Lymphocyte subset analyses in healthy adults vaccinated with yellow fever 17DD virus

Submitted by Sandra Infurna ([email protected]) on 2019-11-28T13:17:55Z No. of bitstreams: 1 JaciaraRamos_AlvaroBertho_etal_IOC_2005.pdf: 249028 bytes, checksum: 1b8458b7ca216f02f62f536bcc52ba4f (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2019-11-28T13:34:33Z (GMT) No. of bitstreams: 1 JaciaraRamos_AlvaroBertho_etal_IOC_2005.pdf: 249028 bytes, checksum: 1b8458b7ca216f02f62f536bcc52ba4f (MD5)Made available in DSpace on 2019-11-28T13:34:33Z (GMT). No. of bitstreams: 1 JaciaraRamos_AlvaroBertho_etal_IOC_2005.pdf: 249028 bytes, checksum: 1b8458b7ca216f02f62f536bcc52ba4f (MD5) Previous issue date: 2005Fundação Oswaldo Cruz. Bio-Manguinhos. Laboratório de Tecnologia Imunológica. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Imunologia. Laboratório de Imunoparasitologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Bio-Manguinhos. Laboratório de Tecnologia Imunológica. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Imunologia. Laboratório de Imunoparasitologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Bio-Manguinhos. Laboratório de Tecnologia Imunológica. Rio de Janeiro, RJ, Brasil.In this study the kinetics of humoral and cellular immune responses in first-time vaccinees and re-vaccinees with the yellow fever 17DD vaccine virus was analyzed. Flow cytometric analyses were used to determine percentual values of T and B cells in parallel to the yellow fever neutralizing antibody production. All lymphocyte subsets analyzed were augmented around the 30th post vaccination day, both for first-time vaccinees and re-vaccinees. CD3+ T cells increased from 30.8% (SE +/- 4%) to 61.15% (SE +/- 4.2%), CD4+ T cells from 22.4% (SE +/- 3.6%) to 39.17% (SE +/- 2%) with 43% of these cells corresponding to CD4+CD45RO+ T cells, CD8+ T cells from 15.2% (SE +/- 2.9%) to 27% (SE +/- 3%) with 70% corresponding to CD8+CD45RO+ T cells in first-time vaccinees. In re-vaccinees, the CD3+ T cells increased from 50.7% (SE +/- 3%) to 80% (SE +/- 2.3%), CD4+ T cells from 24.9% (SE +/- 1.4%) to 40% (SE +/- 3%) presenting a percentage of 95% CD4+CD45RO+ T cells, CD8+ T cells from 19.7% (SE +/- 1.8%) to 25% (SE +/- 2%). Among CD8+CD38+ T cells there could be observed an increase from 15 to 41.6% in first-time vaccinees and 20.7 to 62.6% in re-vaccinees. Regarding neutralizing antibodies, the re-vaccinees presented high titers even before re-vaccination. The levels of neutralizing antibodies of first-time vaccinees were similar to those presented by re-vaccinees at day 30 after vaccination, indicating the success of primary vaccination. Our data provide a basis for further studies on immunological behavior of the YF 17DD vaccine

Viability Analysis of Biomphalaria glabrata Hemocytes During Schistosoma mansoni Infection And Glyphosate-Based Herbicide Exposure

Pesticides are chemical agents that have a range of harmful effects on human health and the environment. They can causedrastic changes in natural communities, such as macroinvertebrates, plankton and fish. Snails, including Biomphalariaglabrata, are often present in aquatic communities and have multiple roles in limnic ecosystems. B. glabrata is an intermediatehost of several species of helminths of medical and veterinary importance, such as Schistosoma mansoni, the etiological agentof schistosomiasis. The original Roundup® herbicide can affect snails and directly affects S. mansoni hemocytes, cells thatact in the snails’ immune defense. Here we analyzed the effect of herbicide exposure on B. glabrata hemocytes divided intofour groups: control group, infected-only group, treated-only group and infected+treated group. For this, flow cytometry andNeubauer-chamber counting were used to determine the morphology, viability and lectin expression profiles of hemocytes.We observed that the group infected by S. mansoni and treated with herbicide had a higher concentration of dead hemocytesin relation to the other groups. The treated group showed similar results to the control group, suggesting that the herbicide(Roundup™) alone does not interfere with the snails’ immune system. Regarding cellular-morphological-characterizationanalysis, hyalinocytes were the cells most commonly found in all groups studied. These findings suggest that S. mansoniinfection and exposure to pesticides directly the immune system of the snails, stimulating the production of hemocytes,especially hyalinocytes, which have a high phagocytic power to quell the infection, but with toxic effects on the snail

