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Tuberculosis remains one of the leading public health problems in the world. The mechanisms that lead to the activation of the immune response against Mycobacterium tuberculosis have been extensively studied, with a focus on the role of cytokines as the main signals for immune cell communication. However, less is known about the role of other signals, such as extracellular vesicles, in the communication between immune cells, particularly during the activation of the adaptive immune response. In this study, we determined that extracellular vesicles released by human neutrophils infected with M. tuberculosis contained several host proteins that are ectosome markers. In addition, we demonstrated that extracellular vesicles released by human neutrophils infected with M. tuberculosis released after only 30 min of infection carried mycobacterial antigens and pathogen-associated molecular patterns, and we identified 15 mycobacterial proteins that were consistently found in high concentrations in extracellular vesicles released by human neutrophils infected with M. tuberculosis; these proteins contain epitopes for CD4 T-cell activation. We found that extracellular vesicles released by human neutrophils infected with M. tuberculosis increased the expression of the costimulatory molecule CD80 and of the coinhibitory molecule PD-L1 on immature monocyte-derived dendritic cells. We also found that immature and mature dendritic cells treated with extracellular vesicles released by human neutrophils infected with M. tuberculosis were able to induce IFN-γ production by autologous M. tuberculosis antigen-specific CD4 T cells, indicating that these extracellular vesicles acted as antigen carriers and transferred mycobacterial proteins to the antigen-presenting cells. Our results provide evidence that extracellular vesicles released by human neutrophils infected with M. tuberculosis participate in the activation of the adaptive immune response against M. tuberculosis.

Cancer mice models are critical for immune-oncology research; they provide conditions to explore tumor immunoenviroment aiming knowledge advance and treatment development. Often, research groups breed their own mice colonies. To assess the effect of C57BL/6 mice breeding nuclei in prostate cancer development and intratumoral macrophage populations, a isotransplantation experiment was performed. C57BL/6J mice from two breeding nuclei (nA and nB) were employed for prostate adenocarcinoma TRAMP-C1 cells implantation, tumor growth period and intratumoral macrophage profile were measured. 54% of BL/6nB mice showed tumor implantation and growth period was 69 days, meanwhile BL/6nA implantation reached 100% and tumor growth occur in 28 days. No difference of total macrophage populations was observed between groups within several tumoral regions, significant higher M2 macrophage profile were founded in tumor microenviroment from both mice groups. Nevertheless, BL/6nB tumors showed around twice populations of M1 profile (11% -27%) than BL6nA (4% -15%) and less non-polarized macrophages. M2:M1 average ratio was 8:1 for A group and 4.2:1 for B. Our results demonstrate different tumor progression and intratumoral macrophage populations among mice from same substrain. Data obtained in this study shows the relevance of animal sources renewing for better control of murine cancer model variables.

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.

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-1β, 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.

hScrib and hDlg belong to the PDZ family of proteins. Since the identification of these highly phylogenetically conserved scaffolds, an increasing amount of experiments has elucidated the roles of hScrib and hDlg in a variety of cell functions. Remarkably, their participation during the establishment of polarity in epithelial cells is well documented. Although the role of both proteins in the immune system is scantly known, it has become a growing field of investigation. Here, we summarize the interactions and functions of hScrib and hDlg1, which participate in diverse functions involving cell polarization in immune cells, and discuss their relevance in the immune cell biology. The fundamental role of hScrib and hDlg1 during the establishment of the immunological synapse, hence T cell activation, and the recently described role of hScrib in reactive oxygen species production in macrophages and of hDlg1 in cytokine production by dendritic cells highlight the importance of both proteins in immune cell biology. The expression of these proteins in other leukocytes can be anticipated and needs to be confirmed. Due to their multiple interaction domains, there is a wide range of possible interactions of hScrib and hDlg1 that remains to be explored in the immune system.

