Dengue Virus Increases the Expression of TREM-1 and CD10 on Human Neutrophils.

Every year, dengue is responsible for 400 million infections worldwide. Inflammation is related to the development of severe forms of dengue. Neutrophils are a heterogeneous cell population with a key role in the immune response. During viral infection, neutrophils are mainly recruited to the infection site; however, their excessive activation is linked to deleterious results. During dengue infection, neutrophils are involved in the pathogenesis through neutrophils extracellular traps production, tumor necrosis factor-alpha, and interleukin-8 secretion. However, other molecules regulate the neutrophil role during viral infection. TREM-1 is expressed on neutrophils and its activation is related to increased production of inflammatory mediators. CD10 is expressed on mature neutrophils and has been associated with the regulation of neutrophil migration and immunosuppression. However, the role of both molecules during viral infection is limited, particularly during dengue infection. Here, we report for the first time that DENV-2 can significantly increase TREM-1 and CD10 expression as well as sTREM-1 production in cultured human neutrophils. Furthermore, we observed that treatment with granulocyte-macrophage colony stimulating factor, a molecule mostly produced in severe cases of dengue, is capable of inducing the overexpression of TREM-1 and CD10 on human neutrophils. These results suggest the participation of neutrophil CD10 and TREM-1 in the pathogenesis of dengue infection.

IL-6, IL-10, sFas, granulysin and indicators of intestinal permeability as early biomarkers for a fatal outcome in COVID-19.

The clinical presentation of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), ranges between mild respiratory symptoms and a severe disease that shares many of the features of sepsis. Sepsis is a deregulated response to infection that causes life-threatening organ failure. During sepsis, the intestinal epithelial cells are affected, causing an increase in intestinal permeability and allowing microbial translocation from the intestine to the circulation, which exacerbates the inflammatory response. Here we studied patients with moderate, severe and critical COVID-19 by measuring a panel of molecules representative of the innate and adaptive immune responses to SARS-CoV-2, which also reflect the presence of systemic inflammation and the state of the intestinal barrier. We found that non-surviving COVID-19 patients had higher levels of low-affinity anti-RBD IgA antibodies than surviving patients, which may be a response to increased microbial translocation. We identified sFas and granulysin, in addition to IL-6 and IL-10, as possible early biomarkers with high sensitivity (>73 %) and specificity (>51 %) to discriminate between surviving and non-surviving COVID-19 patients. Finally, we found that the microbial metabolite d-lactate and the tight junction regulator zonulin were increased in the serum of patients with severe COVID-19 and in COVID-19 patients with secondary infections, suggesting that increased intestinal permeability may be a source of secondary infections in these patients. COVID-19 patients with secondary infections had higher disease severity and mortality than patients without these infections, indicating that intestinal permeability markers could provide complementary information to the serum cytokines for the early identification of COVID-19 patients with a high risk of a fatal outcome.

Differential activation of innate and adaptive lymphocytes during latent or active infection with Mycobacterium tuberculosis.

Most individuals infected with Mycobacterium tuberculosis (Mtb) have latent tuberculosis (TB), which can be diagnosed with tests (such as the QuantiFERON-TB Gold test [QFT]) that detect the production of IFN-γ by memory T cells in response to the Mtb-specific antigens 6 kDa early secretory antigenic target EsxA (Rv3875) (ESAT-6), 10 kDa culture filtrate antigen EsxB (Rv3874) (CFP-10), and Mtb antigen of 7.7 kDa (Rv2654c) (TB7.7). However, the immunological mechanisms that determine if an individual will develop latent or active TB remain incompletely understood. Here we compared the response of innate and adaptive peripheral blood lymphocytes from healthy individuals without Mtb infection (QFT negative) and from individuals with latent (QFT positive) or active TB infection, to determine the characteristics of these cells that correlate with each condition. In active TB patients, the levels of IFN-γ that were produced in response to Mtb-specific antigens had high positive correlations with IL-1ß, TNF-α, MCP-1, IL-6, IL-12p70, and IL-23, while the proinflammatory cytokines had high positive correlations between themselves and with IL-12p70 and IL-23. These correlations were not observed in QFT-negative or QFT-positive healthy volunteers. Activation with Mtb-soluble extract (a mixture of Mtb antigens and pathogen-associated molecular patterns [PAMPs]) increased the percentage of IFN-γ-/IL-17-producing NK cells and of IL-17-producing innate lymphoid cell 3 (ILC3) in the peripheral blood of active TB patients, but not of QFT-negative or QFT-positive healthy volunteers. Thus, active TB patients have both adaptive and innate lymphocyte subsets that produce characteristic cytokine profiles in response to Mtb-specific antigens or PAMPs. These profiles are not observed in uninfected individuals or in individuals with latent TB, suggesting that they are a response to active TB infection.

