cold extremities and hypotension. Estimated ORs tended to be higher for signs and symptoms over the course of illness compared to the early phase of illness. CONCLUSIONS: Day-by-day analysis of clinical signs and symptoms together with longitudinal statistical analysis showed significant associations with testing dengue-positive and important differences during the early phase of illness compared to the entire course of illness. These findings stress the importance of considering day of illness when developing prediction algorithms for real-time clinical management.

Influenza A H1N1 2009 (Swine Flu) and Pregnancy

The Influenza A H1N1 pandemic (A H1N1) occurred between June 2009 and August 2010. Although the pandemic is now over, the virus has emerged as the predominant strain in the current seasonal influenza phase in the northern hemisphere. The A H1N1 influenza is a novel strain of the influenza A virus and is widely known as swine flu. The virus contains a mixture of genetic material from human, pig and bird flu virus. It is a new variety of flu which people have not had much immunity to. Much has been learnt from the Pandemic of 2009/2010 but the messages about vaccination and treatment seem to be taken slowly by the clinical profession. Most people affected by the virus, including pregnant women, suffer a mild viral illness, and make a full recovery. The median duration of illness is around seven days. This influenza typically affects the younger age group i.e. from the ages of 5–65 years. Current experience shows that the age group experiencing increased morbidity and mortality rates are in those under 65 years of age. Pregnant women, because of their altered immunity and physiological adaptations, are at higher risk of developing pulmonary complications, especially in the second and third trimesters. In the United Kingdom, twelve maternal deaths were reported to be associated with the H1N1 virus during the pandemic and clear avoidable factors were identified (Modder, Review of Maternal Deaths in the UK related to A H1N1 2009 influenza (CMACE). www.cmace.org.uk, 2010). The pregnancy outcomes were also poor for women who were affected by the virus with a fivefold increase in the perinatal mortality rate and threefold increase in the preterm delivery rate (Yates et al. Health Technol Assess 14(34):109–182, 2010). There continues to be a low uptake of the flu vaccine and commencement of antiviral treatment for pregnant women.

Use and Evaluation of Molecular Diagnostics for Pneumonia Etiology Studies

Comprehensive microbiological testing will be a core function of the Pneumonia Etiology Research for Child Health (PERCH) project. The development stage of PERCH provided the time and resources necessary for us to conduct a comprehensive review of the current state of respiratory diagnostics. These efforts allowed us to articulate the unique requirements of PERCH, establish that molecular methods would be central to our testing strategy, and focus on a short list of candidate platforms. This process also highlighted critical challenges in the general design and interpretation of diagnostic evaluation studies, particularly in the field of respiratory infections. Although our final molecular diagnostic platform was ultimately selected on the basis of operational and strategic considerations determined by the specific context of PERCH, our review highlighted several conceptual and practical challenges in respiratory diagnostics that have broader relevance for the performance and interpretation of pneumonia research studies.

Resveratrol Inhibits KSHV Reactivation by Lowering the Levels of Cellular EGR-1

In the field of herpesvirus research, the exact molecular mechanism by which such viruses reactivate from latency remains elusive. Kaposi's sarcoma-associated herpesvirus (KSHV) primarily exists in a latent state, while only 1–3% of cells support lytic infection at any specific time. KSHV reactivation from latency is an exceedingly intricate process mediated by the integration of viral and cellular factors. Previously, our lab has described early growth response-1 (Egr-1) as an essential component for the KSHV reactivation process via its ability to mediate transcription of KSHV ORF50, the gene encoding for replication and transcription activator (RTA), a viral component known to control the switch from latent to lytic infection. In here, electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) experiments revealed that Egr-1 binds KSHV ORF50 promoter (ORF50P) in at least two different GC-rich binding domains. Expression profiles of cellular egr-1 and KSHV-encoded ORF50 follow a similar pattern during de novo KSHV infection. Over-expressing Egr-1, a signaling component downstream of Raf>MEK>ERK1/2, in KSHV-infected cells activates KSHV lytic replication. Through performing more physiologically relevant experiments, we analyzed the effect of a dietary supplement containing resveratrol on KSHV-infected cells. Our results, for the first time, demonstrate resveratrol to act in lowering ERK1/2 activity and expression of Egr-1 in KSHV-infected cells, resulting in the suppression of virus reactivation from latency. Taken together, these findings will undoubtedly contribute to future studies on not only combating KSHV related disease conditions, but also on other herpesviruses-induced pathogenesis.

Common Variants in CDKN2B-AS1 Associated with Optic-Nerve Vulnerability of Glaucoma Identified by Genome-Wide Association Studies in Japanese

BACKGROUND: To date, only a small portion of the genetic variation for primary open-angle glaucoma (POAG), the major type of glaucoma, has been elucidated. METHODS AND PRINCIPAL FINDINGS: We examined our two data sets of the genome-wide association studies (GWAS) derived from a total of 2,219 Japanese subjects. First, we performed a GWAS by analyzing 653,519 autosomal common single-nucleotide polymorphisms (SNPs) in 833 POAG patients and 686 controls. As a result, five variants that passed the Bonferroni correction were identified in CDKN2B-AS1 on chromosome 9p21.3, which was already reported to be a significant locus in the Caucasian population. Moreover, we combined the data set with our previous GWAS data set derived from 411 POAG patients and 289 controls by the Mantel-Haenszel test, and all of the combined variants showed stronger association with POAG (P<5.8×10(−10)). We then subdivided the case groups into two subtypes based on the value of intraocular pressure (IOP)—POAG with high IOP (high pressure glaucoma, HPG) and that with normal IOP (normal pressure glaucoma, NPG)—and performed the GWAS using the two data sets, as the prevalence of NPG in Japanese is much higher than in Caucasians. The results suggested that the variants from the same CDKN2B-AS1 locus were likely to be significant for NPG patients. CONCLUSIONS AND SIGNIFICANCE: In this study, we successfully identified POAG-associated variants in the CDKN2B-AS1 locus using a Japanese population, i.e., variants originally reported as being associated with the Caucasian population. Although we cannot rule out that the significance could be due to the differences in sample size between HPG and NPG, the variants could be associated specifically with the vulnerability of the optic nerve to IOP, which is useful for investigating the etiology of glaucoma.

Interleukin-18 expression and the response to treatment in patients with psoriasis

INTRODUCTION: The aim of the study was to demonstrate Interleukin-18 (IL-18) expression in keratinocytes from psoriatic lesions in comparison to keratinocytes from uninvolved skin and to study the change of expression after therapeutic interventions. MATERIAL AND METHODS: This study included 16 patients of different clinical subtypes of psoriasis. IL-18 gene expression analysis was performed using real-time quantitative PCR. Three biopsies were obtained from each patient. Two were taken from the lesional psoriatic skin and from uninvolved skin before starting treatment. A third lesional skin biopsy was taken at the end of two months' treatment course. The treatment was in the form of topical steroids or oral systemic methotrexate. RESULTS: Of all 16 studied patients significantly increased IL-18 expression was noted in keratinocytes from psoriatic lesions before and after treatment when compared to keratinocytes from uninvolved skin (P = 0.001 and 0.002 respectively). The IL-18 expression in the skin lesions after treatment was significantly lower than lesional skin before treatment (P = 0.023). In psoriatic skin lesions of all studied patients IL-18 expression was significantly correlated with disease duration (r = 0.40 and P = 0.01) and clinical severity of psoriasis (r = 0.72 and P = 0.001). CONCLUSIONS: Increased IL-18 expression in keratinocytes from psoriatic lesions of our patients and its correlation with disease duration and severity supported the concept which views psoriasis as a T-cell-mediated autoimmune disease. This could establish therapeutic and preventive approaches for psoriasis that ultimately lead to improved outcomes for patients.

Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus

We provide experimental evidence of a replication enhancer element (REE) within the capsid gene of tick-borne encephalitis virus (TBEV, genus Flavivirus). Thermodynamic and phylogenetic analyses predicted that the REE folds as a long stable stem–loop (designated SL6), conserved among all tick-borne flaviviruses (TBFV). Homologous sequences and potential base pairing were found in the corresponding regions of mosquito-borne flaviviruses, but not in more genetically distant flaviviruses. To investigate the role of SL6, nucleotide substitutions were introduced which changed a conserved hexanucleotide motif, the conformation of the terminal loop and the base-paired dsRNA stacking. Substitutions were made within a TBEV reverse genetic system and recovered mutants were compared for plaque morphology, single-step replication kinetics and cytopathic effect. The greatest phenotypic changes were observed in mutants with a destabilized stem. Point mutations in the conserved hexanucleotide motif of the terminal loop caused moderate virus attenuation. However, all mutants eventually reached the titre of wild-type virus late post-infection. Thus, although not essential for growth in tissue culture, the SL6 REE acts to up-regulate virus replication. We hypothesize that this modulatory role may be important for TBEV survival in nature, where the virus circulates by non-viraemic transmission between infected and non-infected ticks, during co-feeding on local rodents.