Porcine epidemic diarrhoea (PED) is a disease caused by an alphacoronavirus and the symptoms include watery diarrhoea and vomiting, with more than 80% mortality amongst newborn piglets. The placentation in sows hinders the transference of antibodies to the foetus, therefore, the vaccination of pregnant females and transference of antibodies to piglets through colostrum are essential to protect them against virus particles. The aim of the study was to determine whether vaccination of nulliparous gilts could induce a high colostrum antibody titre and lower litter mortality, in comparison with vaccinated multiparous sows previously exposed to the virus. Samples of colostrum were obtained from 11 nulliparous gilts with two previous vaccinations (inactivated vaccine) and from 9 multiparous sows with three or more vaccinations (inactivated vaccine) that had been exposed to the virus. The IgG antibody titre was determined through anti-PED enzyme-linked immunosorbent assays (ELISA) and the neutralisation of antibodies was evaluated through plaque reduction neutralisation tests (PRNT). The colostrum of nulliparous gilts, when compared to the multiparous sows, presented a lower anti-PED IgG antibody titre as well as fewer neutralising antibodies. Furthermore, the piglets of multiparous sows experienced higher survival in comparison with those of nulliparous gilts (P

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.

The immune response plays a critical role in the pathophysiology of SARS‐CoV‐2 infection ranging from protection to tissue damage and all occur in the development of acute respiratory distress syndrome (ARDS). ARDS patients display elevated levels of inflammatory cytokines and innate immune cells, and T and B cell lymphocytes have been implicated in this dysregulated immune response. Mast cells are abundant resident cells of the respiratory tract and are able to release different inflammatory mediators rapidly following stimulation. Recently, mast cells have been associated with tissue damage during viral infections, but their role in SARS‐CoV‐2 infection remains unclear. In this study, we examined the profile of mast cell activation markers in the serum of COVID‐19 patients. We noticed that SARS‐CoV‐2‐infected patients showed increased carboxypeptidase A3 (CPA3) and decreased serotonin levels in their serum when compared with symptomatic SARS‐CoV‐2‐negative patients. CPA3 levels correlated with C‐reactive protein, the number of circulating neutrophils, and quick SOFA. CPA3 in serum was a good biomarker for identifying severe COVID‐19 patients, whereas serotonin was a good predictor of SARS‐CoV‐2 infection. In summary, our results show that serum CPA3 and serotonin levels are relevant biomarkers during SARS‐CoV‐2 infection. This suggests that mast cells and basophils are relevant players in the inflammatory response in COVID‐19 and may represent targets for therapeutic intervention., Graphical Abstract Serum levels of CPA3 and serotonin are affected during SARS‐CoV‐2 infection and can be considered as biomarkers during COVID‐19.

Persistent infection with high-risk human papillomavirus (HR-HPV) is the main factor in the development of cervical cancer (CC). The presence of immunosuppressive factors plays an important role in the development of this type of cancer. To determine whether CD39 and CD73, which participate in the production of immunosuppressive adenosine (Ado), are involved in the progression of CC, we compared the concentrations and hydrolytic activity of these ectonucleotidases in platelet-free plasma (PFP) samples between patients with low-grade squamous intraepithelial lesions (LSILs) ( n = 18 ), high-grade squamous intraepithelial lesions (HSILs) ( n = 12 ), and CC ( n = 19 ) and normal donors (NDs) ( n = 15 ). The concentrations of CD39 and CD73 in PFP increased with disease progression ( r = 0.5929 , p < 0.001 ). The PFP of patients with HSILs or CC showed the highest concentrations of CD39 (2.3 and 2.2 times that of the NDs, respectively) and CD73 (1.7 and 2.68 times that of the NDs, respectively), which were associated with a high capacity to generate Ado from the hydrolysis of adenosine diphosphate (ADP) and adenosine monophosphate (AMP). The addition of POM-1 and APCP, specific inhibitors of CD39 and CD73, respectively, inhibited the ADPase and AMPase activity of PFP by more than 90%. A high level of the 90 kD isoform of CD73 was detected in the PFP of patients with HSILs or CC. Digestion with endoglycosidase H and N-glycanase generated CD73 with weights of approximately 90 kD, 85 kD, 80 kD, and 70 kD. In addition, the levels of transforming grow factor-β (TGF-β) in the PFPs of patients with LSIL, HSIL and CC positively correlated with those of CD39 ( r = 0.4432 , p < 0.001 ) and CD73 ( r = 0.5786 , p < 0.001 ). These results suggest that persistent infection by HR-HPV and the concomitant production of TGF-β promote the expression of CD39 and CD73 to favor CC progression through Ado generation.