CD3+CD4negCD8neg (double negative) T lymphocytes and NKT cells as the main cytotoxic-related-CD107a+ cells in lesions of cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis

Abstract Background Cutaneous leishmaniasis (CL) is caused by Leishmania (Viannia) braziliensis, which infects dermal macrophages and dendritic cells, causing an intense immune-mediated-tissue inflammation and a skin ulcer with elevated borders that can heal spontaneously or after antimonial therapy. The resolution of lesions depends on an adaptive immune response, and cytotoxic cells seem to have a fundamental role in this process. The aim of this study is to better understand the role of cytotoxicity mediated mechanisms that occur during the immune response in the CL lesion milieu, considering distinct cytotoxic-related CD107a+ cells, such as CD8+, CD4+, CD4neg CD8neg (double-negative, DN) and CD4+CD8+ (double-positive, DP) T lymphocytes, as well as NK and NKT cells. Methods Lesion derived cells were assessed for T cell subpopulations and NK cells, as well as CD107a expression by flow cytometry. In addition, cytometric bead array (CBA) was used to quantify cytokines and granzyme B concentrations in supernatants from macerated lesions. Results Flow cytometry analyses revealed that NKT cells are the major CD107a-expressing cell population committed to cytotoxicity in CL lesion, although we also observed high frequencies of CD4+ and DN T cells expressing CD107a. Analysing the pool of CD107a+-cell populations, we found a higher distribution of DN T cells (44%), followed by approximately 25% of NKT cells. Interestingly, NK and CD8+ T cells represented only 3 and 4% of the total-CD107a+-cell pool, respectively. Conclusions The cytotoxicity activity that occurs in the lesion milieu of CL patients seems to be dominated by DN T and NKT cells. These findings suggest the need for a reevaluation of the role of classical-cytotoxic NK and CD8+ T cells in the pathogenesis of CL, implicating an important role for other T cell subpopulations

T-cell receptor Vβ repertoire of CD8⁺ T-lymphocyte subpopulations in cutaneous leishmaniasis patients from the state of Rio de Janeiro, Brazil

In human cutaneous leishmaniasis (CL), the immune response is mainly mediated by T-cells. The role of CD8+ T-lymphocytes, which are related to healing or deleterious functions, in affecting clinical outcome is controversial. The aim of this study was to evaluate T-cell receptor diversity in late-differentiated effector (LDE) and memory CD8+ T-cell subsets in order to create a profile of specific clones engaged in deleterious or protective CL immune responses. Healthy subjects, patients with active disease (PAD) and clinically cured patients were enrolled in the study. Total CD8+ T-lymphocytes showed a disturbance in the expression of the Vβ2, Vβ9, Vβ13.2, Vβ18 and Vβ23 families. The analyses of CD8+T-lymphocyte subsets showed high frequencies of LDE CD8+T-lymphocytes expressing Vβ12 and Vβ22 in PAD, as well as effector-memory CD8+ T-cells expressing Vβ22. We also observed low frequencies of effector and central-memory CD8+ T-cells expressing Vβ2 in PAD, which correlated with a greater lesion size. Particular Vβ expansions point to CD8+ T-cell clones that are selected during CL immune responses, suggesting that CD8+ T-lymphocytes expressing Vβ12 or Vβ22 are involved in a LDE response and that Vβ2 contractions in memory CD8+T-cells are associated with larger lesions.</p

Flow Cytometry as a Tool for Quality Control of Fluorescent Conjugates Used in Immunoassays

