Increased TNF-α production in response to IL-6 in patients with systemic inflammation without infection.

Acute systemic inflammation can lead to life-threatening organ dysfunction. In patients with sepsis, systemic inflammation is triggered in response to infection, but in other patients, a systemic inflammatory response syndrome (SIRS) is triggered by non-infectious events. IL-6 is a major mediator of inflammation, including systemic inflammatory responses. In homeostatic conditions, when IL-6 engages its membrane-bound receptor on myeloid cells, it promotes pro-inflammatory cytokine production, phagocytosis, and cell migration. However, under non-physiologic conditions, such as SIRS and sepsis, leucocyte dysfunction could modify the response of these cells to IL-6. So, our aim was to evaluate the response to IL-6 of monocytes from patients diagnosed with SIRS or sepsis. We observed that monocytes from patients with SIRS, but not from patients with sepsis, produced significantly more TNF-α than monocytes from healthy volunteers, after stimulation with IL-6. Monocytes from SIRS patients had a significantly increased baseline phosphorylation of the p65 subunit of NF-κB, with no differences in STAT3 phosphorylation or SOCS3 levels, compared with monocytes from septic patients, and this increased phosphorylation was maintained during the IL-6 activation. We found no significant differences in the expression levels of the membrane-bound IL-6 receptor, or the serum levels of IL-6, soluble IL-6 receptor, or soluble gp130, between patients with SIRS and patients with sepsis. Our results suggest that, during systemic inflammation in the absence of infection, IL-6 promotes TNF-α production by activating NF-κB, and not the canonical STAT3 pathway.

Innate Lymphoid Cells Have Decreased HLA-DR Expression but Retain Their Responsiveness to TLR Ligands during Sepsis.

Sepsis, one of the leading causes of death in intensive care units, is caused by a dysregulated host response to infection that leads to life-threatening organ dysfunction. The proinflammatory and anti-inflammatory responses activated by the infecting microorganism become systemic, and the sustained anti-inflammatory response induces a state of immunosuppression that is characterized by decreased expression of HLA-DR on monocytes, T cell apoptosis, and reduced production of TNF-α by monocytes and macrophages in response to TLR ligands. Innate lymphoid cells (ILCs) are lymphocytes that lack Ag-specific receptors and lineage-specific markers; they express HLA-DR and are activated by cytokines and by direct recognition of microbial molecules. In this study, we evaluated if ILCs are affected by the anti-inflammatory response during sepsis. We found that the number of peripheral blood ILCs was decreased in septic patients compared with healthy volunteers; this decrease was caused by a reduction in ILC1 and ILC3 and is associated with apoptosis, because ILCs from septic patients expressed active caspase 3. ILCs from septic patients had decreased HLA-DR expression but increased expression of the activating receptors NKp46 and NKp44; they also showed a sustained expression of CD127 (IL-7R α-chain) and retained their capacity to produce TNF-α in response to TLR ligands. These results indicate that during sepsis, ILCs have decreased HLA-DR expression and die via apoptosis, similar to monocytes and T cells, respectively. However, other effector functions of ILCs (activation through NKp46 and NKp44, TNF-α production) may remain unaffected by the immunosuppressive environment prevailing in septic patients.

IgG1 Is Required for Optimal Protection after Immunization with the Purified Porin OmpD from Salmonella Typhimurium.