Background More than 3 million infections were attributed to Chikungunya virus (CHIKV) in the 2014–2016 outbreak in Mexico, Central and South America, with over 500 deaths directly or indirectly related to this viral disease. CHIKV outbreaks are recurrent and no vaccine nor approved therapeutics exist to prevent or treat CHIKV infection. Reliable and robust diagnostic methods are thus critical to control future CHIKV outbreaks. Direct CHIKV detection in serum samples via highly specific and high affinity anti-CHIKV antibodies has shown to be an early and effective clinical diagnosis. Methods To isolate highly specific and high affinity anti-CHIKV, Chikungunya virions were isolated from serum of a patient in Veracruz, México. After purification and characterization via electron microscopy, SDS-PAGE and binding to well-characterized anti-CHIKV antibodies, UV-inactivated particles were utilized as selector in a solid-phase panning in combination with ALTHEA Gold Libraries™, as source of antibodies. The screening was based on ELISA and Next-Generation Sequencing. Results The CHIKV isolate showed the typical morphology of the virus. Protein bands in the SDS-PAGE were consistent with the size of CHIKV capsid proteins. UV-inactivated CHIKV particles bound tightly the control antibodies. The lead antibodies here obtained, on the other hand, showed high expression yield, > 95% monomeric content after a single-step Protein A purification, and importantly, had a thermal stability above 75 °C. Most of the antibodies recognized linear epitopes on E2, including the highest affinity antibody called C7. A sandwich ELISA implemented with C7 and a potent neutralizing antibody isolated elsewhere, also specific for E2 but recognizing a discontinuous epitope, showed a dynamic range of 0.2–40.0 mg/mL of UV-inactivated CHIKV purified preparation. The number of CHIKV particles estimated based on the concentration of E2 in the extract suggested that the assay could detect clinically meaningful amounts of CHIKV in serum. Conclusions The newly discovered antibodies offer valuable tools for characterization of CHIKV isolates. Therefore, the strategy here followed using whole viral particles and ALTHEA Gold Libraries™ could expedite the discovery and development of antibodies for detection and control of emergent and quickly spreading viral outbreaks.

Valproic acid (VPA) is a drug commonly used for epileptic seizure control. Recently, it has been shown that VPA alters the activation of several immune cells, including Natural Killer (NK) cells, which play an important role in the containment of viruses and intracellular bacteria. Although VPA can increase susceptibility to extracellular pathogens, it is unknown whether the suppressor effect of VPA could affect the course of intracellular bacterial infection. This study aimed to evaluate the role of VPA during Listeria monocytogenes (L.m) infection, and whether NK cell activation was affected. We found that VPA significantly augmented mortality in L.m infected mice. This effect was associated with increased bacterial load in the spleen, liver, and blood. Concurrently, decreased levels of IFN-γ in serum and lower splenic indexes were observed. Moreover, in vitro analysis showed that VPA treatment decreased the frequency of IFN-γ-producing NK cells within L.m infected splenocytes. Similarly, VPA inhibited the production of IFN-γ by NK cells stimulated with IL-12 and IL-18, which is a crucial system for early IFN-γ production in listeriosis. Finally, VPA decreased the phosphorylation of STAT4, p65, and p38, without affecting the expression of IL-12 and IL-18 receptors. Altogether, our results indicate that VPA increases the susceptibility to Listeria monocytogenes infection and suggest that NK cell is one of the main targets of VPA, but further work is needed to ascertain this effect.

Gaining knowledge of the neoplastic side of the three main cells—B cells, Follicular Helper T (Tfh) cells, and follicular dendritic cells (FDCs) —involved in the germinal center (GC) reaction can shed light toward further understanding the microuniverse that is the GC, opening the possibility of better treatments. This paper gives a review of the more complex underlying mechanisms involved in the malignant transformations that take place in the GC. Whilst our understanding of the biology of the GC-related B cell lymphomas has increased—this is not reviewed in detail here—the dark side involving neoplasms of Tfh cells and FDCs are poorly studied, in great part, due to their low incidence. The aggressive behavior of Tfh lymphomas and the metastatic potential of FDCs sarcomas make them clinically relevant, merit further attention and are the main focus of this review. Tfh cells and FDCs malignancies can often be misdiagnosed. The better understanding of these entities linked to their molecular and genetic characterization will lead to prediction of high-risk patients, better diagnosis, prognosis, and treatments based on molecular profiles.

