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The arthropod melanization immune response is activated by extracellular protease cascades predominantly comprised of CLIP-domain serine proteases (CLIP-SPs) and serine protease homologs (CLIP-SPHs). In the malaria vector, Anopheles gambiae, the CLIP-SPHs SPCLIP1, CLIPA8, and CLIPA28 form the core of a hierarchical cascade downstream of mosquito complement that is required for microbial melanization. However, our understanding of the regulatory relationship of the CLIP-SPH cascade with the catalytic CLIP-SPs driving melanization is incomplete. Here, we report on the development of a novel screen to identify melanization pathway components based on the quantitation of melanotic mosquito excreta, eliminating the need for microdissections or hemolymph enzymatic assays. Using this screen, we identified CLIPC9 and subsequent functional analyses established that this protease is essential for the melanization of both Escherichia coli and the rodent malaria parasite Plasmodium berghei. Mechanistically, septic infection with E. coli promotes CLIPC9 cleavage and both full-length and cleaved CLIPC9 localize to this bacterium in a CLIPA8-dependent manner. The steady state level of CLIPC9 in the hemolymph is regulated by thioester-containing protein 1 (TEP1), suggesting it functions downstream of mosquito complement. In support, CLIPC9 cleavage is inhibited following SPCLIP1, CLIPA8, and CLIPA28 knockdown positioning it downstream of the CLIP-SPH cascade. Moreover, like CLIPA8 and CLIPA28, CLIPC9 processing is negatively regulated by serine protease inhibitor 2 (SRPN2). This report demonstrates how our novel excretion-based approach can be utilized to dissect the complex protease networks regulating mosquito melanization. Collectively, our findings establish that CLIPC9 is required for microbial melanization in An. gambiae and shed light on how the CLIP-SPH cascade regulates this potent immune response., Author summary Mosquito vector competence for Plasmodium, antifungal defense, and lifespan are all influenced by the melanization response. Despite its importance, our understanding of the proteins comprising the An. gambiae melanization cascade is incomplete. To streamline the discovery of melanization pathway components in this disease vector, we developed a screening method that is able to identify proteins with far fewer mosquitoes than other approaches. This technique facilitated our discovery that the serine protease CLIPC9 is required for the melanization of E. coli bacteria and malaria parasites. CLIPC9 activation and localization to bacteria is regulated by members of the CLIPA subfamily, highlighting that these serine protease homologs broadly regulate the melanization immune response. Traditionally viewed as ‘co-factors’ for the prophenoloxidase activating CLIPBs, this work demonstrates that CLIPAs can also control the activation of the catalytic proteases driving melanization. This work identifies a new player in the melanization hierarchy and provides the field with an efficient approach for dissecting the complex protease cascades controlling this important immune response.

BACKGROUND & AIMS:Extended esophageal endoscopic submucosal dissection (ESD) is highly responsible for esophageal stricture. We conducted a comparative study in a porcine model to evaluate the effectiveness of adipose tissue-derived stromal cell (ADSC) double cell sheet transplantation. METHODS:Twelve female pigs were treated with 5 cm long hemi-circumferential ESD and randomized in two groups. ADSC group (n = 6) received 4 double cell sheets of allogenic ADSC on a paper support membrane and control group (n = 6) received 4 paper support membranes. ADSC were labelled with PKH-67 fluorophore to allow probe-based confocal laser endomicroscopie (pCLE) monitoring. After 28 days follow-up, animals were sacrificed. At days 3, 14 and 28, endoscopic evaluation with pCLE and esophagography were performed. RESULTS:One animal from the control group was excluded (anesthetic complication). Animals from ADSC group showed less frequent alimentary trouble (17% vs 80%; P = 0.08) and higher gain weight on day 28. pCLE demonstrated a compatible cell signal in 4 animals of the ADSC group at day 3. In ADSC group, endoscopy showed that 1 out of 6 (17%) animals developed a severe esophageal stricture comparatively to 100% (5/5) in the control group; P = 0.015. Esophagography demonstrated a decreased degree of stricture in the ADSC group on day 14 (44% vs 81%; P = 0.017) and day 28 (46% vs 90%; P = 0.035). Histological analysis showed a decreased fibrosis development in the ADSC group, in terms of surface (9.7 vs 26.1 mm²; P = 0.017) and maximal depth (1.6 vs 3.2 mm; P = 0.052). CONCLUSION:In this model, transplantation of allogenic ADSC organized in double cell sheets after extended esophegeal ESD is strongly associated with a lower esophageal stricture's rate.

In this essay, we show that 3 distinct approaches to immunological exhaustion coexist and that they only partially overlap, generating potential misunderstandings. Exploring cases ranging from viral infections to cancer, we propose that it is crucial, for experimental and therapeutic purposes, to clarify these approaches and their interconnections so as to make the concept of exhaustion genuinely operational., Author summary In this essay, we have written a critical review on immunological exhaustion. We believe that this widely used concept often remains in fact imprecise because there exist 3 different approaches to exhaustion, namely, in terms of dysfunction, cause, and marker, and those are not sufficiently well distinguished and articulated in most scientific papers. We also propose to talk about “exhaustion” for and only for the phenomena in which all 3 approaches are aligned. This can be called the “convergence strategy”: T cells would be described as “exhausted” if and only if they are simultaneously dysfunctional, express given markers such as programmed cell death protein 1 (PD-1), and induced by a specific cause such as chronicity. This strategy could be perfectly reasonable, and we see much value in adopting it, because it would force everyone to be more specific and rigorous when talking about exhaustion. Clarifying the characteristics of cells defined as “exhausted,” and using a convergent strategy to define T-cell exhaustion could have major experimental and clinical consequences, including for viral infections such as the Coronavirus Disease 2019 (COVID-19).

Funder: National Institute for Health Research; funder-id: http://dx.doi.org/10.13039/501100000272, Background: Understanding the T cell response to SARS-CoV-2 is critical to vaccine development, epidemiological surveillance and disease control strategies. This systematic review critically evaluates and synthesises the relevant peer-reviewed and pre-print literature published from 01/01/2020-26/06/2020. Methods: For this systematic review, keyword-structured literature searches were carried out in MEDLINE, Embase and COVID-19 Primer. Papers were independently screened by two researchers, with arbitration of disagreements by a third researcher. Data were independently extracted into a pre-designed Excel template and studies critically appraised using a modified version of the MetaQAT tool, with resolution of disagreements by consensus. Findings were narratively synthesised. Results: 61 articles were included. 55 (90%) studies used observational designs, 50 (82%) involved hospitalised patients with higher acuity illness, and the majority had important limitations. Symptomatic adult COVID-19 cases consistently show peripheral T cell lymphopenia, which positively correlates with increased disease severity, duration of RNA positivity, and non-survival; while asymptomatic and paediatric cases display preserved counts. People with severe or critical disease generally develop more robust, virus-specific T cell responses. T cell memory and effector function has been demonstrated against multiple viral epitopes, and, cross-reactive T cell responses have been demonstrated in unexposed and uninfected adults, but the significance for protection and susceptibility, respectively, remains unclear. Conclusion: A complex pattern of T cell response to SARS-CoV-2 infection has been demonstrated, but inferences regarding population level immunity are hampered by significant methodological limitations and heterogeneity between studies, as well as a striking lack of research in asymptomatic or pauci-symptomatic individuals. In contrast to antibody responses, population-level surveillance of the T cell response is unlikely to be feasible in the near term. Focused evaluation in specific sub-groups, including vaccine recipients, should be prioritised.

Pain and inflammation are unpleasant experiences that usually occur as a result of tissue damage. Despite the number of existing analgesic drugs, side effects limit their use, stimulating the search for new therapeutic agents. In this sense, five hydrazone derivatives (H1, H2, H3, H4, and H5), with general structure R1R2C = NNR3R4, were synthesized with molecular modification strategies. In this paper, we describe the ability of hydrazone derivatives to attenuate nociceptive behavior and the inflammatory response in mice. Antinociceptive activity was evaluated through acetic acid-induced writhing and formalin-induced nociception tests. In both experimental models, the hydrazone with the greatest potency (H5) significantly (p < 0.05) reduced nociceptive behavior. Additionally, methods of acute and chronic inflammation induced by different chemicals (carrageenan and histamine) were performed to evaluate the anti-inflammatory effect of H5. Moreover, molecular docking analysis revealed that H5 can block the COX-2 enzyme, reducing arachidonic acid metabolism and consequently decreasing the production of prostaglandins, which are important inflammatory mediators. H5 also changes locomotor activity. In summary, H5 exhibited relevant antinociceptive and anti-inflammatory potential and acted on several targets, making it a candidate for a new multi-target oral anti-inflammatory drug.