Both TLR2 and TRIF Contribute to Interferon-β Production during Listeria Infection

Synthesis of interferon-β (IFN-β) is an innate response to cytoplasmic infection with bacterial pathogens. Our recent studies showed that Listeria monocytogenes limits immune detection and IFN-β synthesis via deacetylation of its peptidoglycan, which renders the bacterium resistant to lysozyme degradation. Here, we examined signaling requirements for the massive IFN-β production resulting from the infection of murine macrophages with a mutant strain of L. monocytogenes, ΔpgdA, which is unable to modify its peptidoglycan. We report the identification of unconventional signaling pathways to the IFN-β gene, requiring TLR2 and bacterial internalization. Induction of IFN-β was independent of the Mal/TIRAP adaptor protein but required TRIF and the transcription factors IRF3 and IRF7. These pathways were stimulated to a lesser degree by wild-type L. monocytogenes. They operated in both resident and inflammatory macrophages derived from the peritoneal cavity, but not in bone marrow-derived macrophages. The novelty of our findings thus lies in the first description of TLR2 and TRIF as two critical components leading to the induction of the IFN-β gene and in uncovering that individual macrophage populations adopt different strategies to link pathogen recognition signals to IFN-β gene expression.

Protein Reporter Bioassay Systems for the Phenotypic Screening of Candidate Drugs: A Mouse Platform for Anti-Aging Drug Screening

Recent drug discovery efforts have utilized high throughput screening (HTS) of large chemical libraries to identify compounds that modify the activity of discrete molecular targets. The molecular target approach to drug screening is widely used in the pharmaceutical and biotechnology industries, because of the amount of knowledge now available regarding protein structure that has been obtained by computer simulation. The molecular target approach requires that the structure of target molecules, and an understanding of their physiological functions, is known. This approach to drug discovery may, however, limit the identification of novel drugs. As an alternative, the phenotypic- or pathway-screening approach to drug discovery is gaining popularity, particularly in the academic sector. This approach not only provides the opportunity to identify promising drug candidates, but also enables novel information regarding biological pathways to be unveiled. Reporter assays are a powerful tool for the phenotypic screening of compound libraries. Of the various reporter genes that can be used in such assays, those encoding secreted proteins enable the screening of hit molecules in both living cells and animals. Cell- and animal-based screens enable simultaneous evaluation of drug metabolism or toxicity with biological activity. Therefore, drug candidates identified in these screens may have increased biological efficacy and a lower risk of side effects in humans. In this article, we review the reporter bioassay systems available for phenotypic drug discovery.

Suppression of Adenosine-Activated Chloride Transport by Ethanol in Airway Epithelia

Alcohol abuse is associated with increased lung infections. Molecular understanding of the underlying mechanisms is not complete. Airway epithelial ion transport regulates the homeostasis of airway surface liquid, essential for airway mucosal immunity and lung host defense. Here, air-liquid interface cultures of Calu-3 epithelial cells were basolaterally exposed to physiologically relevant concentrations of ethanol (0, 25, 50 and 100 mM) for 24 hours and adenosine-stimulated ion transport was measured by Ussing chamber. The ethanol exposure reduced the epithelial short-circuit currents (I(SC)) in a dose-dependent manner. The ion currents activated by adenosine were chloride conductance mediated by cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel. Alloxazine, a specific inhibitor for A(2B) adenosine receptor (A(2B)AR), largely abolished the adenosine-stimulated chloride transport, suggesting that A(2B)AR is a major receptor responsible for regulating the chloride transport of the cells. Ethanol significantly reduced intracellular cAMP production upon adenosine stimulation. Moreover, ethanol-suppression of the chloride secretion was able to be restored by cAMP analogs or by inhibitors to block cAMP degradation. These results imply that ethanol exposure dysregulates CFTR-mediated chloride transport in airways by suppression of adenosine-A(2B)AR-cAMP signaling pathway, which might contribute to alcohol-associated lung infections.

High Fidelity Processing and Activation of the Human α-Defensin HNP1 Precursor by Neutrophil Elastase and Proteinase 3

The azurophilic granules of human neutrophils contain four α-defensins called human neutrophil peptides (HNPs 1–4). HNPs are tridisulfide-linked antimicrobial peptides involved in the intracellular killing of organisms phagocytosed by neutrophils. The peptides are produced as inactive precursors (proHNPs) which are processed to active microbicides by as yet unidentified convertases. ProHNP1 was expressed in E. coli and the affinity-purified propeptide isolated as two species, one containing mature HNP1 sequence with native disulfide linkages (“folded proHNP1”) and the other containing non-native disulfide linked proHNP1 conformers (misfolded proHNP1). Native HNP1, liberated by CNBr treatment of folded proHNP1, was microbicidal against Staphylococcus aureus, but the peptide derived from misfolded proHNP1 was inactive. We hypothesized that neutrophil elastase (NE), proteinase 3 (PR3) or cathepsin G (CG), serine proteases that co-localize with HNPs in azurophil granules, are proHNP1 activating convertases. Folded proHNP1 was converted to mature HNP1 by both NE and PR3, but CG generated an HNP1 variant with an N-terminal dipeptide extension. NE and PR3 cleaved folded proHNP1 to produce a peptide indistinguishable from native HNP1 purified from neutrophils, and the microbicidal activities of in vitro derived and natural HNP1 peptides were equivalent. In contrast, misfolded proHNP1 conformers were degraded extensively under the same conditions. Thus, NE and PR3 possess proHNP1 convertase activity that requires the presence of the native HNP1 disulfide motif for high fidelity activation of the precursor in vitro.

Foxp3(+) Regulatory T Cells Control Persistence of Viral CNS Infection

We earlier established a model of a persistent viral CNS infection using two week old immunologically normal (genetically unmodified) mice and recombinant measles virus (MV). Using this model infection we investigated the role of regulatory T cells (Tregs) as regulators of the immune response in the brain, and assessed whether the persistent CNS infection can be modulated by manipulation of Tregs in the periphery. CD4(+) CD25(+) Foxp3(+) Tregs were expanded or depleted during the persistent phase of the CNS infection, and the consequences for the virus-specific immune response and the extent of persistent infection were analyzed. Virus-specific CD8(+) T cells predominantly recognising the H-2D(b)-presented viral hemagglutinin epitope MV-H(22–30) (RIVINREHL) were quantified in the brain by pentamer staining. Expansion of Tregs after intraperitoneal (i.p.) application of the superagonistic anti-CD28 antibody D665 inducing transient immunosuppression caused increased virus replication and spread in the CNS. In contrast, depletion of Tregs using diphtheria toxin (DT) in DEREG (depletion of regulatory T cells)-mice induced an increase of virus-specific CD8(+) effector T cells in the brain and caused a reduction of the persistent infection. These data indicate that manipulation of Tregs in the periphery can be utilized to regulate virus persistence in the CNS.

Superimposed high-frequency jet ventilation combined with continuous positive airway pressure/assisted spontaneous breathing improves oxygenation in patients with H1N1-associated ARDS

BACKGROUND: Numerous cases of swine-origin 2009 H1N1 influenza A virus (H1N1)-associated acute respiratory distress syndrome (ARDS) bridged by extracorporeal membrane oxygenation (ECMO) therapy have been reported; however, complication rates are high. We present our experience with H1N1-associated ARDS and successful bridging of lung function using superimposed high-frequency jet ventilation (SHFJV) in combination with continuous positive airway pressure/assisted spontaneous breathing (CPAP/ASB). METHODS: We admitted five patients with H1N1 infection and ARDS to our intensive care unit. Although all patients required pure oxygen and controlled ventilation, oxygenation was insufficient. We applied SHFJV/CPAP/ASB to improve oxygenation. RESULTS: Initial PaO(2)/FiO(2 )ratio prior SHFJV was 58-79 mmHg. In all patients, successful oxygenation was achieved by SHFJV (PaO(2)/FiO(2 )ratio 105-306 mmHg within 24 h). Spontaneous breathing was set during first hours after admission. SHFJV could be stopped after 39, 40, 72, 100, or 240 h. Concomitant pulmonary herpes simplex virus (HSV) infection was observed in all patients. Two patients were successfully discharged. The other three patients relapsed and died within 7 weeks mainly due to combined HSV infection and in two cases reoccurring H1N1 infection. CONCLUSIONS: SHFJV represents an alternative to bridge lung function successfully and improve oxygenation in the critically ill.

Serious Invasive Saffold Virus Infections in Children, 2009

The first human virus in the genus Cardiovirus was described in 2007 and named Saffold virus (SAFV). Cardioviruses can cause severe infections of the myocardium and central nervous system in animals, but SAFV has not yet been convincingly associated with disease in humans. To study a possible association between SAFV and infections in the human central nervous system, we designed a real-time PCR for SAFV and tested cerebrospinal fluid (CSF) samples from children <4 years of age. SAFV was detected in 2 children: in the CSF and a fecal sample from 1 child with monosymptomatic ataxia caused by cerebellitis; and in the CSF, blood, and myocardium of another child who died suddenly with no history of illness. Virus from each child was sequenced and shown to be SAFV type 2. These findings demonstrate that SAFV can cause serious invasive infection in children.

