Use of topical rSm29 in combination with intravenous meglumine antimoniate in the treatment of cutaneous leishmaniasis: A randomized controlled trial.

BACKGROUND: Cutaneous leishmaniasis (CL) caused by Leishmania (Viannia) braziliensis is associated with an inflammatory response. Granzyme (GzmB) and IL-1ß play a key role in the pathology. Meglumine antimoniate (MA) is the first-choice drug for the treatment of CL, but therapy failure is observed in up to 50% of the cases. The protein, rSm29 of Schistosoma mansoni, down-modulates pro-inflammatory cytokine production. We evaluate if the combination of topical rSm29 plus MA increases the cure rate of CL. METHODS: In this randomized clinical trial, 91 CL patients were allocated in 3 groups. All cases received MA (20 mg/kg/weight) for 20 days. Group 1 used topical rSm29 (10 µg), group 2 a placebo topically applied, and group 3 received only MA. RESULTS: The cure rate on day 90 was 71% in subjects treated with rSm29 plus MA, and 43% in patients who received MA plus placebo or MA alone (P < 0.05). There was a decrease in GzmB and an increase in IFN-γ (P < 0.05) in supernatants of skin biopsies of the lesions obtained on D7 of therapy (P < 0.05) in patients who received rSm29. CONCLUSION: rSm29 associated with MA reduces GzmB levels, is more effective than MA alone, and decreases CL healing time. CLINICAL TRIALS REGISTRATION: ClinicalTrial.gov under NCT06000514.

Modulation of granzymes mRNA expression in neural tissue of BoAHV-1 and BoAHV-5-infected cattle.

Bovine alphaherpesviruses (BoAHV)-1 and 5 are neurotropic viruses of cattle which differ in their neuropathogenic potential. BoAHV-5 is responsible for non-suppurative meningoencephalitis in calves while BoAHV-1 can occasionally cause encephalitis. Granzymes (GZMs) are serine-proteases that participate in CD8+ T cells-mediated killing of virally-infected cells upon release through perforin (PFN)-formed pores in the cell membrane. Recently, six GZMs have been identified in cattle (A, B, K, H, M and O). However, their expression in bovine tissues has not been evaluated. In this study, the mRNA expression levels of PFN and GZMs A, B, K, H and M in the nervous system of calves experimentally-inoculated with BoAHV-1 or BoAHV-5 were analyzed at the three distinct stages of the infectious cycle of alphaherpesviruses: acute infection, latency and reactivation. This is the first report describing the expression of GZMs in bovine neural tissue and the first analysis of GZM expression in the context of bovine alphaherpesviruses neuropathogenesis. The findings revealed that PFN and GZM K are upregulated during BoAHV-1 or BoAHV-5 acute infection. In contrast to BoAHV-1, during BoAHV-5 latency a significant up-regulation of PFN, GZM K and GZM H was detected. PFN and GZM A, K and H expression was also up-regulated during BoAHV-5 reactivation. Therefore, a distinct pattern of PFN and GZM expression is evident along the infectious cycle of each alphaherpesvirus and this might contribute to the differences in BoAHV-1 and BoAHV-5 neuropathogenesis.

Differential regulatory T cell signature after recovery from mild COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is characterized by a range of symptoms in which host immune response have been associated with disease progression. However, the putative role of regulatory T cells (Tregs) in determining COVID-19 outcomes has not been thoroughly investigated. Here, we compared peripheral Tregs between volunteers not previously infected with SARS-CoV-2 (healthy control [HC]) and volunteers who recovered from mild (Mild Recovered) and severe (Severe Recovered) COVID-19. Peripheral blood mononuclear cells (PBMC) were stimulated with SARS-CoV-2 synthetic peptides (Pool Spike CoV-2 and Pool CoV-2) or staphylococcal enterotoxin B (SEB). Results of a multicolor flow cytometric assay showed higher Treg frequency and expression of IL-10, IL-17, perforin, granzyme B, PD-1, and CD39/CD73 co-expression in Treg among the PBMC from the Mild Recovered group than in the Severe Recovered or HC groups for certain SARS-CoV-2 related stimulus. Moreover, Mild Recovered unstimulated samples presented a higher Tregs frequency and expression of IL-10 and granzyme B than did that of HC. Compared with Pool CoV-2 stimuli, Pool Spike CoV-2 reduced IL-10 expression and improved PD-1 expression in Tregs from volunteers in the Mild Recovered group. Interestingly, Pool Spike CoV-2 elicited a decrease in Treg IL-17+ frequency in the Severe Recovered group. In HC, the expression of latency-associated peptide (LAP) and cytotoxic granule co-expression by Tregs was higher in Pool CoV-2 stimulated samples. While Pool Spike CoV-2 stimulation reduced the frequency of IL-10+ and CTLA-4+ Tregs in PBMC from volunteers in the Mild Recovered group who had not experienced certain symptoms, higher levels of perforin and perforin+granzyme B+ co-expression by Tregs were found in the Mild Recovered group in volunteers who had experienced dyspnea. Finally, we found differential expression of CD39 and CD73 among volunteers in the Mild Recovered group between those who had and had not experienced musculoskeletal pain. Collectively, our study suggests that changes in the immunosuppressive repertoire of Tregs can influence the development of a distinct COVID-19 clinical profile, revealing that a possible modulation of Tregs exists among volunteers of the Mild Recovered group between those who did and did not develop certain symptoms, leading to mild disease.