In mice, the IgG subclass induced after Ag encounter can reflect the nature of the Ag. Th2 Ags such as alum-precipitated proteins and helminths induce IgG1, whereas Th1 Ags, such as Salmonella Typhimurium, predominantly induce IgG2a. The contribution of different IgG isotypes to protection against bacteria such as S. Typhimurium is unclear, although as IgG2a is induced by natural infection, it is assumed this isotype is important. Previously, we have shown that purified S. Typhimurium porins including outer membrane protein OmpD, which induce both IgG1 and IgG2a in mice, provide protection to S. Typhimurium infection via Ab. In this study we report the unexpected finding that mice lacking IgG1, but not IgG2a, are substantially less protected after porin immunization than wild-type controls. IgG1-deficient mice produce more porin-specific IgG2a, resulting in total IgG levels that are similar to wild-type mice. The decreased protection in IgG1-deficient mice correlates with less efficient bacterial opsonization and uptake by macrophages, and this reflects the low binding of outer membrane protein OmpD-specific IgG2a to the bacterial surface. Thus, the Th2-associated isotype IgG1 can play a role in protection against Th1-associated organisms such as S. Typhimurium. Therefore, individual IgG subclasses to a single Ag can provide different levels of protection and the IgG isotype induced may need to be a consideration when designing vaccines and immunization strategies.

LLT1 and CD161 Expression in Human Germinal Centers Promotes B Cell Activation and CXCR4 Downregulation.

Germinal centers (GCs) are microanatomical structures critical for the development of high-affinity Abs and B cell memory. They are organized into two zones, light and dark, with coordinated roles, controlled by local signaling. The innate lectin-like transcript 1 (LLT1) is known to be expressed on B cells, but its functional role in the GC reaction has not been explored. In this study, we report high expression of LLT1 on GC-associated B cells, early plasmablasts, and GC-derived lymphomas. LLT1 expression was readily induced via BCR, CD40, and CpG stimulation on B cells. Unexpectedly, we found high expression of the LLT1 ligand, CD161, on follicular dendritic cells. Triggering of LLT1 supported B cell activation, CD83 upregulation, and CXCR4 downregulation. Overall, these data suggest that LLT1-CD161 interactions play a novel and important role in B cell maturation within the GC in humans.

Impaired selective cytokine production by CD4(+) T cells in Common Variable Immunodeficiency associated with the absence of memory B cells.

Common Variable Immunodeficiency (CVID) is a primary immunodeficiency characterized by B cell dysfunction and decreased serum immunoglobulin. CVID patients are classified by the absence or presence of memory B cells. In addition, T cell defects have been demonstrated in only a proportion of CVID patients. The aim of this study was to evaluate the function of CD4(+) T cells from CVID patients and its association with memory B cells. Patients were classified according to their Freiburg groups: group Ia and Ib, with decreased switched memory B cells (<0.4 of PBL), and group II, with normal B cell subsets. Their T cell function was evaluated after stimulation. We observed normal and even increased CD4(+) T cell proliferation in group Ia (p=0.0277). The proliferation positively correlated with the clinical severity score (r=0.4796). We observed lower levels of IL-17A and IL-10 in group Ia (p=0.0177, 0.0109) and Ib (p=0.0009, 0.0084) patients. Group Ib patients also had low levels of IL-13 and IL-9 (p=0.0169, 0.010). Group II patients had similar cytokine production to that of the controls. BAFFR expression was reduced in groups Ia (p=0.0001) and Ib (p=0.0002) and showed an inverse correlation with the severity score (p=0.0262; r=0.5371). ICOS expression was reduced in group Ia (p=0.0364), and PD-1 was increased in group Ib (p=0.0432) patients. This study shows a selective impairment in cytokine production in group Ia patients, which was more extensive than in group Ib patients. The impairment was associated with BAFFR expression in B cells, with ICOS and PD-1 in T cells and, remarkably, with the absence of memory B cells and with the disease severity. Our results suggest that the evaluation of cytokine expression by T cells in combination with the study of B cell memory could be important for understand the pathogenesis of CVID patients.

IFN-γ-producing CD4+ T cells promote generation of protective germinal center-derived IgM+ B cell memory against Salmonella Typhi.