Liposomes Bearing Non-Bilayer Phospholipid Arrangements Induce Specific IgG Anti-Lipid Antibodies by Activating NK1.1+, CD4+ T Cells in Mice.

Liposomes are artificial models of cellular membranes that are used as delivery systems for genes, drugs and protein antigens. We have previously used them to study the antigenic properties of their phospholipids. Here, we used them to induce the production of IgG anti-non-bilayer phospholipid arrangements (NPAs) antibodies in mice; these antibodies cause cell lysis and trigger a lupus-like disease in mice. We studied the mechanisms that lead to the production of these antibodies, and provide evidence that NK1.1+, CD4+ T cells respond to NPA-bearing liposomes and deliver the help required for specific B cell activation and antibody class-switching to IgG. We found increased numbers of IL-4-producing NK1.1+, CD4+ T cells in the secondary lymphoid organs of mice administered with NPAs, and these cells also expressed CD40L, which is required for B cell activation. Additionally, we isolated and purified NK1.1+, CD4+ T cells from spleens and determined that they over-expressed 40 genes, which are key players in inflammatory processes and B cell stimulation and have TRAF6 and UNC39B1 as key nodes in their network. These results show that liposomes are membrane models that can be used to analyze the immunogenicity of lipids.

Intra-host genetic population diversity: Role in emergence and persistence of drug resistance among Mycobacterium tuberculosis complex minor variants.

Drug resistant tuberculosis (DR-TB) is an important public health issue in different parts of the world. Mycobacterium tuberculosis complex variants (MTBC vars) preferentially infect certain hosts, limiting their distribution to different ecosystems. However, MTBC vars can infect other hosts beyond their preferred target potentially contributing to persistence of drug resistance (DR) in other niches. Here, we performed a comprehensive intra-host genetic analysis for the identification of DR-related mutations among all MTBC minor vars whole genome sequences (8,095 strains) publicly available worldwide. High confidence drug-resistance mutations in katG (isoniazid), rpsL (streptomycin), pncA (pyrazinamide), rpoB (rifampicin) and gyrA (fluoroquinolones) genes were identified among intrahost minor sub-populations in 197 different strains (2.43%) belonging to vars africanum, bovis, caprae, microti, orygis and pinnipedii. In addition, a three-dimensional structure modeling analysis to assess the role of novel mutations was also performed. Our findings highlight the importance of detecting discrete intra-host populations carrying DR mutations.

Interleukin 4 deficiency limits the development of a lupus-like disease in mice triggered by phospholipids in a non-bilayer arrangement.

Non-bilayer phospholipids arrangements (NPAs) are transient molecular associations different from lipid bilayers. When they become stable, they can trigger a disease in mice resembling human lupus, which is mainly characterized by the production of anti-NPA IgG antibodies. NPAs are stabilized on liposomes or cell bilayers by the drugs procainamide or chlorpromazine, which produce drug-induced lupus in humans. Here, we evaluated the participation of the TH 2 response, through its hallmark cytokine IL-4, on the development of the lupus-like disease in mice. Wild-type or IL-4 knockout BALB/c mice received liposomes bearing drug-induced NPAs, the drugs alone, or an anti-NPA monoclonal antibody (H308) to induce the lupus-like disease (the last two procedures stabilize NPAs on mice cells). IL-4 KO mice showed minor disease manifestations, compared to wild-type mice, with decreased production of anti-NPA IgG antibodies, no anti-cardiolipin, anti-histones and anticoagulant antibodies, and no kidney or skin lesions. In these mice, H308 was the only inducer of anti-NPA IgG antibodies. These findings indicate that IL-4 has a central role in the development of the murine lupus-like disease induced by NPA stabilization.

The First exploratory spatial distribution analysis of tuberculosis and associated factors in Tonala, Mexico.