We recently reported, for the first time, the expression and regulation of the PDZ polarity proteins Scrib and Dlg1 in human APCs, and also described the viral targeting of these proteins by NS1 of influenza A virus in human dendritic cells (DCs). Scrib plays an important role in reactive oxygen species (ROS) production in Mϕs and uropod formation and migration in T cells, while Dlg1 is important for T cell downstream activation after Ag recognition. Nevertheless, the functions of these proteins in human DCs remain unknown. Here, we knocked-down the expression of both Scrib and Dlg1 in human DCs and then evaluated the expression of co-stimulatory molecules and cytokine production during maturation. We demonstrated that Scrib is necessary for adequate CD86 expression, while Dlg1 is important for CD83 up-regulation and IL-6 production upon maturation, suggesting that Scrib and Dlg1 participate in separate pathways in DCs. Additionally, both proteins are required for adequate IL-12 production after maturation. Furthermore, we showed that the inefficient maturation of DCs induced by Scrib or Dlg1 depletion leads to impaired T cell activation. Our results revealed the previously unknown contribution of Scrib and Dlg1 in human DCs pivotal functions, which may be able to impact innate and adaptive immune response.

Mast cell activation through the high-affinity IgE receptor (FcεRI) plays a central role in allergic reactions. FcεRI-mediated activation triggers multiple signaling pathways leading to degranulation and synthesis of different inflammatory mediators. IgE-mediated mast cell activation can be modulated by different molecules, including several drugs. Herein, we investigated the immunomodulatory activity of the histone deacetylase inhibitor valproic acid (VPA) on IgE-mediated mast cell activation. To this end, bone marrow-derived mast cells (BMMC) were sensitized with IgE and treated with VPA followed by FcεRI cross-linking. The results indicated that VPA reduced mast cell IgE-dependent degranulation and cytokine release. VPA also induced a significant reduction in the cell surface expression of FcεRI and CD117, but not other mast cell surface molecules. Interestingly, VPA treatment inhibited the phosphorylation of PLCγ2, a key signaling molecule involved in IgE-mediated degranulation and cytokine secretion. However, VPA did not affect the phosphorylation of other key components of the FcεRI signaling pathway, such as Syk, Akt, ERK1/2, or p38. Altogether, our data demonstrate that VPA affects PLCγ2 phosphorylation, which in turn decreases IgE-mediated mast cell activation. These results suggest that VPA might be a key modulator of allergic reactions and might be a promising therapeutic candidate.

Mast cells play an essential role in different immunological phenomena including allergy and infectious diseases. Several bacteria induce mast cell activation leading to degranulation and the production of several cytokines and chemokines. However, mast cells also have different microbicidal activities such as phagocytosis and the release of DNA with embedded granular proteins known as Mast Cell Extracellular Traps (MCETs). Although previous reports indicate that extracellular bacteria are able to induce MCETs little is known if intracellular bacteria can induce these structures. In this work, we evaluated MCETs induction by the intracellular bacteria Listeria monocytogenes. We found that mast cells released DNA after stimulation with L. monocytogenes, and this DNA was complexed to histone and tryptase. Before extracellular DNA release, L. monocytogenes induced modifications to the mast cell nuclear envelope and DNA was detected outside the nucleus. L. monocytogenes stimulated mast cells to produce significant amounts of reactive oxygen species (ROS) and blocking NADPH oxidase diminished DNA release by mast cells. Finally, MCETs showed antimicrobial activity against L. monocytogenes that was partially blocked when β-hexosaminidase activity was inhibited. These results show that L. monocytogenes induces mast cells to produce microbicidal MCETs, suggesting a role for mast cells in containing infection beyond the induction of inflammation.

SETTING Mexico City, Mexico. OBJECTIVE To identify proteins synthetised by Mycobacterium tuberculosis in hypoxic culture, which resemble more closely a granuloma environment than aerobic culture, and to determine if they are recognised by antibodies from patients with active pulmonary tuberculosis (PTB). DESIGN Soluble extracts from M. tuberculosis H37Rv cultured under aerobic or hypoxic conditions were analysed using two-dimensional polyacrylamide gel electrophoresis, and proteins over-expressed under hypoxia were identified by mass spectrometry. The presence of immunoglobulin (Ig) G, IgA and IgM antibodies against these proteins was determined in the serum of 42 patients with active PTB and 42 healthy controls. RESULTS We selected three M. tuberculosis H37Rv proteins (alpha-crystallin protein [Acr, Rv2031c], universal stress protein Rv2623 and isocitrate lyase [ICL, RV0467]) that were over-expressed under hypoxia. Titres of anti-Acr and anti-ICL IgA antibodies were higher in patients than in healthy controls, with an area under the receiver operating characteristic curve of 0.71 for anti-ICL IgA antibodies. CONCLUSION ICL could be used in combination with other M. tuberculosis antigens to improve the sensitivity and specificity of current serological TB diagnostic methods.