Abs play a significant role in protection against the intracellular bacterium Salmonella Typhi. In this article, we investigated how long-term protective IgM responses can be elicited by a S. Typhi outer-membrane protein C- and F-based subunit vaccine (porins). We found that repeated Ag exposure promoted a CD4(+) T cell-dependent germinal center reaction that generated mutated IgM-producing B cells and was accompanied by a strong expansion of IFN-γ-secreting T follicular helper cells. Genetic ablation of individual cytokine receptors revealed that both IFN-γ and IL-17 are required for optimal germinal center reactions and production of porin-specific memory IgM(+) B cells. However, more profound reduction of porin-specific IgM B cell responses in the absence of IFN-γR signaling indicated that this cytokine plays a dominant role. Importantly, mutated IgM mAbs against porins exhibited bactericidal capacity and efficiently augmented S. Typhi clearance. In conclusion, repeated vaccination with S. Typhi porins programs type I T follicular helper cell responses that contribute to the diversification of B cell memory and promote the generation of protective IgM Abs.

[Purification of Salmonella Typhimurium OmpD porin induces long-term high levels of antibodies: implications on the development of vaccines against non-typhoid salmonella]. / La porina OmpD de Salmonella Typhimurium induce altos títulos de anticuerpos de larga duración: implicaciones en el desarrollo de vacunas contra la salmonelosis no tifoídica.

In the present work, we report, for the first time, on the purification of the Salmonella Typhimurium OmpD porin. We assessed the integrity and purity of the protein and evaluated the immunogenicity of the protein and its ability to induce antibody without exogenous adjuvant. We observed that 10 µg OmpD induced high antibody levels of IgM and IgG, which were maintained for more than 260 days after immunization. Immunization with OmpD induced multiple IgG antibody isotypes including IgG1, IgG2a, IgG2b, and IgG3 subclasses. Furthermore, these antibodies were able to recognize and bind to the bacterial surface. Our results demonstrate the high immunogenicity of S. Typhimurium OmpD porin, which induces long-lasting antibodies which may be and important target of the immune response against Salmonella infection. In conclusion, we propose the OmpD porin could be used within novel subunit vaccine formulations that do not need additional adjuvant and that confer long lasting humoral immunity against Salmonella infections.

Activated endothelial cells limit inflammatory response, but increase chemoattractant potential and bacterial clearance by human monocytes.

Inflammation is the normal immune response of vascularized tissues to damage and bacterial products, for which leukocyte transendothelial migration (TEM) is critical. The effects of cell-to-cell contact seen in both leukocyte and endothelial cells include cytoskeleton rearrangement, and dynamic expression of adhesion molecules and metalloproteinases. TEM induces expression of anti-apoptotic molecules, costimulatory molecules associated with antigen presentation, and pattern recognition receptors (PRR), such as TLR-4, in monocytes. However, little is known about how TLR-4 increment operates in monocytes during an inflammatory response. To understand it better, we used an in vitro model in which monocytes crossed a layer of IL-1ß stimulated Human Umbilical Vein Endothelial Cells (HUVEC). After TEM, monocytes were tested for the secretion of inflammatory cytokines and chemokines, their phenotype (CD14, CD16, TLR-4 expression), and TLR-4 canonical [Nuclear Factor kappa B, (NF-κB) pathway] and non-canonical [p38, extracellular signal-regulated kinases (ERK) 1/2 pathway] signal transduction induced by lipopolysaccharide (LPS). Phagocytosis and bacterial clearance were also measured. There was diminished secretion of LPS-induced inflammatory cytokines (IL-1ß, IL-6, and TNF-α) and higher secretion of chemokines (CXCL8/IL-8 and CCL2/MCP-1) in supernatant of TEM monocytes. These changes were accompanied by increases in TLR-4, CD14 (surfaces expression), p38, and ERK1/2 phosphorylated cytoplasmic forms, without affecting NF-κB activation. It also increased bacterial clearance after TEM by an O2 -independent mechanism. The data suggest that interaction between endothelial cells and monocytes fine-tunes the inflammatory response and promotes bacterial elimination.

The capsular polysaccharide Vi from Salmonella typhi is a B1b antigen.