INTRODUCTION: The US-Mexico region is at high risk of elevated tuberculosis (TB) incidence due to mobility and migration. Knowledge of how socio-demographic factors varies geographically, provides clues to understanding the determinants of tuberculosis and may provide guidance for regional prevention and control strategies to improve public health in Mexico. The aim of the present study was to describe the epidemiologic characteristics and spatial patterns of the incidence of tuberculosis in Tonala, Jalisco (Mexico) from 2013-2015. METHODOLOGY: The Surveillance System Database from the Health Department, complemented by information from the National Institute of Statistics and Geography, was used to obtain data for a spatial-temporal analysis of TB cases. For the geographical analysis map creation and geoinformation storing, ArcGIS software was used. RESULTS: This study sought to characterize problem areas and jurisdictional locations of TB via a spatial approach based on analyses of case distributions and individual patient variables. The study found that tuberculosis cases were dispersed throughout Tonala County and were mainly concentrated on the Guadalajara city border. The TB cases were mainly individuals between 31 and 45 years old. Most of the cases reported during the observation period were male patients, and most cases primarily had lung involvement; however, there were quite a few cases with lymph node and intestinal disease. CONCLUSION: Our findings show that TB cases are essentially located in areas close to the city of Guadalajara and that most TB cases were pulmonary cases spread throughout the whole jurisdiction.

A unique immune signature of serum cytokine and chemokine dynamics in patients with Zika virus infection from a tropical region in Southern Mexico.

OBJECTIVES: To describe the kinetics of circulating cytokines and chemokines in humans with ZIKAV infection. METHODS: Serum levels of different immune mediators in patients with ZIKAV infection were measured at distinct stages of the disease, as well as in culture supernatants from human monocytes infected with a clinical ZIKAV isolate. We also looked for clinical features associated with specific immune signatures among symptomatic patients. RESULTS: We evaluated 23 ZIKAV-infected patients. Their mean age was 32 ± 8.3 years and 65% were female. ZIKAV patients showed elevated IL-9, IL-17A, and CXCL10 levels at acute stages of the disease. At day 28, levels of CCL4 and CCL5 were increased, whereas IL-1RA, CXCL8 and CCL2 were decreased. At baseline, IL-7 was increased among patients with headache, whereas CCL2, and CCL3 were decreased in patients with bleeding and rash, respectively. Our clinical ZIKAV isolate induced a broad immune response in monocytes that did not resemble the signature observed in ZIKAV patients. CONCLUSIONS: We showed a unique immune signature in our cohort of ZIKAV-infected patients. Our study may provide valuable evidence helpful to identify immune correlates of protection against ZIKAV.

Genetic polymorphisms present in IL10, IL23R, NOD2, and ATG16L1 associated with susceptibility to inflammatory bowel disease in Mexican population.

OBJECTIVE: Ulcerative colitis and Crohn's disease are the two clinical forms of inflammatory bowel disease (IBD). Diverse studies have shown the association of single nucleotide polymorphism (SNP) in molecules of the immune system and the occurrence of IBD. Here, several SNPs of the immune system with controversial results for their association with UC and CD were evaluated in a Mexican population. METHODS: SNPs rs1800896, rs3024505 (IL-10); rs11209026 (IL23R); rs2066844, rs2066845 (NOD-2), and rs2241880 (ATG16L1) were assessed in 93 patients with IBD and 200 healthy controls by hybridization probes and quantitative PCR. RESULTS: The AG genotype for rs1800896 was associated with an increased risk for both UC and CD (P = 0.005 and P = 0.026, respectively); whereas the AA genotype presents a negative association (P = 0.011 for UC, and 0.0038 for CD). For this SNP, G allele was associated with risk of UC (P = 0-043) but not for CD. For the rs3024505 in IL-10, T allele was associated with UC (P = 0.011). Moreover, this allele was associated with early onset of UC (P = 0.033) and with the use of steroid treatment (P = 0.019). No significant differences for NOD2 (rs2066844T and rs2066845C), IL23R (rs11209026), and ATG16L1 (rs22411880) were found between cases and controls and the homozygous TT genotype for rs2066844 and CC for rs2066845 were not observed. CONCLUSION: Our results show both genotypic and phenotypic associations of IL-10 SNPs with IBD but not with the other immune-related SNPs studied in this Mexican cohort.