SARS-CoV-2 uses the human ACE2 (hACE2) receptor for cell attachment and entry, with mouse ACE2 (mACE2) unable to support infection. Herein we describe an ACE2-lentivirus system and illustrate its utility for in vitro and in vivo SARS-CoV-2 infection models. Transduction of non-permissive cell lines with hACE2 imparted replication competence, and transduction with mACE2 containing N30D, N31K, F83Y and H353K substitutions, to match hACE2, rescued SARS-CoV-2 replication. Intrapulmonary hACE2-lentivirus transduction of C57BL/6J mice permitted significant virus replication in lung epithelium. RNA-Seq and histological analyses illustrated that this model involved an acute inflammatory disease followed by resolution and tissue repair, with a transcriptomic profile similar to that seen in COVID-19 patients. hACE2-lentivirus transduction of IFNAR-/- and IL-28RA-/- mouse lungs was used to illustrate that loss of type I or III interferon responses have no significant effect on virus replication. However, their importance in driving inflammatory responses was illustrated by RNA-Seq analyses. We also demonstrate the utility of the hACE2-lentivirus transduction system for vaccine evaluation in C57BL/6J mice. The ACE2-lentivirus system thus has broad application in SARS-CoV-2 research, providing a tool for both mutagenesis studies and mouse model development., Author summary SARS-CoV-2 uses the human ACE2 (hACE2) receptor to infect cells, but cannot infect mice because the virus cannot bind mouse ACE2 (mACE2). We use an ACE2-lentivirus system in vitro to identify four key amino acids in mACE2 that explain why SARS-CoV-2 cannot infect mice. hACE2-lentivirus was used to express hACE2 in mouse lungs in vivo, with the inflammatory responses after SARS-CoV-2 infection similar to those seen in human COVID-19. Genetically modified mice were used to show that type I and III interferon signaling is required for the inflammatory responses. We also show that the hACE2-lentivirus mouse model can be used to test vaccines. Overall this paper demonstrates that our hACE2-lentivirus system has multiple applications in SARS-CoV-2 and COVID-19 research.

The aim of the paper was to assess indicators of muscle and intestinal damage in triathletes. The study involved 15 triathletes whose objective for the season was to start in the XTERRA POLAND 2017 event (1,500-m swimming, 36-km cycling, and 10-km mountain running). Before the 14-week preparatory period, the competitors' body composition was measured, aerobic capacity was tested (graded treadmill test) and blood samples were collected to determine markers showing the level of muscle and intestinal damage. Subsequent tests for body composition were carried out before and after the competition. Blood samples for biochemical indicators were collected the day before the competition, after the completed race, and 24 and 48 hours later. A significant decrease in body mass was observed after completing the race (-3.1±1.5%). The mean maximal oxygen uptake level among the studied athletes equalled 4.9±0.4 L·min-1, 58.8±4.5 mL·kg-1·min-1. The significant increase in concentrations of cortisol, c-reactive protein and myoglobin after the competition, significantly correlated with the significant increase in zonulin concentration (post 1h: r = 0.88, p = 0.007, r = 0,79, p = 0.001, r = 0.78, p = 0.001, and post 12h: r = 0.75, p = 0.01, r = 0.71, p = 0.011, r = 0.83, p = 0.02). No significant changes in the concentration of tumour necrosis factor alpha among the examined competitors were noted at following stages of the study. The results of our research showed that in order to monitor overload in the training of triathletes, useful markers reflecting the degree of muscle and intestinal damage include cortisol, testosterone, testosterone to cortisol ratio, c-reactive protein, myoglobin and zonulin. Changes in muscle cell damage markers strongly correlated with changes in zonulin concentration at particular stages of the study. Thus, one can expect that the concentrations of markers depicting the level of muscle cell damage after an intense and long-lasting effort will significantly influence the level of the intestinal barrier.

Streptococcus pneumonia is an important human pathogen that causes various severe diseases such as pneumonia, otitis and meningitis. Vaccination against S. pneumoniae is implemented in many developed countries. The presently used vaccines are safe, well tolerated but relatively expensive and require modification due to the immunological changes of the epidemic strains. This paper describes the development of a new pneumococcal vaccine candidate for immunization on mucosal surfaces. For this purpose the antigens of chimeric protein PSPF, previously suggested for an injectable S. pneumoniae vaccine, were expressed on the surface of the live probiotic strain Enterococcus faecium L3. Experiments on laboratory mice vaccinated with live bacteria demonstrated the appearance of the specific IgA and IgG which provide protection against the lethal S. pneumoniae infection.

Introduction Reliable measurements of the protein content of biological fluids like serum or plasma can provide valuable input for the development of personalized medicine tests. Standard MALDI analysis typically only shows high abundance proteins, which limits its utility for test development. It also exhibits reproducibility issues with respect to quantitative measurements. In this paper we show how the sensitivity of MALDI profiling of intact proteins in unfractionated human serum can be substantially increased by exposing a sample to many more laser shots than are commonly used. Analytical reproducibility is also improved. Methods To assess what is theoretically achievable we utilized spectra from the same samples obtained over many years and combined them to generate MALDI spectral averages of up to 100,000,000 shots for a single sample, and up to 8,000,000 shots for a set of 40 different serum samples. Spectral attributes, such as number of peaks and spectral noise of such averaged spectra were investigated together with analytical reproducibility as a function of the number of shots. We confirmed that results were similar on MALDI instruments from different manufacturers. Results We observed an expected decrease of noise, roughly proportional to the square root of the number of shots, over the whole investigated range of the number of shots (5 orders of magnitude), resulting in an increase in the number of reliably detected peaks. The reproducibility of the amplitude of these peaks, measured by CV and concordance analysis also improves with very similar dependence on shot number, reaching median CVs below 2% for shot numbers > 4 million. Measures of analytical information content and association with biological processes increase with increasing number of shots. Conclusions We demonstrate that substantially increasing the number of laser shots in a MALDI-TOF analysis leads to more informative and reliable data on the protein content of unfractionated serum. This approach has already been used in the development of clinical tests in oncology.

Galanin is a neuropeptide widely expressed in central and peripheral nerves and is known to be engaged in neuronal responses to pathological changes. Stomach ulcerations are one of the most common gastrointestinal disorders. Impaired stomach function in peptic ulcer disease suggests changes in autonomic nerve reflexes controlled by the inferior vagal ganglion, resulting in stomach dysfunction. In this paper, changes in the galaninergic response of inferior vagal neurons to gastric ulceration in a pig model of the disease were analyzed based on the authors’ previous studies. The study was performed on 24 animals (12 control and 12 experimental). Gastric ulcers were induced by submucosal injections of 40% acetic acid solution into stomach submucosa and bilateral inferior vagal ganglia were collected one week afterwards. The number of galanin-immunoreactive perikarya in each ganglion was counted to determine fold-changes between both groups of animals and Q-PCR was applied to verify the changes in relative expression level of mRNA encoding both galanin and its receptor subtypes: GalR1, GalR2, GalR3. The results revealed a 2.72-fold increase in the number of galanin-immunoreactive perikarya compared with the controls. Q-PCR revealed that all studied genes were expressed in examined ganglia in both groups of animals. Statistical analysis revealed a 4.63-fold increase in galanin and a 1.45-fold increase in GalR3 mRNA as compared with the controls. No differences were observed between the groups for GalR1 or GalR2. The current study confirmed changes in the galaninergic inferior vagal ganglion response to stomach ulcerations and demonstrated, for the first time, the expression of mRNA encoding all galanin receptor subtypes in the porcine inferior vagal ganglia.