Pandemic (H1N1) 2009 among Quarantined Close Contacts, Beijing, People’s Republic of China

We estimated the attack rate of pandemic (H1N1) 2009 and assessed risk factors for infection among close contacts quarantined in Beijing, People’s Republic of China. The first 613 confirmed cases detected between May 16 and September 15, 2009, were investigated; 7,099 close contacts were located and quarantined. The attack rate of confirmed infection in close contacts was 2.4% overall, ranging from 0.9% among aircraft passengers to >5% among household members. Risk factors for infection among close contacts were younger age, being a household member of an index case-patient, exposure during the index case-patient’s symptomatic phase, and longer exposure. Among close contacts with positive test results at the start of quarantine, 17.2% had subclinical infection. Having contact with a household member and younger age were the major risk factors for acquiring pandemic (H1N1) 2009 influenza virus infection. One person in 6 with confirmed pandemic (H1N1) 2009 was asymptomatic.

Dengue Virus Infection Perturbs Lipid Homeostasis in Infected Mosquito Cells

Dengue virus causes ∼50–100 million infections per year and thus is considered one of the most aggressive arthropod-borne human pathogen worldwide. During its replication, dengue virus induces dramatic alterations in the intracellular membranes of infected cells. This phenomenon is observed both in human and vector-derived cells. Using high-resolution mass spectrometry of mosquito cells, we show that this membrane remodeling is directly linked to a unique lipid repertoire induced by dengue virus infection. Specifically, 15% of the metabolites detected were significantly different between DENV infected and uninfected cells while 85% of the metabolites detected were significantly different in isolated replication complex membranes. Furthermore, we demonstrate that intracellular lipid redistribution induced by the inhibition of fatty acid synthase, the rate-limiting enzyme in lipid biosynthesis, is sufficient for cell survival but is inhibitory to dengue virus replication. Lipids that have the capacity to destabilize and change the curvature of membranes as well as lipids that change the permeability of membranes are enriched in dengue virus infected cells. Several sphingolipids and other bioactive signaling molecules that are involved in controlling membrane fusion, fission, and trafficking as well as molecules that influence cytoskeletal reorganization are also up regulated during dengue infection. These observations shed light on the emerging role of lipids in shaping the membrane and protein environments during viral infections and suggest membrane-organizing principles that may influence virus-induced intracellular membrane architecture.

Human Cardioviruses, Meningitis, and Sudden Infant Death Syndrome in Children

Cardioviruses cause myocarditis and encephalomyelitis in rodents; human cardioviruses have not been ascribed to any disease. We screened 6,854 cerebrospinal fluid and 10 myocardium specimens from children and adults. A genotype 2 cardiovirus was detected from a child who died of sudden infant death syndrome, and 2 untypeable cardioviruses were detected from 2 children with meningitis.

Knowledge of Avian Influenza (H5N1) among Poultry Workers, Hong Kong, China

In 2009, a cross-sectional survey of 360 poultry workers in Hong Kong, China, showed that workers had inadequate levels of avian influenza (H5N1) risk knowledge, preventive behavior, and outbreak preparedness. The main barriers to preventive practices were low perceived benefits and interference with work. Poultry workers require occupation-specific health promotion.

A Pilot Study of Host Genetic Variants Associated with Influenza-associated Deaths among Children and Young Adults

We compared the prevalence of 8 polymorphisms in the tumor necrosis factor and mannose-binding lectin genes among 105 children and young adults with fatal influenza with US population estimates and determined in subanalyses whether these polymorphisms were associated with sudden death and bacterial co-infection among persons with fatal influenza. No differences were observed in genotype prevalence or minor allele frequencies between persons with fatal influenza and the reference sample. Fatal cases with low-producing MBL2 genotypes had a 7-fold increased risk for invasive methicillin-resistant Staphylococcus aureus (MRSA) co-infection compared with fatal cases with high- and intermediate-producing MBL2 genotypes (odds ratio 7.1, 95% confidence interval 1.6–32.1). Limited analysis of 2 genes important to the innate immune response found no association between genetic variants and fatal influenza infection. Among children and young adults who died of influenza, low-producing MBL2 genotypes may have increased risk for MRSA co-infection.

Clinical features and risk factors for severe and critical pregnant women with 2009 pandemic H1N1 influenza infection in China

BACKGROUND: 2009 pandemic H1N1 (pH1N1) influenza posed an increased risk of severe illness among pregnant women. Data on risk factors associated with death of pregnant women and neonates with pH1N1 infections are limited outside of developed countries. METHODS: Retrospective observational study in 394 severe or critical pregnant women admitted to a hospital with pH1N1 influenza from Sep. 1, 2009 to Dec. 31, 2009. rRT-PCR testing was used to confirm infection. In-hospital mortality was the primary endpoint of this study. Univariable logistic analysis and multivariate logistic regression analysis were used to investigate the potential factors on admission that might be associated with the maternal and neonatal mortality. RESULTS: 394 pregnant women were included, 286 were infected with pH1N1 in the third trimester. 351 had pneumonia, and 77 died. A PaO(2)/FiO(2 )≤ 200 (odds ratio (OR), 27.16; 95% confidence interval (CI), 2.64-279.70) and higher BMI (i.e. ≥ 30) on admission (OR, 1.26; 95% CI, 1.09 to 1.47) were independent risk factors for maternal death. Of 211 deliveries, 146 neonates survived. Premature delivery (OR, 4.17; 95% CI, 1.19-14.56) was associated neonatal mortality. Among 186 patients who received mechanical ventilation, 83 patients were treated with non-invasive ventilation (NIV) and 38 were successful with NIV. The death rate was lower among patients who initially received NIV than those who were initially intubated (24/83, 28.9% vs 43/87, 49.4%; p = 0.006). Septic shock was an independent risk factor for failure of NIV. CONCLUSIONS: Severe hypoxemia and higher BMI on admission were associated with adverse outcomes for pregnant women. Preterm delivery was a risk factor for neonatal death among pregnant women with pH1N1 influenza infection. NIV may be useful in selected pregnant women without septic shock.

A scientometric analysis of Indian research output in medicine during 1999–2008

OBJECTIVE: This study analyzes the research activities of India in medicine during 1999–2008, based on the total publication output, its growth rate, quality of papers published and rank of India in the global context. Patterns of international collaborative research output and the major partner countries of India are also discussed. This study also evaluates the research performance of different types of Indian medical colleges, hospitals, research institutes, universities and research foundations and the characteristics of published literature in Indian and foreign journals. It also analyzes the medical research output by disease and organs. MATERIALS AND METHODS: The publication data on medicine has been retrieved by using SCOPUS database. RESULTS: India holds 12th rank among the productive countries in medicine research consisting of 65,745 papers with a global publication share of 1.59% and registering a growth rate of 76.68% for the papers published during 1999–2003 to 2004–2008. CONCLUSION: High quality research in India is grossly inadequate and requires strategic planning, investment and resource support. There is also a need to improve the existing medical education system, which should foster research culture.

Membrane Fusion and Cell Entry of XMRV Are pH-Independent and Modulated by the Envelope Glycoprotein's Cytoplasmic Tail

Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus that was originally identified from human prostate cancer patients and subsequently linked to chronic fatigue syndrome. Recent studies showed that XMRV is a recombinant mouse retrovirus; hence, its association with human diseases has become questionable. Here, we demonstrated that XMRV envelope (Env)-mediated pseudoviral infection is not blocked by lysosomotropic agents and cellular protease inhibitors, suggesting that XMRV entry is not pH-dependent. The full length XMRV Env was unable to induce syncytia formation and cell-cell fusion, even in cells overexpressing the viral receptor, XPR1. However, truncation of the C-terminal 21 or 33 amino acid residues in the cytoplasmic tail (CT) of XMRV Env induced substantial membrane fusion, not only in the permissive 293 cells but also in the nonpermissive CHO cells that lack a functional XPR1 receptor. The increased fusion activities of these truncations correlated with their enhanced SU shedding into culture media, suggesting conformational changes in the ectodomain of XMRV Env. Noticeably, further truncation of the CT of XMRV Env proximal to the membrane-spanning domain severely impaired the Env fusogenicity, as well as dramatically decreased the Env incorporations into MoMLV oncoretroviral and HIV-1 lentiviral vectors resulting in greatly reduced viral transductions. Collectively, our studies reveal that XMRV entry does not require a low pH or low pH-dependent host proteases, and that the cytoplasmic tail of XMRV Env critically modulates membrane fusion and cell entry. Our data also imply that additional cellular factors besides XPR1 are likely to be involved in XMRV entry.