Association of Single-Nucleotide Polymorphisms in Immune-Related Genes with Development of Dengue Hemorrhagic Fever in a Mexican Population.

Single-nucleotide polymorphisms (SNPs) occurring in immune-related genes have been associated with risk or protection for development of dengue, depending on ethnicity. Here, we genotyped seven SNPs located in immune response-related genes to identify their association with severe forms of dengue in patients from an endemic region in Mexico. One hundred and thirty-eight patients with dengue fever (DF), thirty-one dengue hemorrhagic fever (DHF) patients, as well as 304 healthy donors were genotyped by using a TaqMan-based approach. SNP analysis, including rs1800629 (TNF), rs4804803 (CD209), rs2780831 (JAK1), rs1801274 (FCGR2A), rs231775 (CTLA4), rs12979860, and rs8099917 (IFNL3), was performed. The rs1800629 A-allele in the TNF gene was associated with an increased risk of DHF (OR = 3.4, CI = 1.235-9.284 p = 0.0212) whereas SNPs rs4804803, rs2780831, rs1801274, rs231775, rs12979860, and rs8099917 showed no association in this cohort. These results show that allelic variations in TNF can play an important role in the development of DHF. However, the lack of association between all remaining SNPs and DHF suggests that the genetic background might directly modify the role of these immune-related molecules, leading to the milder illness often observed in a Mexican population.

Comparison of DOT-ELISA and Standard-ELISA for Detection of the Vibrio cholerae Toxin in Culture Supernatants of Bacteria Isolated from Human and Environmental Samples.

A comparison of DOT-ELISA and Standard-ELISA was made for detection of Vibrio cholerae toxin in culture supernatants of bacteria isolated from human and environmental samples. A total of 293 supernatants were tested in a double blind assay. A correlation of 100 % was obtained between both techniques. The cholera toxin was found in 20 Inaba and 3 Ogawa strains. Positive samples were from seafood (17 samples), potable water (1 sample) and sewage (5 samples). The DOT-ELISA was useful as the standard-ELISA to confirm the presence of cholera toxin in the environmental samples.

The Cellular Bases of Antibody Responses during Dengue Virus Infection.

Dengue virus (DENV) is one of the most significant human viral pathogens transmitted by mosquitoes and can cause from an asymptomatic disease to mild undifferentiated fever, classical dengue, and severe dengue. Neutralizing memory antibody (Ab) responses are one of the most important mechanisms that counteract reinfections and are therefore the main aim of vaccination. However, it has also been proposed that in dengue, some of these class-switched (IgG) memory Abs might worsen the disease. Although these memory Abs derive from B cells by T-cell-dependent processes, we know rather little about the (acute, chronic, or memory) B cell responses and the complex cellular mechanisms generating these Abs during DENV infections. This review aims to provide an updated and comprehensive perspective of the B cell responses during DENV infection, starting since the very early events such as the cutaneous DENV entrance and the arrival into draining lymph nodes, to the putative B cell activation, proliferation, and germinal centers (GCs) formation (the source of affinity-matured class-switched memory Abs), till the outcome of GC reactions such as the generation of plasmablasts, Ab-secreting plasma cells, and memory B cells. We discuss topics very poorly explored such as the possibility of B cell infection by DENV or even activation-induced B cell death. The current information about the nature of the Ab responses to DENV is also illustrated.

Multiregion deep sequencing of hepatitis C virus: An improved approach for genetic relatedness studies.

Hepatitis C virus (HCV) is a major public health problem that affects more than 180 million people worldwide. Identification of HCV transmission networks is of critical importance for disease control. HCV related cases are often difficult to identify due to the characteristic long incubation period and lack of symptoms during the acute phase of the disease, making it challenging to link related cases to a common source of infection. Additionally, HCV transmission chains are difficult to trace back since viral variants from epidemiologically linked cases are genetically related but rarely identical. Genetic relatedness studies primarily rely on information obtained from the rapidly evolving HCV hypervariable region 1 (HVR1). However, in some instances, the rapid divergence of this region can lead to loss of genetic links between related isolates, which represents an important challenge for outbreak investigations and genetic relatedness studies. Sequencing of multiple and longer sub-genomic regions has been proposed as an alternative to overcome the limitations imposed by the rapid molecular evolution of the HCV HVR1. Additionally, conventional molecular approaches required to characterize the HCV intra-host genetic variation are laborious, time-consuming, and expensive while providing limited information about the composition of the viral population. Next generation sequencing (NGS) approaches enormously facilitate the characterization of the HCV intra-host population by detecting rare variants at much lower frequencies. Thus, NGS approaches using multiple sub-genomic regions should improve the characterization of the HCV intra-host population. Here, we explore the usefulness of multiregion sequencing using a NGS platform for genetic relatedness studies among HCV cases.