Background SARS-Cov2 infection may trigger lung inflammation and acute-respiratory-distress-syndrome (ARDS) that requires active ventilation and may have fatal outcome. Considering the severity of the disease and the lack of active treatments, 14 patients with Covid-19 and severe lung inflammation received inhaled adenosine in the attempt to therapeutically compensate for the oxygen-related loss of the endogenous adenosine!A2A adenosine receptor (A2AR)-mediated mitigation of the lung-destructing inflammatory damage. This off label-treatment was based on preclinical studies in mice with LPS-induced ARDS, where inhaled adenosine/A2AR agonists protected oxygenated lungs from the deadly inflammatory damage. The treatment was allowed, considering that adenosine has several clinical applications. Patients and treatment Fourteen consecutively enrolled patients with Covid19-related interstitial pneumonitis and PaO2/FiO2 ratio 30%) in the PaO2/FiO2-ratio was reported in 13 out of 14 patients treated with adenosine compared with that observed in 7 out of52 patients in the control within 15 days. Additionally, we recorded a mean PaO2/FiO2-ratio increase (215 ± 45 vs. 464 ± 136, P = 0.0002) in patients receiving adenosine and no change in the control group (210±75 vs. 250±85 at 120 hours, P>0.05). A radiological response was demonstrated in 7 patients who received adenosine, while SARS-CoV-2 RNA load rapidly decreased in 13 cases within 7 days while no changes were recorded in the control group within 15 days. There was one Covid-19 related death in the experimental group and 11in the control group. Conclusion Our short-term analysis suggests the overall safety and beneficial therapeutic effect of inhaled adenosine in patients with Covid-19-inflammatory lung disease suggesting further investigation in controlled clinical trials. Background Covid-19 outbreak has been declared as pandemic by the WHO reporting more than 4 million new cases worldwide with 300,000 related deaths [1]. Almost 20% of these patients developed interstitial pneumonitis that may evolve in ARDS requiring hyperoxic active ventilation, with mostly fatal outcomes [2–5]. The pathogenesis of the Covid-19-related lung injury is still controversial; however, a massive and uncoordinated release of inflammatory cytokines and a post-ischemic reaction to micro-vascular damage and micro-embolization seem to be involved [5–10]. Due to the acute medical need, different drugs are tested in ongoing trials aimed to hamper the effects of the cytokines involved the first phases of the inflammatory process [11–14]. However, mAbs to IL1β (Kanakinumab), IL6-Receptor (Tocilizumab) and inhibitors of Janus kinases (JAK)-1/2 (Baricitinib and Ruxolitinib) did not produce satisfactory clinical outcomes in patients with Covid19-related interstitial pneumonitis [14–16]. The most recent clinical evidences highlight a 20–30% mortality rate in patients with Covid19-related lung injury requiring active oxygen ventilation and depending on the classification of patients, time of intervention and how critical their illness is [17–19]. These reports are leading to the suspicions that some iatrogenic complication other than a mechanical lung damage does occur [17–19]. Accordingly, we carried out a clinical investigation based on insights and therapeutic suggestions offered in preclinical studies and paper with the self-explanatory title: “Oxygenation inhibits the physiological tissue-protecting mechanism and thereby exacerbates acute inflammatory lung injury” [20]. Based on the results of those preclinical studies in mice we hypothesized that mechanical ventilation and hyper-oxygenation lead to the unacceptable inflammatory side effects in patients with Covid19-related severe pulmonary complications. We further assumed, that the otherwise life-saving oxygenation also weakens the local tissue hypoxia-driven and adenosine A2A receptor (A2AR)-mediated anti-inflammatory mechanism [21–23]. Without this major physiological anti-inflammatory lung tissue protection, the neutrophils, lung macrophages and pulmonary natural killer cells in lungs are no longer inhibited and are unleashed to destroy the still healthy lung [20, 24–26].

The placenta is a dynamic organ which undergoes extensive remodeling throughout pregnancy to support, protect and nourish the developing fetus. Despite the importance of the placenta, very little is known about its gene expression beyond very early pregnancy and post-partum. Therefore, we utilized RNA-sequencing to characterize the transcriptome from the fetal (chorioallantois) and maternal (endometrium) components of the placenta from mares throughout gestation (4, 6, 10, 11 m). Within the endometrium, 47% of genes changed throughout pregnancy, while in the chorioallantois, 29% of genes underwent significant changes in expression. Further bioinformatic analyses of both differentially expressed genes and highly expressed genes help reveal similarities and differences between tissues. Overall, the tissues were more similar than different, with ~ 95% of genes expressed in both tissues, and high similarities between the most highly expressed genes (9/20 conserved), as well as marked similarities between the PANTHER pathways identified. The most highly expressed genes fell under a few broad categories, including endocrine and immune-related transcripts, iron-binding proteins, extracellular matrix proteins, transport proteins and antioxidants. Serine protease inhibitors were particularly abundant, including SERPINA3, 6 and 14, as well as SPINK7 and 9. This paper also demonstrates the ability to effectively separate maternal and fetal components of the placenta, with only a minimal amount of chorioallantoic contamination in the endometrium (~8%). This aspect of equine placentation is a boon for better understanding gestational physiology and allows the horse to be used in areas where a separation of fetal and maternal tissues is essential. Overall, these data represent the first large-scale characterization of placental gene expression in any species and include time points from multiple mid- to late-gestational stages, helping further our understanding of gestational physiology.

We refine and clinically parameterize a mathematical model of the humoral immune response against Shigella, a diarrheal bacteria that infects 80-165 million people and kills an estimated 600,000 people worldwide each year. Using Latin hypercube sampling and Monte Carlo simulations for parameter estimation, we fit our model to human immune data from two Shigella EcSf2a-2 vaccine trials and a rechallenge study in which antibody and B-cell responses against Shigella′s lipopolysaccharide (LPS) and O-membrane proteins (OMP) were recorded. The clinically grounded model is used to mathematically investigate which key immune mechanisms and bacterial targets confer immunity against Shigella and to predict which humoral immune components should be elicited to create a protective vaccine against Shigella. The model offers insight into why the EcSf2a-2 vaccine had low efficacy and demonstrates that at a group level a humoral immune response induced by EcSf2a-2 vaccine or wild-type challenge against Shigella′s LPS or OMP does not appear sufficient for protection. That is, the model predicts an uncontrolled infection of gut epithelial cells that is present across all best-fit model parameterizations when fit to EcSf2a-2 vaccine or wild-type challenge data. Using sensitivity analysis, we explore which model parameter values must be altered to prevent the destructive epithelial invasion by Shigella bacteria and identify four key parameter groups as potential vaccine targets or immune correlates: 1) the rate that Shigella migrates into the lamina propria or epithelium, 2) the rate that memory B cells (BM) differentiate into antibody-secreting cells (ASC), 3) the rate at which antibodies are produced by activated ASC, and 4) the Shigella-specific BM carrying capacity. This paper underscores the need for a multifaceted approach in ongoing efforts to design an effective Shigella vaccine.

Background Due to the surrounding bone, the human inner ear is relatively inaccessible and difficult to reach for cellular and molecular analyses. However, these types of investigations are needed to better understand the etiology, pathophysiology and progression of several inner ear disorders. Moreover, the fluid from the inner ear cannot be sampled for micro-chemical analyses from healthy individuals in vivo. Therefore, in the present paper, we studied patients with vestibular schwannoma (VS) undergoing trans-labyrinthine surgery (TLS). Our primary aim was to identify perilymph proteins in patients with VS on an individual level. Our second aim was to investigate the proteins identified at a functional level and our final aim was to search for biological markers for tumor-associated hearing loss and tumor diameter. Methods and findings Sixteen patients underwent TLS for sporadic VS. Perilymph was aspirated through the round window before opening the labyrinth. One sample was contaminated and excluded resulting in 15 usable samples. Perilymph samples were analyzed with an online tandem LTQ-Orbitrap mass spectrometer. Data were analyzed with MaxQuant software to identify the total number of proteins and to quantify proteins in individual samples. Protein function was analyzed using the PANTHER Overrepresentation tool. Associations between perilymph protein content, clinical parameters, tumor-associated hearing loss and tumor diameter were assessed using Random Forest and Boruta. In total, 314 proteins were identified; 60 in all 15 patients and 130 proteins only once in 15 patients. Ninety-one proteins were detected in at least 12 out of 15 patients. Random Forest followed by Boruta analysis confirmed that alpha-2-HS-glycoprotein (P02765) was an independent variable for tumor-associated hearing loss. In addition, functional analysis showed that numerous processes were significantly increased in the perilymph. The top three enriched biological processes were: 1) secondary metabolic processes; 2) complement activation and 3) cell recognition. Conclusions The proteome of perilymph in patients with vestibular schwannoma has an inter-individual stable section. However, even in a cohort with homogenous disease, the variation between individuals represented the majority of the detected proteins. Alpha-2-HS-glycoprotein, P02765, was shown to be an independent variable for tumor-associated hearing loss, a finding that needs to be verified in other studies. In pathway analysis perilymph had highly enriched functions, particularly in terms of increased immune and metabolic processes.