The Impact of Weather on Influenza and Pneumonia Mortality in New York City, 1975–2002: A Retrospective Study

The substantial winter influenza peak in temperate climates has lead to the hypothesis that cold and/or dry air is a causal factor in influenza variability. We examined the relationship between cold and/or dry air and daily influenza and pneumonia mortality in the cold season in the New York metropolitan area from 1975–2002. We conducted a retrospective study relating daily pneumonia and influenza mortality for New York City and surroundings from 1975–2002 to daily air temperature, dew point temperature (a measure of atmospheric humidity), and daily air mass type. We identified high mortality days and periods and employed temporal smoothers and lags to account for the latency period and the time between infection and death. Unpaired t-tests were used to compare high mortality events to non-events and nonparametric bootstrapped regression analysis was used to examine the characteristics of longer mortality episodes. We found a statistically significant (p = 0.003) association between periods of low dew point temperature and above normal pneumonia and influenza mortality 17 days later. The duration (r = −0.61) and severity (r = −0.56) of high mortality episodes was inversely correlated with morning dew point temperature prior to and during the episodes. Weeks in which moist polar air masses were common (air masses characterized by low dew point temperatures) were likewise followed by above normal mortality 17 days later (p = 0.019). This research supports the contention that cold, dry air may be related to influenza mortality and suggests that warning systems could provide enough lead time to be effective in mitigating the effects.

Finding and removing highly connected individuals using suboptimal vaccines

BACKGROUND: Social networks are often highly skewed, meaning that the vast majority of the population has only few contacts whereas a small minority has a large number of contacts. These highly connected individuals may play an important role in case of an infectious disease outbreak. METHODS: We propose a novel strategy of finding and immunizing highly connected individuals and evaluate this strategy by computer simulations, using a stochastic, individual-and network-based simulation approach. A small random sample of the population is asked to list their acquaintances, and those who are mentioned most frequently are offered vaccination. This intervention is combined with case isolation and contact tracing. RESULTS: Asking only 10% of the population for 10 acquaintances each and vaccinating the most frequently named people strongly diminishes the magnitude of an outbreak which would otherwise have exhausted the available isolation units and gone out of control. It is extremely important to immunize all identified highly connected individuals. Omitting a few of them because of unsuccessful vaccination jeopardizes the overall success, unless non-immunized individuals are taken under surveillance. CONCLUSIONS: The strategy proposed in this paper is particularly successful because it attacks the very point from which the transmission network draws its strength: the highly connected individuals. Current preparedness and containment plans for smallpox and other infectious diseases may benefit from such knowledge.

The impact of media coverage on the transmission dynamics of human influenza

BACKGROUND: There is an urgent need to understand how the provision of information influences individual risk perception and how this in turn shapes the evolution of epidemics. Individuals are influenced by information in complex and unpredictable ways. Emerging infectious diseases, such as the recent swine flu epidemic, may be particular hotspots for a media-fueled rush to vaccination; conversely, seasonal diseases may receive little media attention, despite their high mortality rate, due to their perceived lack of newness. METHODS: We formulate a deterministic transmission and vaccination model to investigate the effects of media coverage on the transmission dynamics of influenza. The population is subdivided into different classes according to their disease status. The compartmental model includes the effect of media coverage on reporting the number of infections as well as the number of individuals successfully vaccinated. RESULTS: A threshold parameter (the basic reproductive ratio) is analytically derived and used to discuss the local stability of the disease-free steady state. The impact of costs that can be incurred, which include vaccination, education, implementation and campaigns on media coverage, are also investigated using optimal control theory. A simplified version of the model with pulse vaccination shows that the media can trigger a vaccinating panic if the vaccine is imperfect and simplified messages result in the vaccinated mixing with the infectives without regard to disease risk. CONCLUSIONS: The effects of media on an outbreak are complex. Simplified understandings of disease epidemiology, propogated through media soundbites, may make the disease significantly worse.

Detection of Mycobacterium ulcerans by the Loop Mediated Isothermal Amplification Method

BACKGROUND: Buruli ulcer (BU) caused by Mycobacterium ulcerans (M. ulcerans) has emerged as an important public health problem in several rural communities in sub-Saharan Africa. Early diagnosis and prompt treatment are important in preventing disfiguring complications associated with late stages of the disease progression. Presently there is no simple and rapid test that is appropriate for early diagnosis and use in the low-resource settings where M. ulcerans is most prevalent. METHODOLOGY: We compared conventional and pocket warmer loop mediated isothermal amplification (LAMP) methods (using a heat block and a pocket warmer respectively as heat source for amplification reaction) for the detection of M. ulcerans in clinical specimens. The effect of purified and crude DNA preparations on the detection rate of the LAMP assays were also investigated and compared with that of IS2404 PCR, a reference assay for the detection of M. ulcerans. Thirty clinical specimens from suspected BU cases were examined by LAMP and IS2404 PCR. PRINCIPAL FINDINGS: The lower detection limit of both LAMP methods at 60°C was 300 copies of IS2404 and 30 copies of IS2404 for the conventional LAMP at 65°C. When purified DNA extracts were used, both the conventional LAMP and IS2404 PCR concordantly detected 21 positive cases, while the pocket warmer LAMP detected 19 cases. Nine of 30 samples were positive by both the LAMP assays as well as IS2404 PCR when crude extracts of clinical specimens were used. CONCLUSION/SIGNIFICANCE: The LAMP method can be used as a simple and rapid test for the detection of M. ulcerans in clinical specimens. However, obtaining purified DNA, as well as generating isothermal conditions, remains a major challenge for the use of the LAMP method under field conditions. With further improvement in DNA extraction and amplification conditions, the pwLAMP could be used as a point of care diagnostic test for BU

A Human PrM Antibody That Recognizes a Novel Cryptic Epitope on Dengue E Glycoprotein

Dengue virus (DENV) is a major mosquito-borne pathogen infecting up to 100 million people each year; so far no effective treatment or vaccines are available. Recently, highly cross-reactive and infection-enhancing pre-membrane (prM)-specific antibodies were found to dominate the anti-DENV immune response in humans, raising concern over vaccine candidates that contain native dengue prM sequences. In this study, we have isolated a broadly cross-reactive prM-specific antibody, D29, during a screen with a non-immunized human Fab-phage library against the four serotypes of DENV. The antibody is capable of restoring the infectivity of virtually non-infectious immature DENV (imDENV) in FcγR-bearing K562 cells. Remarkably, D29 also cross-reacted with a cryptic epitope on the envelope (E) protein located to the DI/DII junction as evidenced by site-directed mutagenesis. This cryptic epitope, while inaccessible to antibody binding in a native virus particle, may become exposed if E is not properly folded. These findings suggest that generation of anti-prM antibodies that enhance DENV infection may not be completely avoided even with immunization strategies employing E protein alone or subunits of E proteins.

Lack of Association between CLEC5A Gene Single-Nucleotide Polymorphisms and Kawasaki Disease in Taiwanese Children

Background. Kawasaki disease is characterized by systemic vasculitis of unknown etiology. Previous genetic studies have identified certain candidate genes associated with susceptibility to KD and coronary artery lesions. Host innate immune response factors are involved in modulating the disease outcome. The aim of this study was to investigate CLEC5A (C-type lectin domain family 5) genetic polymorphisms with regards to the susceptibility and outcome of KD. Methods. A total of 1045 subjects (381 KD patients and 664 controls) were enrolled to identify 4 tagging single-nucleotide polymorphisms (tSNPs) of CLEC5A (rs1285968, rs11770855, rs1285935, rs1285933) by using the TaqMan Allelic Discrimination Assay. The Hardy-Weinberg equilibrium was assessed in cases and controls, and genetic effects were evaluated by the chi-square test. Results. No significant associations were noted between the genotypes and allele frequency of the 4 CLEC5A tSNPs between controls and patients. In the patients, polymorphisms of CLEC5A showed no significant association with coronary artery lesion formation and intravenous immunoglobulin treatment response. Conclusions. This study showed for the first time that polymorphisms of CLEC5A are not associated with susceptibility to KD, coronary artery lesion formation, and intravenous immunoglobulin treatment response in a Taiwanese population.

A Non-VH1-69 Heterosubtypic Neutralizing Human Monoclonal Antibody Protects Mice against H1N1 and H5N1 Viruses

Influenza viruses are among the most important human pathogens and are responsible for annual epidemics and sporadic, potentially devastating pandemics. The humoral immune response plays an important role in the defense against these viruses, providing protection mainly by producing antibodies directed against the hemagglutinin (HA) glycoprotein. However, their high genetic variability allows the virus to evade the host immune response and the potential protection offered by seasonal vaccines. The emergence of resistance to antiviral drugs in recent years further limits the options available for the control of influenza. The development of alternative strategies for influenza prophylaxis and therapy is therefore urgently needed. In this study, we describe a human monoclonal antibody (PN-SIA49) that recognizes a highly conserved epitope located on the stem region of the HA and able to neutralize a broad spectrum of influenza viruses belonging to different subtypes (H1, H2 and H5). Furthermore, we describe its protective activity in mice after lethal challenge with H1N1 and H5N1 viruses suggesting a potential application in the treatment of influenza virus infections.