Vaccination with purified capsular polysaccharide Vi Ag from Salmonella typhi can protect against typhoid fever, although the mechanism for its efficacy is not clearly established. In this study, we have characterized the B cell response to this vaccine in wild-type and T cell-deficient mice. We show that immunization with typhoid Vi polysaccharide vaccine rapidly induces proliferation in B1b peritoneal cells, but not in B1a cells or marginal zone B cells. This induction of B1b proliferation is concomitant with the detection of splenic Vi-specific Ab-secreting cells and protective Ab in Rag1-deficient B1b cell chimeras generated by adoptive transfer-induced specific Ab after Vi immunization. Furthermore, Ab derived from peritoneal B cells is sufficient to confer protection against Salmonella that express Vi Ag. Expression of Vi by Salmonella during infection did not inhibit the development of early Ab responses to non-Vi Ags. Despite this, the protection conferred by immunization of mice with porin proteins from Salmonella, which induce Ab-mediated protection, was reduced postinfection with Vi-expressing Salmonella, although protection was not totally abrogated. This work therefore suggests that, in mice, B1b cells contribute to the protection induced by Vi Ag, and targeting non-Vi Ags as subunit vaccines may offer an attractive strategy to augment current Vi-based vaccine strategies.

Newcastle disease virus vector-based SARS-CoV-2 vaccine candidate AVX/COVID-12 activates T cells and is recognized by antibodies from COVID-19 patients and vaccinated individuals.

Introduction: Several effective vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been developed and implemented in the population. However, the current production capacity falls short of meeting global demand. Therefore, it is crucial to further develop novel vaccine platforms that can bridge the distribution gap. AVX/COVID-12 is a vector-based vaccine that utilizes the Newcastle Disease virus (NDV) to present the SARS-CoV-2 spike protein to the immune system. Methods: This study aims to analyze the antigenicity of the vaccine candidate by examining antibody binding and T-cell activation in individuals infected with SARS-CoV-2 or variants of concern (VOCs), as well as in healthy volunteers who received coronavirus disease 2019 (COVID-19) vaccinations. Results: Our findings indicate that the vaccine effectively binds antibodies and activates T-cells in individuals who received 2 or 3 doses of BNT162b2 or AZ/ChAdOx-1-S vaccines. Furthermore, the stimulation of T-cells from patients and vaccine recipients with AVX/COVID-12 resulted in their proliferation and secretion of interferon-gamma (IFN-γ) in both CD4+ and CD8+ T-cells. Discussion: The AVX/COVID-12 vectored vaccine candidate demonstrates the ability to stimulate robust cellular responses and is recognized by antibodies primed by the spike protein present in SARS-CoV-2 viruses that infected patients, as well as in the mRNA BNT162b2 and AZ/ChAdOx-1-S vaccines. These results support the inclusion of the AVX/COVID-12 vaccine as a booster in vaccination programs aimed at addressing COVID-19 caused by SARS-CoV-2 and its VOCs.

Salmonella Typhi OmpS1 and OmpS2 porins are potent protective immunogens with adjuvant properties.

Salmonella enterica serovar Typhi (S. Typhi) is the causal agent of typhoid fever, a disease that primarily affects developing countries. Various antigens from this bacterium have been reported to be targets of the immune response. Recently, the S. Typhi genome has been shown to encode two porins--OmpS1 and OmpS2--which are expressed at low levels under in vitro culture conditions. In this study, we demonstrate that immunizing mice with either OmpS1 or OmpS2 induced production of specific, long-term antibody titres and conferred protection against S. Typhi challenge; in particular, OmpS1 was more immunogenic and conferred greater protective effects than OmpS2. We also found that OmpS1 is a Toll-like receptor 4 (TLR4) agonist, whereas OmpS2 is a TLR2 and TLR4 agonist. Both porins induced the production of tumour necrosis factor and interleukin-6, and OmpS2 was also able to induce interleukin-10 production. Furthermore, OmpS1 induced the over-expression of MHC II molecules in dendritic cells and OmpS2 induced the over-expression of CD40 molecules in macrophages and dendritic cells. Co-immunization of OmpS1 or OmpS2 with ovalbumin (OVA) increased anti-OVA antibody titres, the duration and isotype diversity of the OVA-specific antibody response, and the proliferation of T lymphocytes. These porins also had adjuvant effects on the antibody response when co-immunized with either the Vi capsular antigen from S. Typhi or inactivated 2009 pandemic influenza A(H1N1) virus [A(H1N1)pdm09]. Taken together, the data indicate that OmpS1 and OmpS2, despite being expressed at low levels under in vitro culture conditions, are potent protective immunogens with intrinsic adjuvant properties.