With the HIV-1 epidemic in southern Africa still rising, a prophylactic vaccine against the region's most prolific subtype (subtype C) would be a significant step forward. In this paper we report on the effect of 2 different adjuvants, AddaVax and AlhydroGel, formulated with HIV-1 subtype C gp140, on the development of binding and neutralising antibody titres in rabbits. AddaVax is a squalene-based oil-in-water nano-emulsion (similar to MF59) which can enhance both cellular and humoral immune responses, whilst AlhydroGel (aluminium hydroxide gel) mainly drives a Th2 response. The gp140 gene tested was derived from the superinfecting virus (SU) from participant CAP256 in the CAPRISA 002 Acute infection cohort. The furin cleavage site of the Env protein was replaced with a flexible linker and an I559P mutation introduced. Lectin affinity purified soluble Env protein was mainly trimeric as judged by molecular weight using BN-PAGE and contained intact broadly neutralising epitopes for the V3-glycan supersite (monoclonal antibodies PGT128 and PGT135), the CD4 binding site (VRC01) and the V2-glycan (PG9) but not for the trimer-specific monoclonal antibodies PG16, PGT145 and CAP256-VRC26_08. When this soluble Env protein was tested in rabbits, AlhydroGel significantly enhanced soluble Env and V1V2 binding antibodies when compared to AddaVax. Finally, AlhydroGel resulted in significantly higher neutralization titres for a subtype C Tier 1A virus (MW965.26) and increased neutralization breadth to Tier 1A and 1B viruses. However, no autologous Tier 2 neutralisation was observed. These data suggest that adjuvant selection is critical for developing a successful vaccine and AlhydroGel should be further investigated. Additional purification of trimeric native-like CAP256 Env and/or priming with DNA or MVA might enhance the induction of neutralizing antibodies and possible Tier 2 HIV-1 neutralisation.

Photorhabdus asymbiotica is one of the three recognized species of the Photorhabdus genus, which consists of gram-negative bioluminescent bacteria belonging to the family Morganellaceae. These bacteria live in a symbiotic relationship with nematodes from the genus Heterorhabditis, together forming a complex that is highly pathogenic for insects. Unlike other Photorhabdus species, which are strictly entomopathogenic, P. asymbiotica is unique in its ability to act as an emerging human pathogen. Analysis of the P. asymbiotica genome identified a novel fucose-binding lectin designated PHL with a strong sequence similarity to the recently described P. luminescens lectin PLL. Recombinant PHL exhibited high affinity for fucosylated carbohydrates and the unusual disaccharide 3,6-O-Me2-Glcβ1–4(2,3-O-Me2)Rhaα-O-(p-C6H4)-OCH2CH2NH2 from Mycobacterium leprae. Based on its crystal structure, PHL forms a seven-bladed β-propeller assembling into a homo-dimer with an inter-subunit disulfide bridge. Investigating complexes with different ligands revealed the existence of two sets of binding sites per monomer—the first type prefers l-fucose and its derivatives, whereas the second type can bind d-galactose. Based on the sequence analysis, PHL could contain up to twelve binding sites per monomer. PHL was shown to interact with all types of red blood cells and insect haemocytes. Interestingly, PHL inhibited the production of reactive oxygen species induced by zymosan A in human blood and antimicrobial activity both in human blood, serum and insect haemolymph. Concurrently, PHL increased the constitutive level of oxidants in the blood and induced melanisation in haemolymph. Our results suggest that PHL might play a crucial role in the interaction of P. asymbiotica with both human and insect hosts., Author summary Photorhabdus asymbiotica, originally isolated in 1989 from a patient in the USA, was revealed to be not only effective insect pathogen but also bacteria causing serious and difficultly treatable diseases in humans. The genome of P. asymbiotica is being intensively studied but little is known about the details of the host-pathogen interaction at the molecular level. In this paper, we focused on the lectin identified in P. asymbiotica genome that may be crucial for the early stage of infection. We designated this lectin as PHL and prepared it in fully functional recombinant form. We identified its specificity and investigated the structural details of ligand binding. We found the PHL structure unique on the basis of number and arrangement of binding sites present in each monomer. Furthermore, we investigated influence of PHL on immune system of human and insect to propose the biological role for this lectin. The antimicrobial activity as well as phenoloxidase activity and reactive oxygen species production which contribute to antimicrobial effect was modulated after exposition to PHL in both blood and haemolymph supporting the idea that PHL might play important role in the host-pathogen interaction.

Rottlerin is a natural polyphenolic compound that was initially indicated as a PKCδ inhibitor. However, it was recently revealed that it may target a number of molecules and have biological effects on various cell types and is considered as a possible agent for tumor and cell proliferative diseases. Psoriasis is a chronic inflammatory cutaneous disorder with undefined etiology and is characterized by abnormal cellular proliferation, angiogenesis, and inflammation. Therefore, this paper investigates the regulatory effects of rottlerin on normal human epidermal keratinocytes (NHEKs) and imiquimod (IMQ)-induced psoriasiform (IPI) lesions. In vitro results showed that rottlerin inhibited cell proliferation in NHEKs through growth arrest and NFκB inhibition. It may also induce apoptosis in an autophagy-dependent pathway. We found that rottlerin inhibited human microvascular endothelial cells tube formation on matrigel. Rottlerin also decreased the cell senescence of keratinocytes and intracellular ROS generation, which indicated its antioxidant effect. We also showed that rottlerin affects the expression of keratinocyte proliferation biomarkers. In 12-O-tetradecanoylphorbol13-acetate (TPA)-induced keratinocytes, rottlerin significantly inhibited the expression of the induced pro-inflammatory cytokines in keratinocytes. An animal experiment provided the corresponding evidence based on this evidence in vitro, by using IPI model, we found that rottlerin could relieve the psoriasiform of BALB/c mice by inhibiting keratinocyte proliferation, inflammatory cell infiltration, and vascular proliferation. In conclusion, our results suggest that rottlerin may prove useful in the development of therapeutic agents against psoriasis. However, the deep mechanism still requires further study.

It is well accepted that influenza A virus predisposes individuals to often more severe superinfections with Streptococcus pneumonia. However, the mechanisms that lead to this synergy are not clearly understood. Recent data suggests that competent Th17 immunity is crucial to clearance and protection from invasive pneumococcal disease of the lung. We demonstrate that early influenza infection significantly reduced levels of pneumococcus driven IL-12p70, IL-23 and IL-27 in human monocytes with significant impairment of IL-17A and IFN-γ in HKSP-treated allogeneic mixed lymphocyte cultures. We also provide evidence to suggest that the hemagglutinin component of the virus is at least partially responsible for this downward pressure on IL-17 responses but surprisingly this suppression occurs despite robust IL-23 levels in hemagglutinin-treated monocyte cultures. This study demonstrates that influenza can directly affect the immunological pathways that promote appropriate responses to Streptococcus pneumonia in human immune cells. Importance Influenza virus is highly contagious and poses substantial public health problems due to its strong association with morbidity and mortality. Approximately 250,000–500,000 deaths are caused by seasonal influenza virus annually, and this figure increases during periods of pandemic infections. Most of these deaths are due to secondary bacterial pneumonia. Influenza-bacterial superinfection can result in hospitalisation and/or death of both patients with pre-existing lung disease or previously healthy individuals. The importance of our research is in determining that influenza and its component haemagglutinin has a direct effect on the classic pneumococcus induced pathways to IL-17A in our human ex vivo model. Our understanding of the mechanism which leaves people exposed to influenza infection during superinfection remain unresolved. This paper demonstrates that early infection of monocytes inhibits an arm of immunity crucial to bacterial clearance. Understanding this mechanism may provide alternative interventions in the case of superinfection with antimicrobial resistant strains of bacteria.