Web-based Investigation of Multistate Salmonellosis Outbreak

We investigated a large outbreak of Salmonella enterica serotype Javiana among attendees of the 2002 U.S. Transplant Games, including 1,500 organ transplant recipients. Web-based survey methods identified pre-diced tomatoes as the source of this outbreak, which highlights the utility of such investigative tools to cope with the changing epidemiology of foodborne diseases.

Dengue Fever, Hawaii, 2001–2002

Autochthonous dengue infections were last reported in Hawaii in 1944. In September 2001, the Hawaii Department of Health was notified of an unusual febrile illness in a resident with no travel history; dengue fever was confirmed. During the investigation, 1,644 persons with locally acquired denguelike illness were evaluated, and 122 (7%) laboratory-positive dengue infections were identified; dengue virus serotype 1 was isolated from 15 patients. No cases of dengue hemorrhagic fever or shock syndrome were reported. In 3 instances autochthonous infections were linked to a person who reported denguelike illness after travel to French Polynesia. Phylogenetic analyses showed the Hawaiian isolates were closely associated with contemporaneous isolates from Tahiti. Aedes albopictus was present in all communities surveyed on Oahu, Maui, Molokai, and Kauai; no Ae. aegypti were found. This outbreak underscores the importance of maintaining surveillance and control of potential disease vectors even in the absence of an imminent disease threat.

Community responses to communication campaigns for influenza A (H1N1): a focus group study

BACKGROUND: This research was a part of a contestable rapid response initiative launched by the Health Research Council of New Zealand and the Ministry of Health in response to the 2009 influenza A pandemic. The aim was to provide health authorities in New Zealand with evidence-based practical information to guide the development and delivery of effective health messages for H1N1 and other health campaigns. This study contributed to the initiative by providing qualitative data about community responses to key health messages in the 2009 and 2010 H1N1 campaigns, the impact of messages on behavioural change and the differential impact on vulnerable groups in New Zealand. METHODS: Qualitative data were collected on community responses to key health messages in the 2009 and 2010 Ministry of Health H1N1 campaigns, the impact of messages on behaviour and the differential impact on vulnerable groups. Eight focus groups were held in the winter of 2010 with 80 participants from groups identified by the Ministry of Health as vulnerable to the H1N1 virus, such as people with chronic health conditions, pregnant women, children, Pacific Peoples and Māori. Because this study was part of a rapid response initiative, focus groups were selected as the most efficient means of data collection in the time available. For Māori, focus group discussion (hui) is a culturally appropriate methodology. RESULTS: Thematic analysis of data identified four major themes: personal and community risk, building community strategies, responsibility and information sources. People wanted messages about specific actions that they could take to protect themselves and their families and to mitigate any consequences. They wanted transparent and factual communication where both good and bad news is conveyed by people who they could trust. CONCLUSIONS: The responses from all groups endorsed the need for community based risk management including information dissemination. Engaging with communities will be essential to facilitate preparedness and build community resilience to future pandemic events. This research provides an illustration of the complexities of how people understand and respond to health messages related to the H1N1 pandemic. The importance of the differences identified in the analysis is not the differences per se but highlight problems with a "one size fits all" pandemic warning strategy.

Quantifying Type-Specific Reproduction Numbers for Nosocomial Pathogens: Evidence for Heightened Transmission of an Asian Sequence Type 239 MRSA Clone

An important determinant of a pathogen's success is the rate at which it is transmitted from infected to susceptible hosts. Although there are anecdotal reports that methicillin-resistant Staphylococcus aureus (MRSA) clones vary in their transmissibility in hospital settings, attempts to quantify such variation are lacking for common subtypes, as are methods for addressing this question using routinely-collected MRSA screening data in endemic settings. Here we present a method to quantify the time-varying transmissibility of different subtypes of common bacterial nosocomial pathogens using routine surveillance data. The method adapts approaches for estimating reproduction numbers based on the probabilistic reconstruction of epidemic trees, but uses relative hazards rather than serial intervals to assign probabilities to different sources for observed transmission events. The method is applied to data collected as part of a retrospective observational study of a concurrent MRSA outbreak in the United Kingdom with dominant endemic MRSA clones (ST22 and ST36) and an Asian ST239 MRSA strain (ST239-TW) in two linked adult intensive care units, and compared with an approach based on a fully parametric transmission model. The results provide support for the hypothesis that the clones responded differently to an infection control measure based on the use of topical antiseptics, which was more effective at reducing transmission of endemic clones. They also suggest that in one of the two ICUs patients colonized or infected with the ST239-TW MRSA clone had consistently higher risks of transmitting MRSA to patients free of MRSA. These findings represent some of the first quantitative evidence of enhanced transmissibility of a pandemic MRSA lineage, and highlight the potential value of tailoring hospital infection control measures to specific pathogen subtypes.

Discovery and Genomic Characterization of a Novel Bat Sapovirus with Unusual Genomic Features and Phylogenetic Position

Sapovirus is a genus of caliciviruses that are known to cause enteric disease in humans and animals. There is considerable genetic diversity among the sapoviruses, which are classified into different genogroups based on phylogenetic analysis of the full-length capsid protein sequence. While several mammalian species, including humans, pigs, minks, and dogs, have been identified as animal hosts for sapoviruses, there were no reports of sapoviruses in bats in spite of their biological diversity. In this report, we present the results of a targeted surveillance study in different bat species in Hong Kong. Five of the 321 specimens from the bat species, Hipposideros pomona, were found to be positive for sapoviruses by RT-PCR. Complete or nearly full-length genome sequences of approximately 7.7 kb in length were obtained for three strains, which showed similar organization of the genome compared to other sapoviruses. Interestingly, they possess many genomic features atypical of most sapoviruses, like high G+C content and minimal CpG suppression. Phylogenetic analysis of the viral proteins suggested that the bat sapovirus descended from an ancestral sapovirus lineage and is most closely related to the porcine sapoviruses. Codon usage analysis showed that the bat sapovirus genome has greater codon usage bias relative to other sapovirus genomes. In summary, we report the discovery and genomic characterization of the first bat calicivirus, which appears to have evolved under different conditions after early divergence from other sapovirus lineages.

Angiotensin Converting Enzyme (ACE) and ACE2 Bind Integrins and ACE2 Regulates Integrin Signalling

The angiotensin converting enzymes (ACEs) are the key catalytic components of the renin-angiotensin system, mediating precise regulation of blood pressure by counterbalancing the effects of each other. Inhibition of ACE has been shown to improve pathology in cardiovascular disease, whilst ACE2 is cardioprotective in the failing heart. However, the mechanisms by which ACE2 mediates its cardioprotective functions have yet to be fully elucidated. Here we demonstrate that both ACE and ACE2 bind integrin subunits, in an RGD-independent manner, and that they can act as cell adhesion substrates. We show that cellular expression of ACE2 enhanced cell adhesion. Furthermore, we present evidence that soluble ACE2 (sACE2) is capable of suppressing integrin signalling mediated by FAK. In addition, sACE2 increases the expression of Akt, thereby lowering the proportion of the signalling molecule phosphorylated Akt. These results suggest that ACE2 plays a role in cell-cell interactions, possibly acting to fine-tune integrin signalling. Hence the expression and cleavage of ACE2 at the plasma membrane may influence cell-extracellular matrix interactions and the signalling that mediates cell survival and proliferation. As such, ectodomain shedding of ACE2 may play a role in the process of pathological cardiac remodelling.

Altered Thymic Function during Interferon Therapy in HCV-Infected Patients

Interferon alpha (IFNα) therapy, despite good efficacy in curing HCV infection, leads to major side effects, in particular inducement of a strong peripheral T-cell lymphocytopenia. We here analyze the early consequences of IFNα therapy on both thymic function and peripheral T-cell homeostasis in patients in the acute or chronic phase of HCV-infection as well as in HIV/HCV co-infected patients. The evolution of T-cell subsets and T-cell homeostasis were estimated by flow cytometry while thymic function was measured through quantification of T-cell receptor excision circles (TREC) and estimation of intrathymic precursor T-cell proliferation during the first four months following the initiation of IFNα therapy. Beginning with the first month of therapy, a profound lymphocytopenia was observed for all T-cell subsets, including naïve T-cells and recent thymic emigrants (RTE), associated with inhibition of intrathymic precursor T-cell proliferation. Interleukin (IL)-7 plasma concentration rapidly dropped while lymphocytopenia progressed. This was neither a consequence of higher consumption of the cytokine nor due to its neutralization by soluble CD127. Decrease in IL-7 plasma concentration under IFNα therapy correlated with the decline in HCV viral load, thymic activity and RTE concentration in blood. These data demonstrate that IFNα-based therapy rapidly impacts on thymopoiesis and, consequently, perturbs T-cell homeostasis. Such a side effect might be detrimental for the continuation of IFNα therapy and may lead to an increased level of infectious risk, in particular in HIV/HCV co-infected patients. Altogether, this study suggests the therapeutic potential of IL-7 in the maintenance of peripheral T-cell homeostasis in IFNα-treated patients.