CD31 is required on CD4+ T cells to promote T cell survival during Salmonella infection.

Hematopoietic cells constitutively express CD31/PECAM1, a signaling adhesion receptor associated with controlling responses to inflammatory stimuli. Although expressed on CD4(+) T cells, its function on these cells is unclear. To address this, we have used a model of systemic Salmonella infection that induces high levels of T cell activation and depends on CD4(+) T cells for resolution. Infection of CD31-deficient (CD31KO) mice demonstrates that these mice fail to control infection effectively. During infection, CD31KO mice have diminished numbers of total CD4(+) T cells and IFN-γ-secreting Th1 cells. This is despite a higher proportion of CD31KO CD4(+) T cells exhibiting an activated phenotype and an undiminished capacity to prime normally and polarize to Th1. Reduced numbers of T cells reflected the increased propensity of naive and activated CD31KO T cells to undergo apoptosis postinfection compared with wild-type T cells. Using adoptive transfer experiments, we show that loss of CD31 on CD4(+) T cells alone is sufficient to account for the defective CD31KO T cell accumulation. These data are consistent with CD31 helping to control T cell activation, because in its absence, T cells have a greater propensity to become activated, resulting in increased susceptibility to become apoptotic. The impact of CD31 loss on T cell homeostasis becomes most pronounced during severe, inflammatory, and immunological stresses such as those caused by systemic Salmonella infection. This identifies a novel role for CD31 in regulating CD4 T cell homeostasis.

PD-L1 expression induced by the 2009 pandemic influenza A(H1N1) virus impairs the human T cell response.

PD-L1 expression plays a critical role in the impairment of T cell responses during chronic infections; however, the expression of PD-L1 on T cells during acute viral infections, particularly during the pandemic influenza virus (A(H1N1)pdm09), and its effects on the T cell response have not been widely explored. We found that A(H1N1)pdm09 virus induced PD-L1 expression on human dendritic cells (DCs) and T cells, as well as PD-1 expression on T cells. PD-L1 expression impaired the T cell response against A(H1N1)pdm09 by promoting CD8⁺ T cell death and reducing cytokine production. Furthermore, we found increased PD-L1 expression on DCs and T cells from influenza-infected patients from the first and second 2009 pandemic waves in Mexico City. PD-L1 expression on CD8⁺ T cells correlated inversely with T cell proportions in patients infected with A(H1N1)pdm09. Therefore, PD-L1 expression on DCs and T cells could be associated with an impaired T cell response during acute infection with A(H1N1)pdm09 virus.

[ABO incompatibility and complications in hematopoietic stem cell transplantation]. / Incompatibilidad ABO en el trasplante de células progenitoras hematopoyéticas y sus complicaciones.

Background: Hematopoietic stem cell transplants (HSCT) can be performed regardless of the ABO group compatibility between donor and recipient. ABO incompatibility in HSCT is related to pure red cell aplasia (PRCA), or passenger lymphocyte syndrome. The impact of ABO incompatibility on graft-versus-host disease and transplant-related mortality is controversial due to the heterogeneity of procedures carried out in different transplant centers. Objective: To determine the prevalence of ABO incompatibility and its complications in a hematopoietic stem transplant unit. Material and methods: An observational, retrospective study was carried out in patients undergoing HSCT from January 2014 to January 2020. All trasplant patients were included. Qualitative variables were analyzed using chi-squared test, and Wilcoxon and Student's t tests were used for quantitative variables. A p < 0.05 was considered significant. Results: 124 patients undergoing HSCT were analyzed, out of which 31 had ABO incompatibility, with a punctual prevalence of 24.4%; among them, 54% presented with major incompatibility, 32% minor incompatibility and 13% bidirectional incompatibility. Three cases of PRCA were reported. There were no differences in survival at one year in both groups. Conclusions: The ABO incompatibility ant its complications were not related to the increase in mortality. Randomized prospective studies are required to define the role of ABO incompatibility in HSCT prognosis.