PD-L1/PD-1 blockage enhanced the cytotoxicity of natural killer cell on the non-small cell lung cancer (NSCLC) by granzyme B secretion.

OBJECTIVE: To explore the role of PD-L1/PD-1 blockage in the cytotoxicity of natural killer cell in NSCLC. METHODS: Two NSCLC cell lines, Calu-1 and H460, were tested for susceptibility to the cytolytic activity of freshly isolated healthy donor NK cells by a non-radioactive cellular cytotoxicity assay kit. Western blot analysis, FACS, ELISA and antibody blockage experiments were conducted to determine the mechanisms. NK cells isolated from NSCLC patients were also collected for functional assays. RESULTS: Calu-1 and H460 cells were lysed by NK cells in a dose-dependent manner. H460 cells showed less susceptibility to NK cell-mediated lysis than Calu-1 cells at all ratios. The expression of PD-L1 on H460 cells was higher than that on Calu-1 cells, as determined by FACS and western blot analysis. The specific lysis of H460 cells by NK cells was enhanced when the PD-L1/PD-1 interaction was blocked by anti-PD-L1 antibody. This finding was also demonstrated in NK cells isolated from NSCLC patients. CONCLUSIONS: The present study revealed that PD-L1/PD-1 blockage enhanced the cytotoxicity of natural killer cells in NSCLC via granzyme B secretion. This study will greatly facilitate the precise treatment of lung cancer through determination of PD-L1 expression in tumors.

Identification of CD8+ T cell subsets that normalize in early-treated people living with HIV receiving antiretroviral therapy.

BACKGROUND: Although combined antiretroviral therapy (cART) has decreased the mortality associated with HIV infection, complete immune reconstitution is not achieved despite viral suppression. Alterations of CD8+ T cells and some of their subpopulations, such as interleukin (IL)-17-producing cells, are evidenced in treated individuals and are associated with systemic inflammation and adverse disease outcomes. We sought to evaluate if different CD8+ T cell subsets are differentially normalized during a clinical follow-up of people living with HIV (PLWH) receiving suppressive cART. METHODS: We explored the changes in the frequencies, activation/exhaustion phenotypes (HLA-DR, CD38, PD-1, and TIM-3), and function (total and HIV-specific cells expressing CD107a, perforin, granzyme B, interferon [IFN]-γ and IL-17) of CD8+ T cells from early-treated PLWH receiving cART in a 1-year follow-up, using a multidimensional flow cytometry approach. RESULTS: Despite continuous cART-induced viral suppression and recovery of CD4+ T cells, after a 1-year follow-up, the CD8+ T cell counts, CD4:CD8 ratio, PD-1 expression, and IL-17 production by CD8+ T cells exhibited incomplete normalization compared with seronegative controls. However, the proportion of CD8+ T cells with an exhausted phenotype (co-expressing PD-1 andTIM-3), and cells co-expressing cytotoxic molecules (Perforin and Granzyme B), reached normalization. CONCLUSIONS: Although suppressive cART achieves normalization of CD4+ T cell counts, only particular subsets of CD8+ T cells are more rapidly normalized in PLWH receiving cART, which could be routinely used as biomarkers for therapy efficiency in these patients.