Influence of levamisole and Freund's adjuvant on mouse immunisation with antigens of adults of the liver fluke Fasciola hepatica Linnaeus, 1758.

We have studied the influence of both levamisole (AL) and Freund's adjuvant (AF) on the immunisation of mice with the secretory antigens of adults of the liver fluke Fasciola hepatica Linnaeus, 1758. Total IgG antibodies were detected in all groups where the F. hepatica antigen was administered, been levels of IgG1 increased respect to IgG2a antibodies. During immunisation, IL-4 and IFN-γ were only detected in AL and AF groups, but after infection, IL-4 boosted in all groups. IFN-γ increased two fold in AF and AL groups compared to the saline solution (AS) group. Worm recovering was of 32-35% in groups administered without antigen whereas in AS, AL and AF groups recovering was of 25%, 12% and 8%, respectively. Macroscopical lesions in the liver were scarce in AL and AF groups. Our data suggest that immunisation of mice with antigens of F. hepatica enhances the immune response avoiding both liver damage and worm establishment after challenge infection. The murine model of fasciolosis has appeared to be useful to elucidate the mechanism by which the parasite modulates immune responses toward a Th2 type but also the development of Th1 type-inducing vaccines.

Human multidrug-resistant Mycobacterium bovis infection in Mexico.

Here, we describe the molecular characterization of six human Mycobacterium bovis clinical isolates, including three multidrug resistant (MDR) strains, collected in Mexico through the National Survey on Tuberculosis Drug Resistance (ENTB-2008), a nationally representative survey conducted during 2008-2009 in nine states with a stratified cluster sampling design. The genetic background of bovine M. bovis strains identified in three different states of Mexico was studied in parallel to assess molecular relatedness of bovine and human strains. Additionally, resistance to first and second line anti-tuberculosis (TB) drugs and molecular identification of mutations conferring drug resistance was also performed. All strains were characterized by spoligotyping and 24-loci MIRU-VNTRs, and analyzed using the SITVIT2 (n = 112,000 strains) and SITVITBovis (n = 25,000 strains) proprietary databases of Institut Pasteur de la Guadeloupe. Furthermore, data from this study (n = 55 isolates), were also compared with genotypes recorded for M. bovis from USA (n = 203), Argentina (n = 726), as well as other isolates from Mexico (independent from the present study; n = 147), to determine any evidence for genetic relatedness between circulating M. bovis strains. The results showed that all human M. bovis cases were not genetically related between them or to any bovine strain. Interestingly, a high degree of genetic variability was observed among bovine strains. Several autochthonous and presumably imported strains were identified. The emergence of drug-resistant M. bovis is an important public health problem that jeopardizes the success of TB control programs in the region.

Advanced molecular surveillance of hepatitis C virus.

Hepatitis C virus (HCV) infection is an important public health problem worldwide. HCV exploits complex molecular mechanisms, which result in a high degree of intrahost genetic heterogeneity. This high degree of variability represents a challenge for the accurate establishment of genetic relatedness between cases and complicates the identification of sources of infection. Tracking HCV infections is crucial for the elucidation of routes of transmission in a variety of settings. Therefore, implementation of HCV advanced molecular surveillance (AMS) is essential for disease control. Accounting for virulence is also important for HCV AMS and both viral and host factors contribute to the disease outcome. Therefore, HCV AMS requires the incorporation of host factors as an integral component of the algorithms used to monitor disease occurrence. Importantly, implementation of comprehensive global databases and data mining are also needed for the proper study of the mechanisms responsible for HCV transmission. Here, we review molecular aspects associated with HCV transmission, as well as the most recent technological advances used for virus and host characterization. Additionally, the cornerstone discoveries that have defined the pathway for viral characterization are presented and the importance of implementing advanced HCV molecular surveillance is highlighted.