Introducción: los trasplantes de células progenitoras hematopoyéticas (TCPH) se pueden hacer independientemente de la compatibilidad de grupo sanguíneo ABO entre donador y receptor. La incompatibilidad ABO (IABO) en los TCPH puede presentar complicaciones, como aplasia pura de serie roja (APSR), o síndrome de linfocito pasajero. El impacto de la IABO en la enfermedad del injerto en contra del huésped y la mortalidad relacionada al trasplante es controversial por la heterogeneidad de procedimientos que se hacen en los distintos centros de trasplante. Objetivo: determinar la prevalencia de la IABO y sus complicaciones en los pacientes trasplantados en una unidad de trasplante de progenitores hematopoyéticos. Material y métodos: se hizo un estudio tipo observacional, descriptivo, en pacientes sometidos a TCPH de enero de 2014 a enero de 2020. Se incluyeron todos los pacientes trasplantados. Las variables cualitativas se analizaron con chi cuadrada y para las variables cuantitativas se usó la prueba de Wilcoxon y t de Student. Una p < 0.05 fue significativa. Resultados: se analizaron 124 pacientes sometidos a TCPH y 31 de ellos presentaron IABO, con una prevalencia puntual de 24.4%; entre ellos, 54% presentaron incompatibilidad mayor, 32% incompatibilidad menor y 13% incompatibilidad bidireccional. Se reportaron tres casos de APSR. No hubo diferencias en la supervivencia global a un año en ambos grupos. Conclusiones: la IABO y sus complicaciones no se relacionaron con aumento en la mortalidad. Se requieren estudios prospectivos aleatorizados para definir el papel de la IABO con el pronóstico del trasplante.

In vivo evaluation of new adjuvant systems based on combination of Salmonella Typhi porins with particulate systems: Liposomes versus polymeric particles.

Subunit vaccines that have weak immunogenic activity require adjuvant systems for enhancedcellular and long-acting humoral immune responses. Both lipid-based and polymeric-based particulate adjuvants have been widely investigated to induce the desired immune responses against the subunit vaccines. The adjuvant efficacy of these particulate adjuvants depends upon their physicochemical properties such as particle size, surface charge, shape and their composition. Previously, we showed in vitro effect of adjuvant systems based on combination of chitosan and Salmonella Typhi porins in microparticle or nanoparticle form, which were spherical with positive surface charge. In the present study, we have further developed an adjuvant system based on combination of porins with liposomes (cationic and neutral) and investigated the adjuvant effect of both the liposomal and polymeric systems in BALB/c mice using a model antigen, ovalbumin. Humoral immune responses were determined following priming and booster dose at 15-day intervals. In overall, IgM and IgG levels were induced in the presence of both the liposomal and polymeric adjuvant systems indicating the positive impact of combination with porins. The highest IgM levels were obtained on Day 8, and liposomal adjuvant systems were found to elicit significantly higher IgM levels compared to polymeric systems. IgG levels were increased significantly after booster, particularly more profound with the micro-sized polymeric system when compared to cationic liposomal system with nano-size. Our results demonstrated that the developed particulate systems are promising both as an adjuvant and delivery system, providing enhanced immune responses against subunit antigens, and have the potential for long-term protection.

Characterization of immunoglobulin loci in the gigantic genome of Ambystoma mexicanum.