Immune characterization of a Colombian family cluster with SARS-CoV-2 infection. / Caracterización inmunológica de un grupo familiar colombiano con infección por SARS-CoV-2

INTRODUCTION: Immunological markers have been described during COVID-19 and persist after recovery. These immune markers are associated with clinical features among SARSCoV-2 infected individuals. Nevertheless, studies reporting a comprehensive analysis of the immune changes occurring during SARS-CoV-2 infection are still limited. OBJECTIVE: To evaluate the production of proinflammatory cytokines, the antibody response, and the phenotype and function of NK cells and T cells in a Colombian family cluster with SARS-CoV-2 infection. MATERIALS AND METHODS: Proinflammatory cytokines were evaluated by RT-PCR and ELISA. The frequency, phenotype, and function of NK cells (cocultures with K562 cells) and T-cells (stimulated with spike/RdRp peptides) were assessed by flow cytometry. Anti-SARS-CoV-2 antibodies were determined using indirect immunofluorescence and plaque reduction neutralization assay. RESULTS: During COVID-19, we observed a high proinflammatory-cytokine production and a reduced CD56bright-NK cell and cytotoxic response. Compared with healthy controls, infected individuals had a higher frequency of dysfunctional CD8+ T cells CD38+HLA-DR-. During the acute phase, CD8+ T cells stimulated with viral peptides exhibited a monofunctional response characterized by high IL-10 production. However, during recovery, we observed a bifunctional response characterized by the co-expression of CD107a and granzyme B or perforin. CONCLUSION: Although the proinflammatory response is a hallmark of SARS-CoV-2 infection, other phenotypic and functional alterations in NK cells and CD8+ T cells could be associated with the outcome of COVID-19. However, additional studies are required to understand these alterations and to guide future immunotherapy strategies.

Introducción. Se han descrito diferentes marcadores inmunológicos durante la COVID-19, los cuales persisten incluso después de la convalecencia y se asocian con los estadios clínicos de la infección. Sin embargo, aún son pocos los estudios orientados al análisis exhaustivo de las alteraciones del sistema inmunológico en el curso de la infección. Objetivo. Evaluar la producción de citocinas proinflamatorias, la reacción de anticuerpos, y el fenotipo y la función de las células NK y los linfocitos T en una familia colombiana con infección por SARS-CoV-2. Materiales y métodos. Se evaluaron las citocinas proinflamatorias mediante RT-PCR y ELISA; la frecuencia, el fenotipo y la función de las células NK (en cocultivos con células K562) y linfocitos T CD8+ (estimulados con péptidos spike/RdRp) mediante citometría de flujo, y los anticuerpos anti-SARS-CoV-2, mediante inmunofluorescencia indirecta y prueba de neutralización por reducción de placa. Resultados. Durante la COVID-19 hubo una producción elevada de citocinas proinflamatorias, con disminución de las células NK CD56bright y reacción citotóxica. Comparados con los controles sanos, los individuos infectados presentaron con gran frecuencia linfocitos T CD8+ disfuncionales CD38+HLA-DR-. Además, en los linfocitos T CD8+ estimulados con péptidos virales, predominó una reacción monofuncional con gran producción de IL-10 durante la fase aguda y una reacción bifuncional caracterizada por la coexpresión de CD107a y granzima B o perforina durante la convalecencia. Conclusión. Aunque la reacción inflamatoria caracteriza la infección por SARS-CoV-2, hay otras alteraciones fenotípicas y funcionales en células NK y linfocitos T CD8+ que podrían asociarse con la progresión de la infección. Se requieren estudios adicionales para entender estas alteraciones y guiar futuras estrategias de inmunoterapia.

PepFun: Open Source Protocols for Peptide-Related Computational Analysis.

Peptide research has increased during the last years due to their applications as biomarkers, therapeutic alternatives or as antigenic sub-units in vaccines. The implementation of computational resources have facilitated the identification of novel sequences, the prediction of properties, and the modelling of structures. However, there is still a lack of open source protocols that enable their straightforward analysis. Here, we present PepFun, a compilation of bioinformatics and cheminformatics functionalities that are easy to implement and customize for studying peptides at different levels: sequence, structure and their interactions with proteins. PepFun enables calculating multiple characteristics for massive sets of peptide sequences, and obtaining different structural observables derived from protein-peptide complexes. In addition, random or guided library design of peptide sequences can be customized for screening campaigns. The package has been created under the python language based on built-in functions and methods available in the open source projects BioPython and RDKit. We present two tutorials where we tested peptide binders of the MHC class II and the Granzyme B protease.