The pathogenesis of Bronchiolitis Obliterans Syndrome (BOS), the main clinical phenotype of chronic lung allograft dysfunction, is poorly understood. Recent studies suggest that epigenetic regulation of microRNAs might play a role in its development. In this paper we present the application of a complex computational pipeline to perform enrichment analysis of miRNAs in pathways applied to the study of BOS. The analysis considered the full set of miRNAs annotated in miRBase (version 21), and applied a sequence of filtering approaches and statistical analyses to reduce this set and to score the candidate miRNAs according to their potential involvement in BOS development. Dysregulation of two of the selected candidate miRNAs–miR-34a and miR-21 –was clearly shown in in-situ hybridization (ISH) on five explanted human BOS lungs and on a rat model of acute and chronic lung rejection, thus definitely identifying miR-34a and miR-21 as pathogenic factors in BOS and confirming the effectiveness of the computational pipeline.

Helminth parasites remain a major constraint upon human health and well-being in many parts of the world. Treatment of these infections relies upon a very small number of therapeutics, most of which were originally developed for use in animal health. A lack of high throughput screening systems, together with limitations of available animal models, has restricted the development of novel chemotherapeutics. This is particularly so for filarial nematodes, which are long-lived parasites with a complex cycle of development. In this paper, we describe attempts to visualise the immune response elicited by filarial parasites in infected mice using a non-invasive bioluminescence imaging reagent, luminol, our aim being to determine whether such a model could be developed to discriminate between live and dead worms for in vivo compound screening. We show that while imaging can detect the immune response elicited by early stages of infection with L3, it was unable to detect the presence of adult worms or, indeed, later stages of infection with L3, despite the presence of worms within the lymphatic system of infected animals. In the future, more specific reagents that detect secreted products of adult worms may be required for developing screens based upon live imaging of infected animals.

For rapid screening and quantification of an antisera antibody, a nanometer bithiophene-based conductive biolinker can enhanced signal performance and can be used to verify the interaction of an anti-IFN-γ antibody with an IFN-γ protein. The experimental measurements take a generic approach which takes advantage of the functionality of thiophene-based linkers for biosensors. Effects associated with using bithiophene as a biolinker for surface plasmon resonance (SPR) spectroscopy are examined in this paper. By using an atomic force microscope (AFM), it was observed that the morphology of the bithiophene modified gold sensor surface became smoother than the original gold surface. We compared the response and concentration of the anti-IFN-γ antibody on a bithiophene-coated and dextran-coated biochip as well as on different thickness-modified surfaces under SPR relevant conditions. The results indicate that a response to IFN-γ molecules immobilized on a sensor using a bithiophene biolinker improved more than 8-fold when compared to that of a sensor using a dextran biolinker. Furthermore, the regeneration ability of the sensor surface shows good repeatability as only less than a 1% decrease was found after repeating the experimental work over 6 cycles. The characteristics provided us with a good platform for rapid screening, real-time monitoring and quantitative concentration of the autoimmune antibody activities.

Due to its strong proliferation-reducing effects on keratinocytes, and also anti-inflammatory properties, the isoflavone genistein has already been proposed as a possible antipsoriatic compound. As there is still no detailed information on this topic, we examined the effects of genistein by using an in vitro model of both, normal and “psoriasis-like” keratinocytes at this stage of our work exhaustively testing the selected flavonoid in a mono-treated experimental design. Gene expression studies revealed transcriptional changes that confirms known disease-associated pathways and highlights many psoriasis-related genes. Our results suggested that aberrant expression of genes contributing to the progress of psoriasis could be improved by the action of genistein. Genistein prevented “cytokine mix” as well as TNF-α-induced NF-κB nuclear translocation, with no effect on the PI3K signaling cascade, indicating the luck of turning this pathway into NF-κB activation. It could have attenuated TNF-α and LPS-induced inflammatory responses by suppressing ROS activation. Regardless of the type of keratinocyte stimulation used, reduction of cytokine IL-8, IL-20 and CCL2 production (both at RNA and protein level) following genistein treatment was visible. Because investigations of other groups supported our commentary on potential administration of genistein as a potential weapon in the armamentarium against psoriasis, it is believed that this paper should serve to encourage researchers to conduct further studies on this subject.

In the present paper, we address by means of mathematical modeling the following main question: How can oncolytic virus infection of some normal cells in the vicinity of tumor cells enhance oncolytic virotherapy? We formulate a mathematical model describing the interactions between the oncolytic virus, the tumor cells, the normal cells, and the antitumoral and antiviral immune responses. The model consists of a system of delay differential equations with one (discrete) delay. We derive the model's basic reproductive number within tumor and normal cell populations and use their ratio as a metric for virus tumor-specificity. Numerical simulations are performed for different values of the basic reproduction numbers and their ratios to investigate potential trade-offs between tumor reduction and normal cells losses. A fundamental feature unravelled by the model simulations is its great sensitivity to parameters that account for most variation in the early or late stages of oncolytic virotherapy. From a clinical point of view, our findings indicate that designing an oncolytic virus that is not 100% tumor-specific can increase virus particles, which in turn, can further infect tumor cells. Moreover, our findings indicate that when infected tissues can be regenerated, oncolytic viral infection of normal cells could improve cancer treatment.

The spread of KPC-type carbapenemases is mainly attributed to the global dissemination of Klebsiella pneumoniae (KP) strains belonging to the clonal group (CG) 258, including sequence type (ST) 258 and other related STs. Two distinct clades of CG258-KP have evolved, which differ mainly for the composition of their capsular polysaccharides, and recent studies indicate that clade 1 evolved from an ancestor of clade 2 by recombination of a genomic fragment carrying the capsular polysaccharide (cps) locus. In this paper, we investigated the ability of two ST258-KP strains, KKBO-1 and KK207-1, selected as representatives of ST258-KP clade 2 and clade 1, respectively, to activate an adaptive immune response using ex vivo-stimulation of PBMC from normal donors as an experimental model. Our data showed that KKBO-1 (clade 2) induces a Th17 response more efficiently than KK207-1 (clade 1): the percentage of CD4+IL17+ cells and the production of IL-17A were significantly higher in cultures with KKBO-1 compared to cultures with KK207-1. While no differences in the rate of bacterial internalization or in the bacteria-induced expression of CD86 and HLA-DR by monocytes and myeloid dendritic cells were revealed, we found that the two strains significantly differ in inducing the production of cytokines involved in the adaptive immune response, as IL-1β, IL-23 and TNF-α, by antigen-presenting cells, with KKBO-1 being a more efficient inducer than KK207-1. The immune responses elicited by KK207-1 were comparable to those elicited by CIP 52.145, a highly virulent K. pneumoniae reference strain known to escape immune-inflammatory responses. Altogether, present results suggest that CG258-KP of the two clades are capable of inducing a different response of adaptive immunity in the human host.

Background and aim: The Montreal classification of disease behaviour in Crohn's disease describes progression of disease towards a stricturing and penetrating phenotype. In the present paper, we propose an alternative representation of the long-term course of Crohn's disease complications, the rolling phenotype. As is commonly observed in clinical practice, this definition allows progression to a more severe phenotype (stricturing, penetrating) but also, regression to a less severe behaviour (inflammatory, or remission) over time. Methods: All patients diagnosed with Crohn's Disease between 01/01/1994 and 01/03/2008, managed at a single centre and observed for a minimum of 5 years, had development and resolution of all complications recorded. A rolling phenotype was defined at each time point based on all observed complications in the three years prior to the time point. Phenotype was defined as B1, B2, B3, or B23 (penetrating and stenotic). The progression over time of the rolling phenotype was compared to that of the cumulative Montreal phenotype. Results: 305 patients were observed a median of 10.0 (Intraquartile range 7.3-13.7) years. Longitudinal progression of rolling phenotype demonstrated a consistent proportion of patients with B1 (70%), B2 (20%), B3 (5%) and B23 (5%) phenotypes. These proportions were observed regardless of initial phenotype. In contrast, the cumulative Montreal phenotype progressed towards a more severe phenotype with time (B1 (39%), B2 (26%), B3(35%) at 10 years). Conclusion: A rolling phenotype provides an alternative view of the longitudinal burden of intra-abdominal complications in Crohn's disease. From this viewpoint, 70% of patients have durable freedom from complication over time (>3 years).