Ursolic and oleanolic acids are natural isomeric triterpenes known for their anticancer activity. Here, we investigated the effect of triterpenes on the viability of A549 human lung cancer cells and the role of autophagy in their activity. The induction of autophagy, the mitochondrial changes and signaling pathway stimulated by triterpenes were systematically explored by confocal microscopy and western blotting. Ursolic and oleanolic acids induce autophagy in A549 cells. Ursolic acid activates AKT/mTOR pathways and oleanolic acid triggers a pathway independent on AKT. Both acids promote many mitochondrial changes, suggesting that mitochondria are targets of autophagy in a process known as mitophagy. The PINK1/Parkin axis is a pathway usually associated with mitophagy, however, the mitophagy induced by ursolic or oleanolic acid is just dependent on PINK1. Moreover, both acids induce an ROS production. The blockage of autophagy with wortmannin is responsible for a decrease of mitochondrial membrane potential (&Delta, &psi, ) and cell death. The wortmannin treatment causes an over-increase of p62 and Nrf2 proteins promote a detoxifying effect to rescue cells from the death conducted by ROS. In conclusion, the mitophagy and p62 protein play an important function as a survival mechanism in A549 cells and could be target to therapeutic control.

Mariano García-Martínez,1,2 Luis Vázquez-Flores,1,2 Violeta D Álvarez-Jiménez,1,3 Jessica Castañeda-Casimiro,1 Miguel Ibáñez-Hernández,4 Luvia Enid Sánchez-Torres,1 Jorge Barrios-Payán,5 Dulce Mata-Espinosa,5 Sergio Estrada-Parra,1 Rommel Chacón-Salinas,1 Jeanet Serafín-López,1 Isabel Wong-Baeza,1 Rogelio Hernández-Pando,5 Iris Estrada-García11Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas (ENCB), Instituto Politécnico Nacional (IPN), Mexico City, Mexico; 2Posgrado en Inmunología, Escuela Nacional de Ciencias Biológicas (ENCB), Instituto Politécnico Nacional (IPN), Mexico City, Mexico; 3Laboratorio de Biología Molecular y Bioseguridad Nivel 3, Centro Médico Naval (CEMENAV), Secretaría de Marina Armada de México (Semar), Mexico City, Mexico; 4Departamento de Bioquímica, Escuela Nacional de Ciencias Biológicas (ENCB), Instituto Politécnico Nacional (IPN), Mexico City, Mexico; 5Sección de Patología Experimental, Departamento de Patología, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, MexicoCorrespondence: Iris Estrada-GarcíaDepartamento de Inmunología, Escuela Nacional de Ciencias Biológicas (ENCB), Instituto Politécnico Nacional (IPN), Prolongación de Carpio y Plan de Ayala s/n, Col. Santo Tomás, Ciudad de México 11340, MéxicoTel +52 555 729 6000 x 62507Fax +52 555 729 6000 x 62369Email iestrada5@hotmail.comBackground: Tuberculosis is the leading cause of death by an infectious microorganism worldwide. Conventional treatment lasts at least six months and has adverse effects; therefore, it is important to find therapeutic alternatives that reduce the bacterial load and may reduce the treatment duration. The immune response against tuberculosis can be modulated by several mechanisms, including extracellular vesicles (EVs), which are nano-sized membrane-bound structures that constitute an efficient communication mechanism among immune cells.Methods: The EVs released by the J774A.1 mouse macrophage cell line, both spontaneously (S-EV) and after infection with Mycobacterium tuberculosis H37Rv (Mtb-EV), were purified by ultra-centrifugation and size-exclusion chromatography. The size distribution and chemical composition of these EVs were evaluated, and their effect on the bacterial load and the production of cytokines was determined in both in vitro and in vivo models of M. tuberculosis infection.Results: Mtb-EV are larger than S-EV, they contain M. tuberculosis-specific antigens (not detected in EVs released from M. fortuitum-infected J774A.1 cells) and are rich in phosphatidylserine, present in their outer membrane layer. S-EV, but not Mtb-EV, reduced the bacterial load and the production of MCP-1 and TNF-α in M. tuberculosis-infected macrophages, and these effects were reversed when phosphatidylserine was blocked with annexin V. Both S-EV and Mtb-EV significantly reduced the lung bacterial load in mice infected with M. tuberculosis after 60days of treatment, but they had no effect on survival or on the lung pneumonic area of these mice.Conclusion: J774A.1 macrophages infected with M. tuberculosis H37Rv released EVs that differed in size and phosphatidylserine content from spontaneously released EVs, and these EVs also had different biological effects: S-EV reduced the mycobacterial load and the cytokine production in vitro (through a phosphatidylserine-dependent mechanism), while both EVs reduced the lung bacterial load in vivo. These results are the basis for further experiments to evaluate whether EVs improve the efficiency of the conventional treatment for tuberculosis.Keywords: tuberculosis, extracellular vesicles, phosphatidylserine, macrophages, lung disease