Background: The axolotl, Ambystoma mexicanum is a unique biological model for complete tissue regeneration. Is a neotenic endangered species and is highly susceptible to environmental stress, including infectious disease. In contrast to other amphibians, the axolotl is particularly vulnerable to certain viral infections. Like other salamanders, the axolotl genome is one of the largest (32 Gb) and the impact of genome size on Ig loci architecture is unknown. To better understand the immune response in axolotl, we aimed to characterize the immunoglobulin loci of A. mexicanum and compare it with other model vertebrates. Methods: The most recently published genome sequence of A. mexicanum (V6) was used for alignment-based annotation and manual curation using previously described axolotl Ig sequences or reference sequences from other vertebrates. Gene models were further curated using A. mexicanum spleen RNA-seq data. Human, Xenopus tropicalis, Danio rerio (zebrafish), and eight tetrapod reference genomes were used for comparison. Results: Canonical A. mexicanum heavy chain (IGH), lambda (IGL), sigma (IGS), and the putative surrogate light chain (SLC) loci were identified. No kappa locus was found. More than half of the IGHV genes and the IGHF gene are pseudogenes and there is no clan I IGHV genes. Although the IGH locus size is proportional to genome size, we found local size restriction in the IGHM gene and the V gene intergenic distances. In addition, there were V genes with abnormally large V-intron sizes, which correlated with loss of gene functionality. Conclusion: The A. mexicanum immunoglobulin loci share the same general genome architecture as most studied tetrapods. Consistent with its large genome, Ig loci are larger; however, local size restrictions indicate evolutionary constraints likely to be imposed by high transcriptional demand of certain Ig genes, as well as the V(D)J recombination over very long genomic distance ranges. The A. mexicanum has undergone an extensive process of Ig gene loss which partially explains a reduced potential repertoire diversity that may contribute to its impaired antibody response.

Mycobacterium bovis naturally infected calves present a higher bacterial load and proinflammatory response than adult cattle.

Granulomas are characteristic bovine tuberculosis lesions; studying this structure has improved our understanding of tuberculosis pathogenesis. However, the immune response that develops in granulomas of young cattle naturally infected with Mycobacterium bovis (M. bovis) has not been fully studied. Our previous work described an atypical pattern in granulomatous lesions of cattle younger than 4 months (calves) naturally infected previously M. bovis that did not correspond to the histological classification previously proposed. Histologically, granulomas from calves lack a connective tissue capsule and have fewer multinucleated giant cells (MGCs) and more acid-fast bacilli (AFB) than the classic tuberculosis lesions found in cattle older than 1 year (adults); this suggests a deficient immune response against M. bovis infection in young animals. Therefore, we used IHC and digital pathology analysis to characterize the in situ immune response of granulomas from young and adult cattle. The immunolabeling quantification showed that granulomas from calves had more mycobacteria, CD3+ cells, IFN-γ, TNF-α, and inducible nitric oxide synthase (iNOS) than those of adult cattle. Furthermore, calf granulomas showed lower immunolabeling of MAC387+, CD79+, and WC1+ cells without connective tissue surrounding the lesion and were associated with less vimentin, Alpha Smooth Muscle Actin (α-SMA), and TGF-ß compared with granulomas from adult cattle. Our results suggest that the immune responses in granulomas of cattle naturally infected with M. bovis may be age dependent. This implies that an exacerbated proinflammatory response may be associated with active tuberculosis, producing more necrosis and a lower microbicidal capacity in the granulomas of calves naturally infected with M. bovis.

Starting from scratch: Step-by-step development of diagnostic tests for SARS-CoV-2 detection by RT-LAMP.