Laboratory-based HIV tests for recent infection (TRIs), which primarily measure a specific serological biomarker(s) that distinguishes recent from long-term HIV infection, have facilitated the estimation of population-based incidence. Dried blood spots (DBS) on filter paper are an attractive sample source for HIV surveillance, given the simplified and cost-effective methods of specimen collection, storage, and shipment. Here, we evaluated the use of DBS in conjunction with an in-house multiplex TRI, the HIV-1-specific Bio-Plex assay, which measures direct antibody binding and avidity to multiple HIV-1 analytes. The assay performance was comparable between matched plasma and DBS samples from HIV-1 infected individuals obtained from diverse sources. The coefficients of variation, comparing the median antibody reactivity for each analyte between plasma and DBS, ranged from 2.78% to 9.40% and the correlation coefficients between the two sample types ranged from 0.89 to 0.97, depending on the analyte. The correlation in antibody reactivity between laboratory and site-prepared DBS for each analyte ranged from 0.87 to 0.98 and from 0.90 to 0.97 between site-prepared DBS and plasma. The correlation in assay measures between plasma and DBS indicate that the sample types can be used interchangeably with the Bio-Plex format, without negatively impacting the misclassification rate of the assay.

Phosphorylation is a fundamental biochemical reaction that modulates protein activity in cells. While a single phosphorylation event is relatively easy to understand, multisite phosphorylation requires systems approaches for deeper elucidation of the underlying molecular mechanisms. In this paper we develop a mechanistic model for single- and multi-site phosphorylation. The proposed model is compared with previously reported studies. We compare the predictions of our model with experiments published in the literature in the context of inflammatory signaling events in order to provide a mechanistic description of the multisite phosphorylation-mediated regulation of Signal Transducer and Activator of Transcription 3 (STAT3) and Interferon Regulatory Factor 5 (IRF-5) proteins. The presented model makes crucial predictions for transcription factor phosphorylation events in the immune system. The model proposes potential mechanisms for T cell phenotype switching and production of cytokines. This study also provides a generic framework for the better understanding of a large number of multisite phosphorylation-regulated biochemical circuits.

Journal Article; Research Support, Non-U.S. Gov't; CD66b is a member of the carcinoembryonic antigen family, which mediates the adhesion between neutrophils and to endothelial cells. Allergen-specific immunotherapy is widely used to treat allergic diseases, and the molecular mechanisms underlying this therapy are poorly understood. The present work was undertaken to analyze A) the in vitro effect of allergens and immunotherapy on cell-surface CD66b expression of neutrophils from patients with allergic asthma and rhinitis and B) the in vivo effect of immunotherapy on cell-surface CD66b expression of neutrophils from nasal lavage fluid during the spring season. Myeloperoxidase expression and activity was also analyzed in nasal lavage fluid as a general marker of neutrophil activation. RESULTS CD66b cell-surface expression is upregulated in vitro in response to allergens, and significantly reduced by immunotherapy (p

Immunotherapy (eg interferon α) in combination with tyrosine kinase inhibitors is currently in clinical trials for treatment of chronic myeloid leukemia (CML). Cancer patients commonly have problems with so called immune escape mechanisms that may hamper immunotherapy. Hence, to study the function of the immune system in CML is of interest. In the present paper we have identified immune escape mechanisms in CML with focus on those that directly hamper T cells since these cells are important to control tumor progression. CML patient samples were investigated for the presence of myeloid-derived suppressor cells (MDSCs), expression of programmed death receptor ligand 1/programmed death receptor 1 (PD-L1/PD-1), arginase 1 and soluble CD25. MDSC levels were increased in samples from Sokal high risk patients (p

Alpha-synuclein protein is strongly implicated in the pathogenesis Parkinson's disease. Increased expression of α-synuclein due to genetic multiplication or point mutations leads to early onset disease. While α-synuclein is known to modulate membrane vesicle dynamics, it is not clear if this activity is involved in the pathogenic process or if measurable physiological effects of α-synuclein over-expression or mutation exist in vivo. Macrophages and microglia isolated from BAC α-synuclein transgenic mice, which overexpress α-synuclein under regulation of its own promoter, express α-synuclein and exhibit impaired cytokine release and phagocytosis. These processes were affected in vivo as well, both in peritoneal macrophages and microglia in the CNS. Extending these findings to humans, we found similar results with monocytes and fibroblasts isolated from idiopathic or familial Parkinson's disease patients compared to age-matched controls. In summary, this paper provides 1) a new animal model to measure α-synuclein dysfunction; 2) a cellular system to measure synchronized mobilization of α-synuclein and its functional interactions; 3) observations regarding a potential role for innate immune cell function in the development and progression of Parkinson's disease and other human synucleinopathies; 4) putative peripheral biomarkers to study and track these processes in human subjects. While altered neuronal function is a primary issue in PD, the widespread consequence of abnormal α-synuclein expression in other cell types, including immune cells, could play an important role in the neurodegenerative progression of PD and other synucleinopathies. Moreover, increased α-synuclein and altered phagocytosis may provide a useful biomarker for human PD.

For the purposes of information retrieval, users must find highly relevant documents from within a system (and often a quite large one comprised of many individual documents) based on input query. Ranking the documents according to their relevance within the system to meet user needs is a challenging endeavor, and a hot research topic–there already exist several rank-learning methods based on machine learning techniques which can generate ranking functions automatically. This paper proposes a parallel B cell algorithm, RankBCA, for rank learning which utilizes a clonal selection mechanism based on biological immunity. The novel algorithm is compared with traditional rank-learning algorithms through experimentation and shown to outperform the others in respect to accuracy, learning time, and convergence rate; taken together, the experimental results show that the proposed algorithm indeed effectively and rapidly identifies optimal ranking functions.

Immunological checkpoints, such as the inhibitory CD200 receptor (CD200R), play a dual role in balancing the immune system during microbial infection. On the one hand these inhibitory signals prevent excessive immune mediated pathology but on the other hand they may impair clearance of the pathogen. We studied the influence of the inhibitory CD200-CD200R axis on clearance and pathology in two different virus infection models. We find that lack of CD200R signaling strongly enhances type I interferon (IFN) production and viral clearance and improves the outcome of mouse hepatitis corona virus (MHV) infection, particularly in female mice. MHV clearance is known to be dependent on Toll like receptor 7 (TLR7)-mediated type I IFN production and sex differences in TLR7 responses previously have been reported for humans. We therefore hypothesize that CD200R ligation suppresses TLR7 responses and that release of this inhibition enlarges sex differences in TLR7 signaling. This hypothesis is supported by our findings that in vivo administration of synthetic TLR7 ligand leads to enhanced type I IFN production, particularly in female Cd200−/− mice and that CD200R ligation inhibits TLR7 signaling in vitro. In influenza A virus infection we show that viral clearance is determined by sex but not by CD200R signaling. However, absence of CD200R in influenza A virus infection results in enhanced lung neutrophil influx and pathology in females. Thus, CD200-CD200R and sex are host factors that together determine the outcome of viral infection. Our data predict a sex bias in both beneficial and pathological immune responses to virus infection upon therapeutic targeting of CD200-CD200R., Author Summary Immune responses need to be carefully orchestrated to prevent disease due to an overactive immune system. Immunological checkpoints are provided by immune inhibitory receptors, which set a threshold for activation and dampen the immune system. In the case of a viral infection, this prevents pathology induced by the immune system, but on the other hand may prevent adequate removal of the virus. In this paper, we show that removal of such an immunological checkpoint in mice leads to rapid removal of corona virus, but also to more immune-induced disease symptoms in case of influenza virus infection. We observe this predominantly in female mice. We demonstrate that this particular checkpoint inhibits anti-viral responses that are naturally stronger in females. Release of this checkpoint enlarges these sex differences. Our findings have major implications for therapeutic use of blockers of this pathway, which are currently in clinical trials for the treatment of cancer, as we predict that female patients will have a stronger response to such therapeutics.