Mesenchymal stromal cells (MSCs) in the tumor microenvironment (TME) participate together with tumor cells to suppress antitumor effector cells through the production of immunosuppressive factors, such as transforming growth factor-beta 1 (TGF-β1). Furthermore, TGF-β1 can induce 5'-nucleotidase (CD73) expression in various cell types; this functional activity is associated with the production of adenosine (Ado), which is an immunosuppressive nucleoside. In this study, we provide evidence that coculture of MSCs derived from cervical tumors (CeCa-MSC) with CeCa tumor cells increases CD73 expression in tumor cells and the capacity of these cells to generate Ado in a MSC ratio-dependent manner. Interestingly, the increase in CD73 in the CeCa cell membrane corresponded to an increase in the TGF-β1 expression level in the tumor cells and the TGF-β1 content in the supernatants of the CeCa/CeCa-MSC cocultures. The addition of anti-hTGF-β neutralizing antibodies strongly reversed CD73 expression in the tumor cells. This phenomenon was not exclusive to CeCa-MSCs; coculture of MSCs derived from the normal cervix with CeCa cells produced similar results. These results suggest that the interaction of MSCs with CeCa tumor cells in the TME may condition higher TGF-β1 production to maintain an immunosuppressive status not only through the activity of this cytokine per se but also through its ability to induce CD73 expression in tumor cells and generate an immunosuppressive microenvironment rich in Ado.

Transferon® is an immunomodulator made of a complex mixture of peptides from human dialyzable leucocyte extracts (hDLEs). Development of surrogate antibodies directed to hDLE is an indispensable tool for studies during process control and preclinical trials. These antibodies are fundamental for different analytical approaches, such as identity test and drug quantitation, as well as to characterize its pharmacokinetic and mechanisms of action. A previous murine study showed the inability of the peptides of Transferon® to induce antibody production by themselves; therefore, in this work, two approaches were tested to increase its immunogenicity: chemical conjugation of the peptides of Transferon® to carrier proteins and the use of a rabbit model. Bioconjugates were generated with Keyhole Limpet Hemocyanin (KLH) or Bovine Serum Albumin (BSA) through maleimide-activated carrier proteins. BALB/c mice and New Zealand rabbits were immunized with Transferon® conjugated to KLH or nonconjugated Transferon®. Animals that were immunized with conjugated Transferon® showed significant production of antibodies as evinced by the recognition of Transferon®-BSA conjugate in ELISA assays. Moreover, rabbits showed higher antibody titers when compared with mice. Neither mouse nor rabbits developed antibodies when immunized with nonconjugated Transferon®. Interestingly, rabbit antibodies were able to partially block IL-2 production in Jurkat cells after costimulation with Transferon®. In conclusion, it is feasible to elicit specific and functional antibodies anti-hDLE with different potential uses during the life cycle of the product.

Transferon® is a complex drug based on a mixture of low molecular weight peptides. This biotherapeutic is employed as a coadjuvant in clinical trials of several diseases, including viral infections and allergies. Given that macrophages play key roles in pathogen recognition, phagocytosis, processing, and antigen presentation, we evaluated the effect of Transferon® on phenotype and function of macrophage-like cells derived from THP-1 monocytes. We determined the surface expression of CD80 and CD86 by flow cytometry and IL-1β, TNF-α, and IL-6 levels by ELISA. Transferon® alone did not alter the steady state of PMA-differentiated macrophage-like THP-1 cells. On the contrary, simultaneous stimulation of cells with Transferon® and LPS elicited a significant increase in CD80 (P≤0.001) and CD86 (P≤0.001) expression, as well as in IL-6 production (P≤0.05) compared to the LPS control. CD80 expression and IL-6 production exhibited a positive correlation (r=0.6, P≤0.05) in cells exposed to Transferon® and LPS. Our results suggest that the administration of Transferon® induces the expression of costimulatory molecules and the secretion of cytokines in LPS-activated macrophages. Further studies are necessary to determine the implication of these findings in the therapeutic properties of Transferon®.