γδ T cells suppress Plasmodium falciparum blood-stage infection by direct killing and phagocytosis.

Activated Vγ9Vδ2 (γδ2) T lymphocytes that sense parasite-produced phosphoantigens are expanded in Plasmodium falciparum-infected patients. Although previous studies suggested that γδ2 T cells help control erythrocytic malaria, whether γδ2 T cells recognize infected red blood cells (iRBCs) was uncertain. Here we show that iRBCs stained for the phosphoantigen sensor butyrophilin 3A1 (BTN3A1). γδ2 T cells formed immune synapses and lysed iRBCs in a contact, phosphoantigen, BTN3A1 and degranulation-dependent manner, killing intracellular parasites. Granulysin released into the synapse lysed iRBCs and delivered death-inducing granzymes to the parasite. All intra-erythrocytic parasites were susceptible, but schizonts were most sensitive. A second protective γδ2 T cell mechanism was identified. In the presence of patient serum, γδ2 T cells phagocytosed and degraded opsonized iRBCs in a CD16-dependent manner, decreasing parasite multiplication. Thus, γδ2 T cells have two ways to control blood-stage malaria-γδ T cell antigen receptor (TCR)-mediated degranulation and phagocytosis of antibody-coated iRBCs.

Evaluation of the Immune Response Induced by CoronaVac 28-Day Schedule Vaccination in a Healthy Population Group.

CoronaVac vaccine from Sinovac Life Science is currently being used in several countries. In Chile, the effectiveness of preventing hospitalization is higher than 80% with a vaccination schedule. However, to date, there are no data about immune response induction or specific memory. For this reason, we recruited 15 volunteers without previous suspected/diagnosed COVID-19 and with negative PCR over time to evaluate the immune response to CoronaVac 28 and 90 days after the second immunization (dpi). The CoronaVac administration induces total and neutralizing anti-spike antibodies in all vaccinated volunteers at 28 and 90 dpi. Furthermore, using ELISpot analysis to assay cellular immune responses against SARS-CoV-2 spike protein, we found an increase in IFN-gamma- and Granzyme B-producing cells in vaccinated volunteers at 28 and 90 dpi. Together, our results indicate that CoronaVac induces a robust humoral immune response and cellular immune memory of at least 90 dpi.

Granzyme B Produced by Natural Killer Cells Enhances Inflammatory Response and Contributes to the Immunopathology of Cutaneous Leishmaniasis.

BACKGROUND: Skin lesions from patients infected with Leishmania braziliensis has been associated with inflammation induced by cytotoxic CD8+ T cells. In addition, CD8+ T cell-mediated cytotoxicity has not been linked to parasite killing. Meanwhile, the cytotoxic role played by natural killer (NK) cells in cutaneous leishmaniasis (CL) remains poorly understood. METHODS: In this study, we observed higher frequencies of NK cells in the peripheral blood of CL patients compared with healthy subjects, and that NK cells expressed more interferon-γ, tumor necrosis factor (TNF), granzyme B, and perforin than CD8+ T cells. RESULTS: We also found that most of the cytotoxic activity in CL lesions was triggered by NK cells, and that the high levels of granzyme B produced in CL lesions was associated with larger lesion size. Furthermore, an in vitro blockade of granzyme B was observed to decrease TNF production. CONCCLUSIONS: Our data, taken together, suggest an important role by NK cells in inducing inflammation in CL, thereby contributing to disease immunopathology.

M1-like macrophage polarization prevails in young children with classic Hodgkin Lymphoma from Argentina.

The microenvironment in classical Hodgkin lymphoma (cHL) comprises a mixture of different types of cells, which are responsible for lymphoma pathogenesis and progression. Even though microenvironment composition in adult cHL has been largely studied, only few groups studied pediatric cHL, in which both Epstein Barr virus (EBV) infection and age may display a role in their pathogenesis. Furthermore, our group described that EBV is significantly associated with cHL in Argentina in patients under the age of 10 years old. For that reason, our aim was to describe the microenvironment composition in 46 pediatric cHL patients. M1-like polarization status prevailed in the whole series independently of EBV association. On the other hand, in children older than 10 years, a tolerogenic environment illustrated by higher FOXP3 expression was proved, accompanied by a macrophage polarization status towards M2. In contrast, in children younger than 10 years, M1-like was prevalent, along with an increase in cytotoxic GrB+ cells. This study supports the notion that pediatric cHL exhibits a particular tumor microenvironment composition.