There is growing concern over the welfare of animals used in research, in particular when these animals develop pathology. The present study aims to identify the main sources of animal distress and to assess the possible implementation of refinement measures in experimental infection research, using mouse models of tuberculosis (TB) as a case study. This choice is based on the historical relevance of mouse studies in understanding the disease and the present and long-standing impact of TB on a global scale. Literature published between 1997 and 2009 was analysed, focusing on the welfare impact on the animals used and the implementation of refinement measures to reduce this impact. In this 12-year period, we observed a rise in reports of ethical approval of experiments. The proportion of studies classified into the most severe category did however not change significantly over the studied period. Information on important research parameters, such as method for euthanasia or sex of the animals, were absent in a substantial number of papers. Overall, this study shows that progress has been made in the application of humane endpoints in TB research, but that a considerable potential for improvement remains., Nuno H. Franco is funded by Fundação para a Ciência e Tecnologia (SFRH/BD/38337/2007). This work is funded by FEDER funds through the Operational Competitiveness Programme - COMPETE and by national funds through FCT - Fundação para a Ciência e Tecnologia under the project FCOMP-01-0124-FEDER-022718 (PEst-C/SAU/LA0002/2011.

Influenza virus infection remains a public health problem worldwide. The mechanisms underlying viral control during an uncomplicated influenza virus infection are not fully understood. Here, we developed a mathematical model including both innate and adaptive immune responses to study the within-host dynamics of equine influenza virus infection in horses. By comparing modeling predictions with both interferon and viral kinetic data, we examined the relative roles of target cell availability, and innate and adaptive immune responses in controlling the virus. Our results show that the rapid and substantial viral decline (about 2 to 4 logs within 1 day) after the peak can be explained by the killing of infected cells mediated by interferon activated cells, such as natural killer cells, during the innate immune response. After the viral load declines to a lower level, the loss of interferon-induced antiviral effect and an increased availability of target cells due to loss of the antiviral state can explain the observed short phase of viral plateau in which the viral level remains unchanged or even experiences a minor second peak in some animals. An adaptive immune response is needed in our model to explain the eventual viral clearance. This study provides a quantitative understanding of the biological factors that can explain the viral and interferon kinetics during a typical influenza virus infection., Author Summary Influenza, commonly referred to as the flu, is a contagious respiratory illness caused by influenza virus infections. Although most infected subjects with intact immune systems are able to clear the virus without developing serious flu complications, the mechanisms underlying viral control are not fully understood. In this paper, we address this question by developing mathematical models that include both innate and adaptive immune responses, and fitting them to experimental data from horses infected with equine influenza virus. We find that the innate immune response, such as natural killer cell-mediated infected cell killing and interferon's antiviral effect, can explain the first rapid viral decline and subsequent second peak viremia, and that the adaptive immune response is needed to eventually clear the virus. This study improves our understanding of influenza virus dynamics and may provide more information for future research in influenza pathogenesis, treatment, and vaccination.

Numerous aspects of the relationship between bacteria and human have been investigated. One aspect that has recently received attention is sequence overlap at the proteomic level. However, there has not yet been a study that comprehensively characterizes the level of sequence overlap between bacteria and human, especially as it relates to bacterial characteristics like pathogenicity, G-C content, and proteome size. In this study, we began by performing a general characterization of the range of bacteria-human similarity at the proteomic level, and identified characteristics of the most- and least-similar bacterial species. We then examined the relationship between proteomic similarity and numerous other variables. While pathogens and nonpathogens had comparable similarity to the human proteome, pathogens causing chronic infections were found to be more similar to the human proteome than those causing acute infections. Although no general correspondence between a bacterium’s proteome size and its similarity to the human proteome was noted, no bacteria with small proteomes had high similarity to the human proteome. Finally, we discovered an interesting relationship between similarity and a bacterium’s G-C content. While the relationship between bacteria and human has been studied from many angles, their proteomic similarity still needs to be examined in more detail. This paper sheds further light on this relationship, particularly with respect to immunity and pathogenicity.

A genetic absence of the common IFN- α/β signaling receptor (IFNAR) in mice is associated with enhanced viral replication and altered adaptive immune responses. However, analysis of IFNAR-/- mice is limited for studying the functions of type I IFN at discrete stages of viral infection. To define the temporal functions of type I IFN signaling in the context of infection by West Nile virus (WNV), we treated mice with MAR1-5A3, a neutralizing, non cell-depleting anti-IFNAR antibody. Inhibition of type I IFN signaling at or before day 2 after infection was associated with markedly enhanced viral burden, whereas treatment at day 4 had substantially less effect on WNV dissemination. While antibody treatment prior to infection resulted in massive expansion of virus-specific CD8+ T cells, blockade of type I IFN signaling starting at day 4 induced dysfunctional CD8+ T cells with depressed cytokine responses and expression of phenotypic markers suggesting exhaustion. Thus, only the later maturation phase of anti-WNV CD8+ T cell development requires type I IFN signaling. WNV infection experiments in BATF3 -/- mice, which lack CD8-α dendritic cells and have impaired priming due to inefficient antigen cross-presentation, revealed a similar effect of blocking IFN signaling on CD8+ T cell maturation. Collectively, our results suggest that cell non-autonomous type I IFN signaling shapes maturation of antiviral CD8+ T cell response at a stage distinct from the initial priming event., Author Summary Although it is well established that type I IFN responses protect against viral infections by inducing expression of antiviral genes and modulators of adaptive immune responses, its function at different stages of viral infections has remained poorly studied. In this paper, we administered a monoclonal antibody that blocks the common type I IFN signaling receptor to mice at different times after WNV infection to dissect the temporal functions of IFN. Administration of the blocking antibody at day -1 resulted in a massive increase in viral replication and the number of WNV-specific -CD8+ T cells. In contrast, treatment with a single dose of antibody at day 4 had limited effects on viral dissemination, but instead promoted development of dysfunctional CD8+ T cells that produced lower levels of cytokines and expressed proteins implicated in T cell exhaustion. Thus, we show a stage-specific effect of type I IFN in optimal maturation of antiviral CD8+ T cell responses. Our study provides new insight as to how and when innate immune signals affect maturation of antiviral CD8+ T cells after the initial priming event with viral antigen.

Leishmaniasis remains one of the world's most devastating neglected tropical diseases. It mainly affects developing countries, where it often co-exists with chronic malnutrition, one of the main risk factors for developing the disease. Few studies have been published, however, on the relationship between leishmaniasis progression and malnutrition. The present paper reports the influence of protein malnutrition on the immune response and visceral disease development in adult hamsters infected with Leishmania infantum fed either standard or low protein diets. The low protein diet induced severe malnutrition in these animals, and upon infection with L. infantum 33% had severe visceral leishmaniasis compared to only 8% of animals fed the standard diet. The infected, malnourished animals showed notable leukocyte depletion, mild specific antibody responses, impairment of lymphoproliferation, presence of parasites in blood (16.67% of the hamsters) and significant increase of the splenic parasite burden. Animals fed standard diet suffered agranulocytosis and monocytopenia, but showed stronger specific immune responses and had lower parasite loads than their malnourished counterparts. The present results show that protein malnutrition promotes visceral leishmaniasis and provide clues regarding the mechanisms underlying the impairment of the immune system. This work was funded by project PI10/00829 from the Fondo de Investigación Sanitaria, http://www.isciii.es/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Sí

Lack of immunogenicity of cancer cells has been considered a major reason for their failure in induction of a tumor specific T cell response. In this paper, we present evidence that decitabine (DAC), a DNA methylation inhibitor that is currently used for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and other malignant neoplasms, is capable of eliciting an anti-tumor cytotoxic T lymphocyte (CTL) response in mouse EL4 tumor model. C57BL/6 mice with established EL4 tumors were treated with DAC (1.0 mg/kg body weight) once daily for 5 days. We found that DAC treatment resulted in infiltration of IFN-γ producing T lymphocytes into tumors and caused tumor rejection. Depletion of CD8(+), but not CD4(+) T cells resumed tumor growth. DAC-induced CTL response appeared to be elicited by the induction of CD80 expression on tumor cells. Epigenetic evidence suggests that DAC induces CD80 expression in EL4 cells via demethylation of CpG dinucleotide sites in the promoter of CD80 gene. In addition, we also showed that a transient, low-dose DAC treatment can induce CD80 gene expression in a variety of human cancer cells. This study provides the first evidence that epigenetic modulation can induce the expression of a major T cell co-stimulatory molecule on cancer cells, which can overcome immune tolerance, and induce an efficient anti-tumor CTL response. The results have important implications in designing DAC-based cancer immunotherapy.