The development of cervical cancer (CeCa) is associated with high-risk human papilloma virus (HR-HPV) infections, mainly HPV-16, which is present in more than 50% of cases. The presence of immunosuppressive factors in the early stages of the disease is also strongly linked to CeCa progression. In this context, it is unknown whether ectonucleotidases CD39 and CD73, which are involved in the production of adenosine (Ado) that suppresses the specific antitumor immune response, are present in precursor lesions of CeCa. In this pilot study, we analyzed the presence of CD39 and CD73 and their capacity to generate Ado in 25 cervical samples from patients with grade 1 cervical intraepithelial neoplasms (CIN-1) and 25 samples from normal donors (NDs) free of HPV infection. Cells obtained from cervical samples of CIN-1 patients positive for HPV-16 showed higher CD39 and CD73 contents compared to samples obtained from CIN-1 patients negative for HPV-16 and NDs. Interestingly, solubilized cervical mucus from these patients also showed higher contents of soluble CD39 and CD73, which were associated with a greater capacity to produce Ado from the hydrolysis of adenosine triphosphate (ATP) and adenosine monophosphate (AMP). In addition, serum samples of these patients showed higher levels of TGF-βthan those of CIN-1 patients negative for HPV-16 and ND. These results suggest that persistent infection with HR-HPV, mostly HPV-16, in CIN-1 patients may promote the expression of CD39 and CD73 through the production of TGF-βin precursor lesions to generate an immunosuppressive microenvironment and allow its progression to CeCa.

Tuberculosis is one of the leading causes of mortality worldwide, it is caused by Mycobacterium tuberculosis (Mtb), a bacteria that employs several strategies to evade the host immune response. For instance, Mtb interferes with the overexpression of class II transactivator (CIITA) in macrophages exposed to IFN-γ by inhibiting histone acetylation at its promoter, which can be reverted by the histone deacetylase inhibitor (HDACi) sodium butyrate. In this work, we evaluated whether a different HDACi, valproic acid (VPA), could revert the inhibition of gene expression induced by Mtb. J774 macrophages treated with VPA and IFN-γ unexpectedly induced a higher expression of the inducible nitric oxide synthase and a higher production of nitric oxide when exposed to the 19 kDa lipoprotein of Mtb or the whole bacteria. However, VPA was unable to revert the inhibition of CIITA expression induced by the 19 kDa lipoprotein of Mtb. Finally, macrophages infected with Mtb and treated with VPA and IFN-γ showed a significant reduction in intracellular bacteria. Our findings suggest a new therapeutic potential of VPA for the treatment of tuberculosis.

Valproic acid (VPA) is widely recognized for its use in the control of epilepsy and other neurological disorders in the past 50 years. Recent evidence has shown the potential of VPA in the control of certain cancers, owed in part to its role in modulating epigenetic changes through the inhibition of histone deacetylases, affecting the expression of genes involved in the cell cycle, differentiation, and apoptosis. The direct impact of VPA in cells of the immune system has only been explored recently. In this review, we discuss the effects of VPA in the suppression of some activation mechanisms in several immune cells that lead to an anti-inflammatory response. As expected, immune cells are not exempt from the effect of VPA, as it also affects the expression of genes of the cell cycle and apoptosis through epigenetic modifications. In addition to inhibiting histone deacetylases, VPA promotes RNA interference, activates histone methyltransferases, or represses the activation of transcription factors. However, during the infectious process, the effectiveness of VPA is subject to the biological nature of the pathogen and the associated immune response; this is because VPA can promote the control or the progression of the infection. Due to its various effects, VPA is a promising alternative for the control of autoimmune diseases and hypersensitivity and needs to be further explored.