Phenotypic and functional changes in gamma delta T lymphocytes from HTLV-1 carriers.

Human T-cell lymphotropic virus type-1 (HTLV-1) is the etiologic agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), which is a chronic inflammatory disease that leads to gradual loss of motor movement as a result of the death of spinal cord cells through immune mediated mechanisms. The risk to develop HAM/TSP disease positively correlates with the magnitude of HTLV-1 proviral load. Gamma-delta T lymphocytes have been recognized as important players in a variety of infectious diseases. Therefore, we have investigated interactions between HTLV-1 infection and γδ T lymphocytes during HAM/TSP. Similar frequencies of total γδ T lymphocytes and their Vγ9δ2+ and Vγ9δ2neg subpopulations were observed in HAM/TSP patients. However, T lymphocytes obtained from HTLV-1 carriers displayed significantly higher rates of spontaneous proliferation and NKp30 expression when compared to cells from uninfected donors. In addition, an important decrease in the frequency of granzyme B+ γδ T lymphocytes (approximately 50%) was observed in HAM/TSP patients. Higher proportion of IFN-γ+ γδ T lymphocytes was found in HTLV-1-infected patients, which positively correlated with the HTLV-1 proviral load in peripheral blood mononuclear cells. Collectively, our data indicates that HTLV-1 infection leads to phenotypic and functional changes in the population of γδ T lymphocyte population, suggesting that HTLV-1 infection modulates functions associated to these cells, which might be involved in controlling the infection or in the development of HTLV-1-associated diseases.

Functional capacity of natural killer cells in HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients.

BACKGROUND: Natural killer (NK) cells are part of the innate immune system and provide surveillance against viruses and cancers. The ability of NK cells to kill virus-infected cells depends on the balance between the effects of inhibitory and activating NK cell receptors. This study aimed to investigate the phenotypic profile and the functional capacity of NK cells in the context of HTLV-1 infection. METHODS: This cross-sectional study sequentially recruited HTLV-1 infected individuals with HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) and asymptomatic HTLV-1 (AS) from the Integrated and Multidisciplinary HTLV Center in Salvador, Brazil. Blood samples from healthy blood donors served as controls. NK cell surface receptors (NKG2D, KIR2DL2/KIR2DL3, NKp30, NKG2A, NKp46, TIM-3 and PD-1), intracellular cytolytic (Granzyme B, perforin) and functional markers (CD107a for degranulation, IFN-γ) were assayed by flow cytometry in the presence or absence of standard K562 target cells. In addition, cytotoxicity assays were performed in the presence or absence of anti-NKp30. RESULTS: The frequency of NKp30+ NK cells was significantly decreased in HAM/TSP patients [58%, Interquartile Range (IQR) 30-61] compared to controls (73%, IQR 54-79, p = 0.04). The production of cytolytic (perforin, granzyme B) and functional markers (CD107a and IFN-γ) was higher in unstimulated NK cells from HAM/TSP and AS patients compared to controls. By contrast, stimulation with K562 target cells did not alter the frequency of CD107a+ NK cells in HAM/TSP subjects compared to the other groups. Blockage of the NKp30 receptor was shown to decrease cytotoxic activity (CD107a) and IFN-γ expression only in asymptomatic HTLV-1-infected individuals. CONCLUSIONS: NK cells from individuals with a diagnosis of HAM/TSP present decreased expression of the activating receptor NKp30, in addition to elevated degranulation activity that remained unaffected after blocking the NKp30 receptor.

Human T Lymphocytes Are Permissive for Dengue Virus Replication.