Hepatitis C virus molecular evolution: transmission, disease progression and antiviral therapy.

Hepatitis C virus (HCV) infection represents an important public health problem worldwide. Reduction of HCV morbidity and mortality is a current challenge owned to several viral and host factors. Virus molecular evolution plays an important role in HCV transmission, disease progression and therapy outcome. The high degree of genetic heterogeneity characteristic of HCV is a key element for the rapid adaptation of the intrahost viral population to different selection pressures (e.g., host immune responses and antiviral therapy). HCV molecular evolution is shaped by different mechanisms including a high mutation rate, genetic bottlenecks, genetic drift, recombination, temporal variations and compartmentalization. These evolutionary processes constantly rearrange the composition of the HCV intrahost population in a staging manner. Remarkable advances in the understanding of the molecular mechanism controlling HCV replication have facilitated the development of a plethora of direct-acting antiviral agents against HCV. As a result, superior sustained viral responses have been attained. The rapidly evolving field of anti-HCV therapy is expected to broad its landscape even further with newer, more potent antivirals, bringing us one step closer to the interferon-free era.

Vertical transmission of hepatitis C virus: a tale of multiple outcomes.

Globally, hepatitis C virus (HCV) infection affects approximately 130 million people and 3 million new infections occur annually. HCV is also recognized as an important cause of chronic liver disease in children. The absence of proofreading properties of the HCV RNA polymerase leads to a highly error prone replication process, allowing HCV to escape host immune response. The adaptive nature of HCV evolution dictates the outcome of the disease in many ways. Here, we investigated the molecular evolution of HCV in three unrelated children who acquired chronic HCV infection as a result of mother-to-child transmission, two of whom were also coinfected with HIV-1. The persistence of discrete HCV variants and their population structure were assessed using median joining network and Bayesian approaches. While patterns of viral evolution clearly differed between subjects, immune system dysfunction related to HIV coinfection or persistent HCV seronegativity stand as potential mechanisms to explain the lack of molecular evolution observed in these three cases. In contrast, treatment of HCV infection with PegIFN, which did not lead to sustained virologic responses in all 3 cases, was not associated with commensurate variations in the complexity of the variant spectrum. Finally, the differences in the degree of divergence suggest that the mode of transmission of the virus was not the main factor driving viral evolution.

Participation of CD161(+) and invariant natural killer T cells in pediatric asthma exacerbations.

Asthma has been defined as a disease of chronic airway inflammation in which many cells and cellular products participate with variable degrees of airflow obstruction and hyperresponsiveness that lead to recurrent episodes of wheezing, breathlessness, chest tightness, and coughing. Prominent among these cellular elements are two cell types referred to as the invariant natural killer T (iNKT) cells and a subpopulation of T cells expressing the molecule CD161, which are both thought to play a role in the pathogenesis of asthma. Although the presence of iNKT and other CD161(+) cells in murine models has been associated with asthma, relatively few studies have been performed in the adult patient with asthma that have been often conflicting and even fewer studies are available in children. The present study was performed to investigate the peripheral blood frequencies of iNKT and CD161(+) T cells in children with asthma. A total of 35 children, 19 stable asthmatic patients, 6 who had experienced an asthmatic attack within 24 hours and had not received any treatment, and 10 healthy controls, aged 6-12 years, were enrolled in the study. iNKT and CD161(+) T-cell frequencies in blood were measured together with quantitative levels of IL-4 and interferon (IFN) γ using a cytofluorimetric approach. The results show that iNKT cells are increased in pediatric asthmatic patients undergoing exacerbations of asthma. These cells also produced less IFN-γ and more IL-4 than children with stable asthma and in healthy control children. These results suggest that iNKT cells might participate in the development of the asthmatic exacerbations. The increased production of IL-4 in conjunction with the decrease of IFN-γ may be mechanistically responsible, at least partially, for the heightening of the immunologic response leading to the asthmatic attack in children. Knowledge of these interactive mechanisms involving the iNKT cell and our understanding of its role in the exacerbation of asthma hold great promise in the development of better diagnostic predictive markers of disease progression as well as new forms of therapeutic interventions.