Baculovirus-based Vaccine Displaying Respiratory Syncytial Virus Glycoprotein Induces Protective Immunity against RSV Infection without Vaccine-Enhanced Disease

BACKGROUND: Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract diseases in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine against RSV yet. The attachment glycoprotein (G) of RSV is a potentially important target for protective antiviral immune responses. Recombinant baculovirus has been recently emerged as a new vaccine vector, since it has intrinsic immunostimulatory properties and good bio-safety profile. METHODS: We have constructed a recombinant baculovirus-based RSV vaccine, Bac-RSV/G, displaying G glycoprotein, and evaluated immunogenicity and protective efficacy by intranasal immunization of BALB/c mice with Bac-RSV/G. RESULTS: Bac-RSV/G efficiently provides protective immunity against RSV challenge. Strong serum IgG and mucosal IgA responses were induced by intranasal immunization with Bac-RSV/G. In addition to humoral immunity, G-specific Th17- as well as Th1-type T-cell responses were detected in the lungs of Bac-RSV/G-immune mice upon RSV challenge. Neither lung eosinophilia nor vaccine-induced weight loss was observed upon Bac-RSV/G immunization and subsequent RSV infection. CONCLUSION: Our data demonstrate that intranasal administration of baculovirus-based Bac-RSV/G vaccine is efficient for the induction of protection against RSV and represents a promising prophylactic vaccination regimen.

Divergent lineage of a novel hantavirus in the banana pipistrelle (Neoromicia nanus) in Côte d'Ivoire

Recently identified hantaviruses harbored by shrews and moles (order Soricomorpha) suggest that other mammals having shared ancestry may serve as reservoirs. To investigate this possibility, archival tissues from 213 insectivorous bats (order Chiroptera) were analyzed for hantavirus RNA by RT-PCR. Following numerous failed attempts, hantavirus RNA was detected in ethanol-fixed liver tissue from two banana pipistrelles (Neoromicia nanus), captured near Mouyassué village in Côte d'Ivoire, West Africa, in June 2011. Phylogenetic analysis of partial L-segment sequences using maximum-likelihood and Bayesian methods revealed that the newfound hantavirus, designated Mouyassué virus (MOUV), was highly divergent and basal to all other rodent- and soricomorph-borne hantaviruses, except for Nova virus in the European common mole (Talpa europaea). Full genome sequencing of MOUV and further surveys of other bat species for hantaviruses, now underway, will provide critical insights into the evolution and diversification of hantaviruses.

Protective Immunity to Listeria Monocytogenes Infection Mediated by Recombinant Listeria innocua Harboring the VGC Locus

In this study we propose a novel bacterial vaccine strategy where non-pathogenic bacteria are complemented with traits desirable for the induction of protective immunity. To illustrate the proof of principle of this novel vaccination strategy, we use the model organism of intracellular immunity Listeria. We introduced a, low copy number BAC-plasmid harbouring the virulence gene cluster (vgc) of L. monocytogenes (Lm) into the non-pathogenic L. innocua (L.inn) strain and examined for its ability to induce protective cellular immunity. The resulting strain (L.inn::vgc) was attenuated for virulence in vivo and showed a strongly reduced host detrimental inflammatory response compared to Lm. Like Lm, L.inn::vgc induced the production of Type I Interferon's and protection was mediated by Listeria-specific CD8(+) T cells. Rational vaccine design whereby avirulent strains are equipped with the capabilities to induce protection but lack detrimental inflammatory effects offer great promise towards future studies using non-pathogenic bacteria as vectors for vaccination.

Transcriptomics of In Vitro Immune-Stimulated Hemocytes from the Manila Clam Ruditapes philippinarum Using High-Throughput Sequencing

BACKGROUND: The Manila clam (Ruditapes philippinarum) is a worldwide cultured bivalve species with important commercial value. Diseases affecting this species can result in large economic losses. Because knowledge of the molecular mechanisms of the immune response in bivalves, especially clams, is scarce and fragmentary, we sequenced RNA from immune-stimulated R. philippinarum hemocytes by 454-pyrosequencing to identify genes involved in their immune defense against infectious diseases. METHODOLOGY AND PRINCIPAL FINDINGS: High-throughput deep sequencing of R. philippinarum using 454 pyrosequencing technology yielded 974,976 high-quality reads with an average read length of 250 bp. The reads were assembled into 51,265 contigs and the 44.7% of the translated nucleotide sequences into protein were annotated successfully. The 35 most frequently found contigs included a large number of immune-related genes, and a more detailed analysis showed the presence of putative members of several immune pathways and processes like the apoptosis, the toll like signaling pathway and the complement cascade. We have found sequences from molecules never described in bivalves before, especially in the complement pathway where almost all the components are present. CONCLUSIONS: This study represents the first transcriptome analysis using 454-pyrosequencing conducted on R. philippinarum focused on its immune system. Our results will provide a rich source of data to discover and identify new genes, which will serve as a basis for microarray construction and the study of gene expression as well as for the identification of genetic markers. The discovery of new immune sequences was very productive and resulted in a large variety of contigs that may play a role in the defense mechanisms of Ruditapes philippinarum.

Distribution and Risk Factors of 2009 Pandemic Influenza A (H1N1) in Mainland China

Data from all reported cases of 2009 pandemic influenza A (H1N1) were obtained from the China Information System for Disease Control and Prevention. The spatiotemporal distribution patterns of cases were characterized through spatial analysis. The impact of travel-related risk factors on invasion of the disease was analyzed using survival analysis, and climatic factors related to local transmission were identified using multilevel Poisson regression, both at the county level. The results showed that the epidemic spanned a large geographic area, with the most affected areas being in western China. Significant differences in incidence were found among age groups, with incidences peaking in school-age children. Overall, the epidemic spread from southeast to northwest. Proximity to airports and being intersected by national highways or freeways but not railways were variables associated with the presence of the disease in a county. Lower temperature and lower relative humidity were the climatic factors facilitating local transmission after correction for the effects of school summer vacation and public holidays, as well as population density and the density of medical facilities. These findings indicate that interventions focused on domestic travel, population density, and climatic factors could play a role in mitigating the public health impact of future influenza pandemics.

Replication, Neurotropism, and Pathogenicity of Avian Paramyxovirus Serotypes 1–9 in Chickens and Ducks

Avian paramyxovirus (APMV) serotypes 1–9 have been isolated from many different avian species. APMV-1 (Newcastle disease virus) is the only well-characterized serotype, because of the high morbidity, mortality, and economic loss caused by highly virulent strains. Very little is known about the pathogenesis, replication, virulence, and tropism of the other APMV serotypes. Here, this was evaluated for prototypes strains of APMV serotypes 2–9 in cell culture and in chickens and ducks. In cell culture, only APMV-1, -3 and -5 induced syncytium formation. In chicken DF1 cells, APMV-3 replicated with an efficiency approaching that of APMV-1, while APMV-2 and -5 replicated to lower, intermediate titers and the others were much lower. Mean death time (MDT) assay in chicken eggs and intracerebral pathogenicity index (ICPI) test in 1-day-old SPF chicks demonstrated that APMV types 2–9 were avirulent. Evaluation of replication in primary neuronal cells in vitro as well as in the brains of 1-day-old chicks showed that, among types 2–9, only APMV-3 was neurotropic, although this virus was not neurovirulent. Following intranasal infection of 1-day-old and 2-week-old chickens, replication of APMV types 2–9 was mostly restricted to the respiratory tract, although APMV-3 was neuroinvasive and neurotropic (but not neurovirulent) and also was found in the spleen. Experimental intranasal infection of 3-week-old mallard ducks with the APMVs did not produce any clinical signs (even for APMV-1) and exhibited restricted viral replication of the APMVs (including APMV-1) to the upper respiratory tract regardless of their isolation source, indicating avirulence of APMV types 1–9 in mallard ducks. The link between the presence of a furin cleavage site in the F protein, syncytium formation, systemic spread, and virulence that has been well-established with APMV-1 pathotypes was not evident with the other APMV serotypes.

Patterns and influencing factor of synonymous codon usage in porcine circovirus

BACKGROUND: Analysis of codon usage can reveal much about the molecular evolution of the viruses. Nevertheless, little information about synonymous codon usage pattern of porcine circovirus (PCV) genome in the process of its evolution is available. In this study, to give a new understanding on the evolutionary characteristics of PCV and the effects of natural selection from its host on the codon usage pattern of the virus, Patterns and the key determinants of codon usage in PCV were examined. METHODS: We carried out comprehensive analysis on codon usage pattern in the PCV genome, by calculating relative synonymous codon usage (RSCU), effective number of codons (ENC), dinucleotides and nucleic acid content of the PCV genome. RESULTS: PCV genomes have relatively much lower content of GC and codon preference, this result shows that nucleotide constraints have a major impact on its synonymous codon usage. The results of the correspondence analysis indicate codon usage patterns of PCV of various genotypes, various subgenotypes changed greatly, and significant differences in codon usage patterns of Each virus of Circoviridae.There is much comparability between PCV and its host in their synonymous codon usage, suggesting that the natural selection pressure from the host factor also affect the codon usage patterns of PCV. In particular, PCV genotype II is in synonymous codon usage more similar to pig than to PCV genotype I, which may be one of the most important molecular mechanisms of PCV genotype II to cause disease. The calculations results of the relative abundance of dinucleotides indicate that the composition of dinucleotides also plays a key role in the variation found in synonymous codon usage in PCV. Furthermore, geographic factors, the general average hydrophobicity and the aromaticity may be related to the formation of codon usage patterns of PCV. CONCLUSION: The results of these studies suggest that synonymous codon usage pattern of PCV genome are the result of interaction between mutation pressure and natural selection from its host. The information from this study may not only have theoretical value in understanding the characteristics of synonymous codon usage in PCV genomes, but also have significant value for the molecular evolution of PCV.