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected millions of people worldwide. Public health strategies to reduce viral transmission are based on widespread diagnostic testing to detect and isolate contagious patients. Several reverse transcription (RT)-PCR tests, along with other SARS-CoV-2 diagnostic assays, are available to attempt to cover the global demand. Loop-mediated isothermal amplification (LAMP) based methods have been established as rapid, accurate, point of care diagnostic tests for viral infections; hence, they represent an excellent alternative for SARS-CoV-2 detection. The aim of this study was to develop and describe molecular detection systems for SARS-CoV-2 based on RT-LAMP. Recombinant DNA polymerase from Bacillus stearothermophilus and thermostable engineered reverse transcriptase from Moloney Murine Leukemia Virus were expressed using a prokaryotic system and purified by fast protein liquid chromatography. These enzymes were used to set up fluorometric real time and colorimetric end-point RT-LAMP assays. Several reaction conditions were optimized such as reaction temperature, Tris-HCl concentration, and pH of the diagnostic tests. The key enzymes for RT-LAMP were purified and their enzymatic activity was determined. Standardized reaction conditions for both RT-LAMP assays were 65°C and a Tris-HCl-free buffer at pH 8.8. Colorimetric end-point RT-LAMP assay was successfully used for viral detection from clinical saliva samples with 100% sensitivity and 100% specificity compared to the results obtained by RT-qPCR based diagnostic protocols with Ct values until 30. The developed RT-LAMP diagnostic tests based on purified recombinant enzymes allowed a sensitive and specific detection of the nucleocapsid gene of SARS-CoV-2.

Chronic Comorbidities in Middle Aged Patients Contribute to Ineffective Emergency Hematopoiesis in Covid-19 Fatal Outcomes.

BACKGROUND AND AIMS: Mexico is among the countries with the highest estimated excess mortality rates due to the COVID-19 pandemic, with more than half of reported deaths occurring in adults younger than 65 years old. Although this behavior is presumably influenced by the young demographics and the high prevalence of metabolic diseases, the underlying mechanisms have not been determined. METHODS: The age-stratified case fatality rate (CFR) was estimated in a prospective cohort with 245 hospitalized COVID-19 cases, followed through time, for the period October 2020-September 2021. Cellular and inflammatory parameters were exhaustively investigated in blood samples by laboratory test, multiparametric flow cytometry and multiplex immunoassays. RESULTS: The CFR was 35.51%, with 55.2% of deaths recorded in middle-aged adults. On admission, hematological cell differentiation, physiological stress and inflammation parameters, showed distinctive profiles of potential prognostic value in patients under 65 at 7 days follow-up. Pre-existing metabolic conditions were identified as risk factors of poor outcomes. Chronic kidney disease (CKD), as single comorbidity or in combination with diabetes, had the highest risk for COVID-19 fatality. Of note, fatal outcomes in middle-aged patients were marked from admission by an inflammatory landscape and emergency myeloid hematopoiesis at the expense of functional lymphoid innate cells for antiviral immunosurveillance, including NK and dendritic cell subsets. CONCLUSIONS: Comorbidities increased the development of imbalanced myeloid phenotype, rendering middle-aged individuals unable to effectively control SARS-CoV-2. A predictive signature of high-risk outcomes at day 7 of disease evolution as a tool for their early stratification in vulnerable populations is proposed.

Interim safety and immunogenicity results from an NDV-based COVID-19 vaccine phase I trial in Mexico.

There is still a need for safe, efficient, and low-cost coronavirus disease 2019 (COVID-19) vaccines that can stop transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we evaluated a vaccine candidate based on a live recombinant Newcastle disease virus (NDV) that expresses a stable version of the spike protein in infected cells as well as on the surface of the viral particle (AVX/COVID-12-HEXAPRO, also known as NDV-HXP-S). This vaccine candidate can be grown in embryonated eggs at a low cost, similar to influenza virus vaccines, and it can also be administered intranasally, potentially to induce mucosal immunity. We evaluated this vaccine candidate in prime-boost regimens via intramuscular, intranasal, or intranasal followed by intramuscular routes in an open-label non-randomized non-placebo-controlled phase I clinical trial in Mexico in 91 volunteers. The primary objective of the trial was to assess vaccine safety, and the secondary objective was to determine the immunogenicity of the different vaccine regimens. In the interim analysis reported here, the vaccine was found to be safe, and the higher doses tested were found to be immunogenic when given intramuscularly or intranasally followed by intramuscular administration, providing the basis for further clinical development of the vaccine candidate. The study is registered under ClinicalTrials.gov identifier NCT04871737.