Background Previous work has shown that disruption of the gene for group X secreted phospholipase A2 (sPLA2-X) markedly diminishes airway hyperresponsiveness and remodeling in a mouse asthma model. With the large number of additional sPLA2s in the mammalian genome, the involvement of other sPLA2s in the asthma model is possible – in particular, the group V sPLA2 (sPLA2-V) that like sPLA2-X is highly active at hydrolyzing membranes of mammalian cells. Methodology and Principal Findings The allergen-driven asthma phenotype was significantly reduced in sPLA2-V-deficient mice but to a lesser extent than observed previously in sPLA2-X-deficient mice. The most striking difference observed between the sPLA2-V and sPLA2-X knockouts was the significant impairment of the primary immune response to the allergen ovalbumin (OVA) in the sPLA2-V−/− mice. The impairment in eicosanoid generation and dendritic cell activation in sPLA2-V−/− mice diminishes Th2 cytokine responses in the airways. Conclusions This paper illustrates the diverse roles of sPLA2s in the immunopathogenesis of the asthma phenotype and directs attention to developing specific inhibitors of sPLA2-V as a potential new therapy to treat asthma and other allergic disorders.

The development and clinical testing of drug combinations for the treatment of Non-Hodgkin Lymphoma (NHL) and other cancers has recently shown great promise. However, determining the optimum combination and its associated dosages for maximum efficacy and minimum side effects is still a challenge. This paper describes a parametric analysis of the dynamics of malignant B-cells and the effects of an anti-sense oligonucleotide targeted to BCL-2 (as-bcl-2), anti-CD-20 (rituximab) and their combination, for a SCID mouse human lymphoma xenograft model of NHL. Our parametric model is straightforward. Several mechanisms of malignant B-cell birth and death in the nodal micro-environment are simulated. Cell death is accelerated by hypoxia and starvation induced by tumor scale, by modification of anti-apoptosis with as-bcl-2, and by direct kill effects of rituximab (cell kill by cytotoxic immune cells is not included, due to the absence of an immune system in the corresponding experiments). We show that the cell population dynamics in the control animals are primarily determined by K*, the ratio of rate constants for malignant cell death, K(d), and cell birth, K(b). Tumor growth with independent treatments is reproduced by the model, and is used to predict their effect when administered in combination. Malignant cell lifetimes are derived to provide a quantitative comparison of the efficacy of these treatments. Future experimental and clinical applications of the model are discussed.

Surgery induced inflammation is a potent promoter of tumour recurrence and metastasis in colorectal cancer. The recently discovered family of Nox enzymes represent a major source of endogenous reactive oxygen species (ROS) and are now heavily implicated in tumour cell metastasis. Interestingly, Nox enzymes can be 'purposefully' activated by inflammatory cytokines and growth factors which are present in abundance in the peri-operative window. As colon cancer cells express Nox enzymes and Toll-like receptor 4 (TLR-4), we hypothesised that LPS may potentiate the ability of colon cancer cells to metastasise via Nox enzyme mediated redox signalling. In support of this hypothesis, this paper demonstrates that LPS induces a significant, transient increase of endogenous ROS in SW480, SW620 and CT-26 colon cancer cells. This increase in LPS-induced ROS activity is completely abrogated by a Nox inhibitor, diphenyleneiodonium (DPI), Nox1 siRNA and an NF-κB inhibitor, Dihydrochloride. A significant increase in Nox1 and Nox2 protein expression occurs following LPS treatment. Inhibition of NF-κB also attenuates the increase of Nox1 and Nox2 protein expression. The sub-cellular location of LPS-induced ROS generation lies mainly in the endoplasmic reticulum. LPS activates the PI3K/Akt pathway via Nox generated ROS and this signal is inhibited by DPI. This LPS activated Nox mechanism facilitates a significant increase in SW480 colon cancer cell adhesion to collagen I, which is inhibited by DPI, Nox1 siRNA and a PI3K inhibitor. Altogether, these data suggest that the LPS-Nox1 redox signalling axis plays a crucial role in facilitation of colon cancer cell adhesion, thus increasing the metastatic potential of colon cancer cells. Nox1 may represent a valuable target in which to prevent colon cancer metastasis.

The ability to examine the behavior of biological systems in silico has the potential to greatly accelerate the pace of discovery in diseases, such as stroke, where in vivo analysis is time intensive and costly. In this paper we describe an approach for in silico examination of responses of the blood transcriptome to neuroprotective agents and subsequent stroke through the development of dynamic models of the regulatory processes observed in the experimental gene expression data. First, we identified functional gene clusters from these data. Next, we derived ordinary differential equations (ODEs) from the data relating these functional clusters to each other in terms of their regulatory influence on one another. Dynamic models were developed by coupling these ODEs into a model that simulates the expression of regulated functional clusters. By changing the magnitude of gene expression in the initial input state it was possible to assess the behavior of the networks through time under varying conditions since the dynamic model only requires an initial starting state, and does not require measurement of regulatory influences at each time point in order to make accurate predictions. We discuss the implications of our models on neuroprotection in stroke, explore the limitations of the approach, and report that an optimized dynamic model can provide accurate predictions of overall system behavior under several different neuroprotective paradigms., Author Summary Computational modeling aims to use mathematical and algorithmic principles to link components of biological systems to predict system behavior. In the past such models have described a small set of carefully studied molecular interactions (proteins in signal transduction pathways) or larger abstract components (cell types or functional processes in the immune system). In this study we use data from global transcriptional analysis of the processes of neuroprotection in a mouse model of stroke to generate functional modules, groups of genes that coherently act to accomplish functions. We then derive equations relating the expression of these modules to one another, treating these individual equations as a closed system, and demonstrate that the model can be used to simulate the gene expression of the system over time. Our work is novel in describing the use of global transcriptomic data to develop dynamic models of expression in an animal model. We believe that the models developed will aid in understanding the complex dynamics of neuroprotection and provide ways to predict outcomes in terms of neuroprotection or injury. This approach will be broadly applicable to other problems and provides an approach to building dynamic models from the bottom up.

Agent-based simulation is a powerful method for investigating the complex interplay of the processes occurring in a lymph node during an adaptive immune response. We have previously established an agent-based modeling framework for the interactions between T cells and dendritic cells within the paracortex of lymph nodes. This model simulates in three dimensions the "random-walk" T cell motility observed in vivo, so that cells interact in space and time as they process signals and commit to action such as proliferation. On-lattice treatment of cell motility allows large numbers of densely packed cells to be simulated, so that the low frequency of T cells capable of responding to a single antigen can be dealt with realistically. In this paper we build on this model by incorporating new numerical methods to address the crucial processes of T cell ingress and egress, and chemotaxis, within the lymph node. These methods enable simulation of the dramatic expansion and contraction of the T cell population in the lymph node paracortex during an immune response. They also provide a novel probabilistic method to simulate chemotaxis that will be generally useful in simulating other biological processes in which chemotaxis is an important feature.

Cell signaling processes involve receptor trafficking through highly connected networks of interacting components. The binding of surface receptors to their specific ligands is a key factor for the control and triggering of signaling pathways. In most experimental systems, ligand concentration and cell density vary within a wide range of values. Dependence of the signal response on cell density is related with the extracellular volume available per cell. This dependence has previously been studied using non-spatial models which assume that signaling components are well mixed and uniformly distributed in a single compartment. In this paper, a mathematical model that shows the influence exerted by cell density on the spatio-temporal evolution of ligands, cell surface receptors, and intracellular signaling molecules is developed. To this end, partial differential equations were used to model ligand and receptor trafficking dynamics through the different domains of the whole system. This enabled us to analyze several interesting features involved with these systems, namely: a) how the perturbation caused by the signaling response propagates through the system; b) receptor internalization dynamics and how cell density affects the robustness of dose-response curves upon variation of the binding affinity; and c) that enhanced correlations between ligand input and system response are obtained under conditions that result in larger perturbations of the equilibrium ligand + surface receptor [Please see text] ligand - receptor complex. Finally, the results are compared with those obtained by considering that the above components are well mixed in a single compartment.