Development of a Humanized Antibody with High Therapeutic Potential against Dengue Virus Type 2

BACKGROUND: Dengue virus (DENV) is a significant public health threat in tropical and subtropical regions of the world. A therapeutic antibody against the viral envelope (E) protein represents a promising immunotherapy for disease control. METHODOLOGY/PRINCIPAL FINDINGS: We generated seventeen novel mouse monoclonal antibodies (mAbs) with high reactivity against E protein of dengue virus type 2 (DENV-2). The mAbs were further dissected using recombinant E protein domain I-II (E-DI-II) and III (E-DIII) of DENV-2. Using plaque reduction neutralization test (PRNT) and mouse protection assay with lethal doses of DENV-2, we identified four serotype-specific mAbs that had high neutralizing activity against DENV-2 infection. Of the four, E-DIII targeting mAb DB32-6 was the strongest neutralizing mAb against diverse DENV-2 strains. Using phage display and virus-like particles (VLPs) we found that residue K310 in the E-DIII A-strand was key to mAb DB32-6 binding E-DIII. We successfully converted DB32-6 to a humanized version that retained potency for the neutralization of DENV-2 and did not enhance the viral infection. The DB32-6 showed therapeutic efficacy against mortality induced by different strains of DENV-2 in two mouse models even in post-exposure trials. CONCLUSIONS/SIGNIFICANCE: We used novel epitope mapping strategies, by combining phage display with VLPs, to identify the important A-strand epitopes with strong neutralizing activity. This study introduced potential therapeutic antibodies that might be capable of providing broad protection against diverse DENV-2 infections without enhancing activity in humans.

CD8(+) T Cells in Leishmania Infections: Friends or Foes?

Host protection against several intracellular pathogens requires the induction of CD8(+) T cell responses. CD8(+) T cells are potent effector cells that can produce high amounts of pro-inflammatory cytokines and kill infected target cells efficiently. However, a protective role for CD8(+) T cells during Leishmania infections is still controversial and largely depends on the infection model. In this review, we discuss the role of CD8(+) T cells during various types of Leishmania infections, following vaccination, and as potential immunotherapeutic targets.

Macroevolutionary Immunology: A Role for Immunity in the Diversification of Animal life

An emerging picture of the nature of immune systems across animal phyla reveals both conservatism of some features and the appearance among and within phyla of novel, lineage-specific defense solutions. The latter collectively represent a major and underappreciated form of animal diversity. Factors influencing this macroevolutionary (above the species level) pattern of novelty are considered and include adoption of different life styles, life histories, and body plans; a general advantage of being distinctive with respect to immune defenses; and the responses required to cope with parasites, many of which afflict hosts in a lineage-specific manner. This large-scale pattern of novelty implies that immunological phenomena can affect microevolutionary processes (at the population level within species) that can eventually lead to macroevolutionary events such as speciation, radiations, or extinctions. Immunologically based phenomena play a role in favoring intraspecific diversification, specialization and host specificity of parasites, and mechanisms are discussed whereby this could lead to parasite speciation. Host switching – the acquisition of new host species by parasites – is a major mechanism that drives parasite diversity and is frequently involved in disease emergence. It is also one that can be favored by reductions in immune competence of new hosts. Mechanisms involving immune phenomena favoring intraspecific diversification and speciation of host species are also discussed. A macroevolutionary perspective on immunology is invaluable in today’s world, including the need to study a broader range of species with distinctive immune systems. Many of these species are faced with extinction, another macroevolutionary process influenced by immune phenomena.

Recent Advances in the Diagnosis and Treatment of Influenza Pneumonia

A potentially fatal complication of influenza infection is the development of pneumonia, caused either directly by the influenza virus, or by secondary bacterial infection. Pneumonia related to the 2009 influenza A pandemic was found to be underestimated by commonly used pneumonia severity scores in many cases, and to be rapidly progressive, leading to respiratory failure. Confirmation of etiology by laboratory testing is warranted in such cases. Rapid antigen and immunofluorescence testing are useful screening tests, but have limited sensitivity. Confirmation of pandemic H1N1 influenza A infection can only be made by real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) or viral culture. The most effective preventive measure is annual influenza vaccination in selected individuals. Decisions to administer antiviral medications for influenza treatment or chemoprophylaxis should be based upon clinical and epidemiological factors, and should not be delayed by confirmatory laboratory testing results. Neuraminidase inhibitors (NI) are the agents of choice.

1,3-Diphenyl-4,5-dihydro-1H-pyrazol-5-one

In the title pyrazolone derivative, C(15)H(12)N(2)O, the five-membered ring is approximately planar (r.m.s. deviation = 0.018 Å), and the N- and C-bound benzene rings are inclined to this plane [dihedral angles = 21.45 (10) and 6.96 (10)°, respectively] and form a dihedral angle of 20.42 (10)° with each other. Supra­molecular layers are formed in the crystal structure via C—H⋯O and C—H⋯N inter­actions, and these are assembled into double layers by C—H⋯π and π–π inter­actions between the pyrazole and C-bound benzene rings [ring centroid–centroid distance = 3.6476 (12) Å]. The double layers stack along the a axis being connected by π–π inter­actions between the N- and C-bound benzene rings [ring centroid–centroid distance = 3.7718 (12) Å].

The CEA/CD3-Bispecific Antibody MEDI-565 (MT111) Binds a Nonlinear Epitope in the Full-Length but Not a Short Splice Variant of CEA

MEDI-565 (also known as MT111) is a bispecific T-cell engager (BiTE®) antibody in development for the treatment of patients with cancers expressing carcinoembryonic antigen (CEA). MEDI-565 binds CEA on cancer cells and CD3 on T cells to induce T-cell mediated killing of cancer cells. To understand the molecular basis of human CEA recognition by MEDI-565 and how polymorphisms and spliced forms of CEA may affect MEDI-565 activity, we mapped the epitope of MEDI-565 on CEA using mutagenesis and homology modeling approaches. We found that MEDI-565 recognized a conformational epitope in the A2 domain comprised of amino acids 326–349 and 388–410, with critical residues F(326), T(328), N(333), V(388), G(389), P(390), E(392), I(408), and N(410). Two non-synonymous single-nucleotide polymorphisms (SNPs) (rs10407503, rs7249230) were identified in the epitope region, but they are found at low homozygosity rates. Searching the National Center for Biotechnology Information GenBank® database, we further identified a single, previously uncharacterized mRNA splice variant of CEA that lacks a portion of the N-terminal domain, the A1 and B1 domains, and a large portion of the A2 domain. Real-time quantitative polymerase chain reaction analysis of multiple cancers showed widespread expression of full-length CEA in these tumors, with less frequent but concordant expression of the CEA splice variant. Because the epitope was largely absent from the CEA splice variant, MEDI-565 did not bind or mediate T-cell killing of cells solely expressing this form of CEA. In addition, the splice variant did not interfere with MEDI-565 binding or activity when co-expressed with full-length CEA. Thus MEDI-565 may broadly target CEA-positive tumors without regard for expression of the short splice variant of CEA. Together our data suggest that MEDI-565 activity will neither be impacted by SNPs nor by a splice variant of CEA.

High Throughput Screening for Small Molecule Enhancers of the Interferon Signaling Pathway to Drive Next-Generation Antiviral Drug Discovery

Most of current strategies for antiviral therapeutics target the virus specifically and directly, but an alternative approach to drug discovery might be to enhance the immune response to a broad range of viruses. Based on clinical observation in humans and successful genetic strategies in experimental models, we reasoned that an improved interferon (IFN) signaling system might better protect against viral infection. Here we aimed to identify small molecular weight compounds that might mimic this beneficial effect and improve antiviral defense. Accordingly, we developed a cell-based high-throughput screening (HTS) assay to identify small molecules that enhance the IFN signaling pathway components. The assay is based on a phenotypic screen for increased IFN-stimulated response element (ISRE) activity in a fully automated and robust format (Z′>0.7). Application of this assay system to a library of 2240 compounds (including 2160 already approved or approvable drugs) led to the identification of 64 compounds with significant ISRE activity. From these, we chose the anthracycline antibiotic, idarubicin, for further validation and mechanism based on activity in the sub-µM range. We found that idarubicin action to increase ISRE activity was manifest by other members of this drug class and was independent of cytotoxic or topoisomerase inhibitory effects as well as endogenous IFN signaling or production. We also observed that this compound conferred a consequent increase in IFN-stimulated gene (ISG) expression and a significant antiviral effect using a similar dose-range in a cell-culture system inoculated with encephalomyocarditis virus (EMCV). The antiviral effect was also found at compound concentrations below the ones observed for cytotoxicity. Taken together, our results provide proof of concept for using activators of components of the IFN signaling pathway to improve IFN efficacy and antiviral immune defense as well as a validated HTS approach to identify small molecules that might achieve this therapeutic benefit.