Dengue virus (DV) infection can cause either a self-limiting flu-like disease or a threatening hemorrhage that may evolve to shock and death. A variety of cell types, such as dendritic cells, monocytes, and B cells, can be infected by DV. However, despite the role of T lymphocytes in the control of DV replication, there remains a paucity of information on possible DV-T cell interactions during the disease course. In the present study, we have demonstrated that primary human naive CD4+ and CD8+ T cells are permissive for DV infection. Importantly, both T cell subtypes support viral replication and secrete viable virus particles. DV infection triggers the activation of both CD4+ and CD8+ T lymphocytes, but preactivation of T cells reduces the susceptibility of T cells to DV infection. Interestingly, the cytotoxicity-inducing protein granzyme A is highly secreted by human CD4+ but not CD8+ T cells after exposure to DV in vitro Additionally, using annexin V and polycaspase assays, we have demonstrated that T lymphocytes, in contrast to monocytes, are resistant to DV-induced apoptosis. Strikingly, both CD4+ and CD8+ T cells were found to be infected with DV in acutely infected dengue patients. Together, these results show that T cells are permissive for DV infection in vitro and in vivo, suggesting that this cell population may be a viral reservoir during the acute phase of the disease.IMPORTANCE Infection by dengue virus (DV) causes a flu-like disease that can evolve to severe hemorrhaging and death. T lymphocytes are important cells that regulate antibody secretion by B cells and trigger the death of infected cells. However, little is known about the direct interaction between DV and T lymphocytes. Here, we show that T lymphocytes from healthy donors are susceptible to infection by DV, leading to cell activation. Additionally, T cells seem to be resistant to DV-induced apoptosis, suggesting a potential role as a viral reservoir in humans. Finally, we show that both CD4+ and CD8+ T lymphocytes from acutely infected DV patients are infected by DV. Our results raise new questions about DV pathogenesis and vaccine development.

TLR3 Is a Negative Regulator of Immune Responses Against Paracoccidioides brasiliensis.

Toll-like receptors (TLRs) comprise the best-characterized pattern-recognition receptor (PRR) family able to activate distinct immune responses depending on the receptor/adaptor set assembled. TLRs, such as TLR2, TLR4 and TLR9, and their signaling were shown to be important in Paracoccidioides brasiliensis infections. However, the role of the endosomal TLR3 in experimental paracoccidioidomycosys remains obscure. In vitro assays, macrophages of the bone marrow of WT or TLR3-/- mice were differentiated for evaluation of their microbicidal activity. In vivo assays, WT or TLR3-/- mice were infected intratracheally with Paracoccidioides brasiliensis yeasts for investigation of the lung response type induced. The cytotoxic activity of CD8+ T cells was assessed by cytotoxicity assay. To confirm the importance of CD8+ T cells in the control of infection in the absence of tlr3, a depletion assay of these cells was performed. Here, we show for the first time that TLR3 modulate the infection against Paracoccidioides brasiliensis by dampening pro-inflammatory response, NO production, IFN+CD8+T, and IL-17+CD8+T cell activation and cytotoxic function, associated with granzyme B and perforin down regulation. As conclusion, we suggest that TLR3 could be used as an escape mechanism of the fungus in an experimental paracoccidioidomycosis.

Evaluation of the cytotoxic response mediated by perforin and granzyme B in patients with non-Hodgkin lymphoma.

This study quantified the perforin and granzyme B in patients with non-Hodgkin lymphoma (NHL) at the time of diagnosis. Protein quantification was performed by flow cytometry. NHL patients had a higher number of cytotoxic T lymphocytes (CTLs) expressing perforin as well as a greater number of activated CTLs than the control group. However, intracellular perforin levels in natural killer cells were lower in the NHL patients compared to the control group. Quantitative real time PCR showed that patients had more expression of perforin and granzyme B transcripts compared to the control group. In addition, patients who had expression of both genes below the median found for the NHL group had lower survival rates. Considering this, we believe that perforin and granzyme B are potential prognostic markers in NHL and thus it is fundamental to pay attention to their expressions in these patients.

Dendritic cells, macrophages, NK and CD8+ T lymphocytes play pivotal roles in controlling HSV-1 in the trigeminal ganglia by producing IL1-beta, iNOS and granzyme B.