The expression of CD73 in tumor cells plays a significant role in the production of adenosine (Ado) that suppresses antitumor effector cells.In this study we analyzed the capability of HPV-positive (HPV+) cervical cancer (CeCa) cell lines CaSki, SiHa, HeLa, and RoVa; and HPV-negative (HPV-) cell lines C33A and ViBo to produce Ado and inhibit effector functions of CD8+ T cells.HPV+ CeCa cells expressed significantly higher levels of CD73 in the membrane (p0.01) than HPV- CeCa cells and this expression was associated with the production of larger amounts of Ado (400μM) compared to HPV-CeCa cells (200μM) in the presence of AMP, as well asa stronger inhibition of (50%) proliferation, activation, and cytotoxic activity of CD8+ T cells via interaction with A2A adenosine receptor. We also provide evidence that silenced E6/E7 expression in CeCa cells, strongly reduced its CD73 expression level and its capability to generate Ado.This results suggest that HPV infection, which is associated with more than 99% of CeCa cases, may present an increased constitutive expression of CD73 in cervical neoplasia to contribute to the suppression of the immune response mediated by the production of large amounts of Ado.

Transferon, a human dialyzable leukocyte extract (hDLE), is a biotherapeutic that comprises a complex mixture of low-molecular-weight peptides (

Lungs are indispensable organs for the respiratory process, and maintaining their homeostasis is essential for human health and survival. However, during the lifetime of an individual, the lungs suffer countless insults that put at risk their delicate organization and function. Many cells of the immune system participate to maintain this equilibrium and to keep functional lungs. Among these cells, mast cells have recently attracted attention because of their ability to rapidly secrete many chemical and biological mediators that modulate different processes like inflammation, angiogenesis, cell proliferation, etc. In this review, we focus on recent advances in the understanding of the role that mast cells play in lung protection during infections, and of the relation of mast cell responses to type I hypersensitivity-associated pathologies. Furthermore, we discuss the potential role of mast cells during wound healing in the lung and its association with lung cancer, and how mast cells could be exploited as therapeutic targets in some diseases.

Immunoglobulin A (IgA) is the main antibody isotype present in the body fluids such as tears, intestinal mucous, colostrum and saliva. There are two subtypes of IgA in humans: IgA1 mainly present in blood and IgA2, the preferentially expressed mucosal antibody. In clinical practice, immunoglobulins are typically measured in venous or capillary blood; however, alternative samples including saliva are now being considered given its non-invasive and easy collection nature. Since IgA deficiency could be frequently detected in patients with autoimmune diseases, we decided to evaluate the levels of both IgA subtypes in serum and saliva of systemic lupus erythematosus (SLE) patients. Specific IgA1 and IgA2 levels were measured by a light chain capture-based ELISA in a cohort of 32 patients with SLE that were compared with antibody levels of healthy volunteers. Surprisingly, our results indicated that in saliva of SLE patients, both IgA subtypes were significantly elevated; however, serum IgA1 levels were decreased when compared with control subjects. Interestingly, we also found that salivary IgA levels, most specifically IgA1, positively correlate with the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) values as well with the amount of serum anti-nucleosomes and anti-dsDNA IgG autoantibodies. Strikingly, we also were able to detect the presence of salivary anti-nucleosome IgA antibodies in SLE patients, a feature that was not previously reported elsewhere. According to our results, IgA characterization in saliva could be used as a pre-diagnostic or follow-up clinical tool in SLE with easier, faster and safer to manipulate than blood samples.

Natural Killer cells (NK) are key for the innate immune response against tumors and viral infections. Several viral proteins evade host immune response and target the NK cell receptor NKG2D and its ligands. Areas covered: This review aimed to describe the viruses and their proteins that interfere with the NKG2D receptor and their ligands, and how these interactions lead to immune evasion, host protection, and tissue damage from acute and chronic viral infections. Expert opinion: The study of viral proteins has already impacted the field of oncology. A prime example is the HBV vaccine and the development of antiviral drugs for HIV, Hepatitis C, and the family of Herpesviridae viruses. The NKG2D system seems to be a rational therapeutic target. Nevertheless, an effective cytotoxic response by NK cells is mediated by a network of activating and inhibitory receptors, the integration of which determines if the NK cell becomes cytotoxic or permissive. Immunotherapeutic agents that increase the antitumor lytic activity of NK cells through modulating activation and inhibitory signaling of NK cells are being developed. Nevertheless, more research is needed to dissect the integrative mechanism of NK cells function to fully exploit their antitumor and antiviral effector mechanisms.