Feline Immunodeficiency Virus in South America

The rapid emergence of AIDS in humans during the period between 1980 and 2000 has led to extensive efforts to understand more fully similar etiologic agents of chronic and progressive acquired immunodeficiency disease in several mammalian species. Lentiviruses that have gene sequence homology with human immunodeficiency virus (HIV) have been found in different species (including sheep, goats, horses, cattle, cats, and several Old World monkey species). Lentiviruses, comprising a genus of the Retroviridae family, cause persistent infection that can lead to varying degrees of morbidity and mortality depending on the virus and the host species involved. Feline immunodeficiency virus (FIV) causes an immune system disease in domestic cats (Felis catus) involving depletion of the CD4+ population of T lymphocytes, increased susceptibility to opportunistic infections, and sometimes death. Viruses related to domestic cat FIV occur also in a variety of nondomestic felids. This is a brief overview of the current state of knowledge of this large and ancient group of viruses (FIVs) in South America.

Chinese Medicine Shenfu Injection for Heart Failure: A Systematic Review and Meta-Analysis

Objective. Heart failure (HF) is a global public health problem. Early literature studies manifested that Shenfu injection (SFI) is one of the most commonly used traditional Chinese patent medicine for HF in China. This article intended to systematically evaluate the efficacy and safety of SFI for HF. Methods. An extensive search was performed within 6 English and Chinese electronic database up to November 2011. Ninety-nine randomized controlled trails (RCTs) were collected, irrespective of languages. Two authors extracted data and assessed the trial quality independently. RevMan 5.0.2 was used for data analysis. Results. Compared with routine treatment and/or device support, SFI combined with routine treatment and/or device support showed better effect on clinical effect rate, mortality, heart rate, NT-proBNP and 6-minute walk distance. Results in ultrasonic cardiography also showed that SFI combined with routine treatment improved heart function of HF patients. There were no significant difference in blood pressure between SFI and routine treatment groups. Adverse events were reported in thirteen trails with thirteen specific symptoms, while no serious adverse effect was reported. Conclusion. SFI appear to be effective for treating HF. However, further rigorously designed RCTs are warranted because of insufficient methodological rigor in the majority of included trials.

Genome Stability of Pandemic Influenza A (H1N1) 2009 Based on Analysis of Hemagglutinin and Neuraminidase Genes

Influenza A virus (H1N1), which arose in 2009, constituted the fourth pandemic after the cases of 1918, 1957, and 1968. This new variant was formed by a triple reassortment, with genomic segments from swine, avian, and human influenza origins. The objective of this study was to analyze sequences of hemagglutinin (n=2038) and neuraminidase (n=1273) genes, in order to assess the extent of diversity among circulating 2009-2010 strains, estimate if these genes evolved through positive, negative, or neutral selection models of evolution during the pandemic phase, and analyze the worldwide percentage of detection of important amino acid mutations that could enhance the viral performance, such as transmissibility or resistance to drugs. A continuous surveillance by public health authorities will be critical to monitor the appearance of new influenza variants, especially in animal reservoirs such as swine and birds, in order to prevent the potential animal-human transmission of viruses with pandemic potential.

Severe imported malaria in an intensive care unit: a review of 59 cases

BACKGROUND: In view of the close relationship of Portugal with African countries, particularly former Portuguese colonies, the diagnosis of malaria is not a rare thing. When a traveller returns ill from endemic areas, malaria should be the number one suspect. World Health Organization treatment guidelines recommend that adults with severe malaria should be admitted to an intensive care unit (ICU). METHODS: Severe cases of malaria in patients admitted to an ICU were reviewed retrospectively (1990-2011) and identification of variables associated with in-ICU mortality performed. Malaria prediction score (MPS), malaria score for adults (MSA), simplified acute physiology score (SAPSII) and a score based on WHO's malaria severe criteria were applied. Statistical analysis was performed using StataV12. RESULTS: Fifty nine patients were included in the study, all but three were adults; 47 (79,6%) were male; parasitaemia on admission, quantified in 48/59 (81.3%) patients, was equal or greater than 2% in 47 of them (97.9%); the most common complications were thrombocytopaenia in 54 (91.5%) patients, associated with disseminated intravascular coagulation (DIC) in seven (11.8%), renal failure in 31 (52.5%) patients, 18 of which (30.5%) oliguric, shock in 29 (49.1%) patients, liver dysfunction in 27 (45.7%) patients, acidaemia in 23 (38.9%) patients, cerebral dysfunction in 22 (37.2%) patients, 11 of whom with unrousable coma, pulmonary oedema/ARDS in 22 (37.2%) patients, hypoglycaemia in 18 (30.5%) patients; 29 (49.1%) patients presented five or more dysfunctions. The case fatality rate was 15.2%. Comparing the four scores, the SAPS II and the WHO score were the most sensitive to death prediction. In the univariate analysis, death was associated with the SAPS II score, cerebral malaria, acute renal and respiratory failure, DIC, spontaneous bleeding, acidosis and hypoglycaemia. Age, partial immunity to malaria, delay in malaria diagnosis and the level of parasitaemia were not associated with death in this cohort. CONCLUSION: Severe malaria cases should be continued monitored in the ICUs. SAPS II and the WHO score are good predictors of mortality in malaria patients, but other specific scores deserve to be studied prospectively.

Use of controlled low dose gamma irradiation to sterilize allograft tendons for ACL reconstruction: biomechanical and clinical perspective

As reviewed here, numerous biomechanical and clinical studies support the use of controlled, low temperature irradiation of allograft tendons, to provide both excellent clinical results and medical-device grade sterile allografts with minimal risk of disease transmission.

Distinct immunoregulatory cytokine pattern in Egyptian patients with occult Hepatitis C infection and unexplained persistently elevated liver transaminases

BACKGROUND/AIM: The immunopathogenesis of occult Hepatitis C virus (HCV) infection is a matter of great controversy and has been suggested to involve a complex balance between cytokines with pro- and anti-inflammatory activity. This work aimed at studying the serum Th1 and Th2 cytokine production in patients with occult HCV infection. MATERIALS AND METHODS: Serum levels of cytokines of Th1 (interleukin [IL]-2, INF-γ) and Th2 (IL-4) were measured in 27 patients with occult HCV infection and 28 patients with chronic hepatitis C infection. RESULTS: The levels of IL-2 and interferon-γ were highly significantly increased in patients with chronic HCV infection (P<0.001). IL-4 was highly significantly increased in occult HCV infection (P<0.001). Significant increases were noted in chronic HCV infection regarding bilirubin (P<0.001), ALT (P = 0.009), AST (P = 0.013), AFP (P<0.001), while serum albumin was significantly higher in occult HCV infection (P<0.001). Necroinflammation (P<0.001), fibrosis (P<0.001), and cirrhosis (P = 0.03) were significantly increased in chronic HCV infection. CONCLUSION: Our data revealed a high prevalence of occult HCV infection (25%) in patients with unexplained persistently abnormal liver function test results. Those patients exhibited a distinct immunoregulatory cytokine pattern, favoring viral persistence and explaining the less aggressive course of this disease entity than chronic HCV infection.

DC-SIGN (CD209) Promoter −336 A/G (rs4804803) Polymorphism Associated with Susceptibility of Kawasaki Disease

Kawasaki disease (KD) is characterized by systemic vasculitis of unknown etiology. High-dose intravenous immunoglobulin (IVIG) is the most effective therapy for KD to reduce the prevalence of coronary artery lesion (CAL) formation. Recently, the α2, 6 sialylated IgG was reported to interact with a lectin receptor, specific intracellular adhesion molecule-3 grabbing nonintegrin homolog-related 1 (SIGN-R1) in mice and dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) in human, and to trigger an anti-inflammatory cascade. This study was conducted to investigate whether the polymorphism of DC-SIGN (CD209) promoter −336 A/G (rs4804803) is responsible for susceptibility and CAL formation in KD patients using Custom TaqMan SNP Genotyping Assays. A total of 521 subjects (278 KD patients and 243 controls) were investigated to identify an SNP of rs4804803, and they were studied and showed a significant association between the genotypes and allele frequency of rs4804803 in control subjects and KD patients (P = 0.004 under the dominant model). However, the promoter variant of DC-SIGN gene was not associated with the occurrence of IVIG resistance, CAL formation in KD. The G allele of DC-SIGN promoter −336 (rs4804803) is a risk allele in the development of KD.