BACKGROUND: Herpes simplex virus type 1 (HSV-1) cause not only mild symptoms but also blindness and encephalitis. It was previously shown that the immune response against HSV-1 occurs mainly in the trigeminal ganglia (TG) and that Toll-like receptors 2 and 9 (TLR2/9) are important in mediating this response. It was also demonstrated that iNOS (nitric oxide synthase) and interleukin 1 beta (IL-1ß) play an essential role in the defense against HSV-1 infection. Importantly, the present work aimed to identify the primary cells responsible for iNOS and IL-1ß production and search for other important molecules and cells that might or might not depend on TLR2/9 receptors to mediate the immune response against HSV-1. METHODS: C57BL/6 (wild type, WT) and TLR2/9-/- mice were infected by the intranasal route with HSV-1 (1 × 106 p.f.u.). Cells were obtained from the TG and spleen tissues and the profile of immune cells was determined by flow cytometry in infected and mock infected WT and knockout mice. The percentage of cells producing iNOS, IL-1ß, granzyme B and perforin was also determined by flow cytometry. Chemokine monocyte chemoattractant protein-1 (MCP1) was measured by Cytometric Bead Array (CBA) in the TG, spleen and lung. Expression of type I interferons (IFNs), interleukins (IL) 5 and 10, IL-1ß and granzyme B were quantified by real time PCR. RESULTS: The results indicate that dendritic cells (DCs) and monocytes/macrophages (Mo/Mϕ) were the main sources of IL-1ß and iNOS, respectively, which, together with type I IFNs, were essential for the immune response against HSV-1. Additionally, we showed that granzyme B produced by CD8+ T and NK lymphocytes and MCP-1 were also important for this immune response. Moreover, our data indicate that the robust production of MCP-1 and granzyme B is either TLR-independent or down regulated by TLRs and occurs in the TG of TLR2/9-/- infected mice. CONCLUSION: Taken together, our data provide strong evidence that the responses mediated by DCs, Mo/Mϕ, NK and CD8+ T lymphocytes through IL-1ß, iNOS and granzyme B production, respectively, together with the production of type I IFN early in the infection, are crucial to host defense against HSV-1.

Regulatory T Cells: Molecular Actions on Effector Cells in Immune Regulation.

T regulatory cells play a key role in the control of the immune response, both in health and during illness. While the mechanisms through which T regulatory cells exert their function have been extensively described, their molecular effects on effector cells have received little attention. Thus, this revision is aimed at summarizing our current knowledge on those regulation mechanisms on the target cells from a molecular perspective.

Increased circulating macrophage migration inhibitory factor levels are associated with coronary artery disease

BACKGROUND: To evaluate the macrophage migration inhibitory factor and E-selectin levels in patients with acute coronary syndrome. MATERIALS/METHODS: We examined the plasma migration inhibitory factor and E-selectin levels in 87 patients who presented with chest pain at our hospital. The patients were classified into two groups according to their cardiac status. Sixty-five patients had acute myocardial infarction, and 22 patients had non-cardiac chest pain (non-coronary disease). We designated the latter group of patients as the control group. The patients who presented with acute myocardial infarction were further divided into two subgroups: ST-elevated myocardial infarction (n = 30) and non-ST elevated myocardial infarction (n = 35). RESULTS: We found higher plasma migration inhibitory factor levels in both acute myocardial infarction subgroups than in the control group. However, the E-selectin levels were similar between the acute myocardial infarction and control patients. In addition, we did not find a significant difference in the plasma migration inhibitory factor levels between the ST elevated myocardial infarction and NST-elevated myocardial infarction subgroups. DISCUSSION: The circulating concentrations of migration inhibitory factor were significantly increased in acute myocardial infarction patients, whereas the soluble E-selectin levels were similar between acute myocardial infarction patients and control subjects. Our results suggest that migration inhibitory factor may play a role in the atherosclerotic process. .

Spontaneous resolution of acute rejection and tolerance induction with IL-2 fusion protein in vascularized composite allotransplantation.

Vascularized composite allotransplantation (VCA) has emerged as a treatment option for treating nonlife-threatening conditions. Therefore, in order to make VCA a safe reconstruction option, there is a need to minimize immunosuppression, develop tolerance-inducing strategies and elucidate the mechanisms of VCA rejection and tolerance. In this study we explored the effects of hIL-2/Fc (a long-lasting human IL-2 fusion protein), in combination with antilymphocyte serum (ALS) and short-term cyclosporine A (CsA), on graft survival, regulatory T cell (Treg) proliferation and tolerance induction in a rat hind-limb transplant model. We demonstrate that hIL-2/Fc therapy tips the immune balance, increasing Treg proliferation and suppressing effector T cells, and permits VCA tolerance as demonstrated by long-term allograft survival and donor-antigen acceptance. Moreover, we observe two distinct types of acute rejection (AR), progressive and reversible, within hIL-2/Fc plus ALS and CsA treated recipients. Our study shows differential gene expression profiles of FoxP3 versus GzmB, Prf1 or interferon-γ in these two types of AR, with reversible rejection demonstrating higher Treg to Teff gene expression. This correlation of gene expression profile at the first clinical sign of AR with VCA outcomes can provide the basis for further inquiry into the mechanistic aspects of VCA rejection and future drug targets.