Submitted by Sandra Infurna ([email protected]) on 2017-03-28T15:46:20Z No. of bitstreams: 1 alvaro_bertho_etal_IOC_2016.pdf: 2901056 bytes, checksum: 7d5255b1b013920484bc8b0918988719 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2017-03-28T16:01:38Z (GMT) No. of bitstreams: 1 alvaro_bertho_etal_IOC_2016.pdf: 2901056 bytes, checksum: 7d5255b1b013920484bc8b0918988719 (MD5)Made available in DSpace on 2017-03-28T16:01:38Z (GMT). No. of bitstreams: 1 alvaro_bertho_etal_IOC_2016.pdf: 2901056 bytes, checksum: 7d5255b1b013920484bc8b0918988719 (MD5) Previous issue date: 2016Fundação Oswaldo Cruz. Instituto de Tecnologia Imunobiológica - Farmanguinhos. Laboratório de Tecnologia de Diagnóstico. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Tecnologia Imunobiológica - Farmanguinhos. Laboratório de Tecnologia de Diagnóstico. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Tecnologia Imunobiológica - Farmanguinhos. Laboratório de Tecnologia de Diagnóstico. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Tecnologia Imunobiológica - Farmanguinhos. Laboratório de Tecnologia de Diagnóstico. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunoparasitologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunologia Clínica. Rio de Janeiro, RJ. Brasil.The use of antibodies in immunodiagnostic kits generally implies the conjugation of these proteins with other molecules such as chromophores or fluorochromes. The development of more sensitive quality control procedures than spectrophotometry is essential to assure the use of better fluorescent conjugates since the fluorescent conjugates are critical reagents for a variety of immunodiagnostic kits. In this article, we demonstrate a new flow cytometric protocol to evaluate conjugates by molecules of equivalent soluble fluorochromes (MESF) and by traditional flow cytometric analysis. We have coupled microspheres with anti-IgG-PE and anti-HBSAg-PE conjugates from distinct manufactures and/or different lots and evaluated by flow cytometry. Their fluorescence intensities were followed for a period of 18 months. Our results showed that there was a great difference in the fluorescence intensities between the conjugates studied. The differences were observed between manufactures and lots from both anti-IgG-PE and anti-HBSAg-PE conjugates. Coefficients of variation (CVs) showed that this parameter can be used to determine better coupling conditions, such as homogenous coupling. The MESF analysis, as well as geometric mean evaluation by traditional flow cytometry, showed a decrease in the values for all conjugates during the study and were indispensable tools to validate the results of stability tests. Our data demonstrated the feasibility of the flow cytometric method as a standard quality control of immunoassay kits

Contribution of Leishmania braziliensis antigen-specific CD4+ T, CD8+ T, NK and CD3+CD56+NKT cells in the immunopathogenesis of cutaneous leishmaniasis patients: Cytotoxic, activation and exhaustion profiles.

The pathogenesis of cutaneous leishmaniasis (CL) caused by Leishmania (Viannia) braziliensis is dictated mainly by the immune-mediated-tissue inflammation developed. The understanding of the immunological mechanisms that generate tissue damage or resolution of lesions is the key to the development of effective vaccine protocols and proper therapeutic schemes. It is clear that the specific immune response mediated by T cells is responsible for the beneficial outcome of the disease, however, the roles of CD4+ T, CD8+ T, NK and NKT cell subpopulations in immunopathogenesis of CL need to be elucidated. Peripheral blood cells from patients before, during and after the antimonial therapy, as well as healthy individuals (HI) were cultured with (LbAgS) or without (NS) L. braziliensis antigens (LbAg). Afterwards, the frequencies of LbAg-specific-cytotoxic CD8+ T, CD4+ T, NK and CD3+CD56+ NKT cells, as well as their activation and exhaustion profiles, were defined by flow cytometry. We observed higher frequencies of CD8+ T, NK and CD3+CD56+ NKT cells and lower frequencies of CD4+ T lymphocytes in LbAgS cell cultures from patients before treatment. The specific response to LbAg resulted in an expansion of cytotoxic-activated CD4+ T, CD8+ T, and NK cells, before and during treatment, indicating specificity in the response by these cells against L. braziliensis. Furthermore, comparing the differences of frequencies of cytotoxic-activated CD4+T, CD8+T, and NK cells, among before and during treatment patients and HI groups, we conclude that these cell populations are in charge of immune response elicited by antimonial therapy. Interestingly, we also observed that NK cells were induced by LbAg to an exhaustion profile during all clinical stages of the disease. The increased antigen-specific activation and cytotoxic activity are in line with the strong inflammatory response described in this disease, a likely cause of tissue damage. These findings reinforce the involvement of these distinct cytotoxic-activated cell populations in the immunopathogenesis of CL, showing a character of specificity in this immune response

Conjugate stability study during 18 months.

<p>A: A1, A2, A3, A4 and MESF bead #4 fluorescence intensities during the months of analysis. B: B1, B2, C, D and MESF bead #4 fluorescence intensities during the months of analysis. C: Conjugates histograms of PE-fluorescence intensity (X axis) vs. count (Y axis) on the first month (black peak) and last month of analysis (white peak). MESF bead #4 was included as an example of the MESF kit used to control instrument performance during the 18 months of evaluation. The A4 conjugate was coupled later than the other conjugates and its intensities evaluation started at month 4.</p

Staining profile of anti-IgG-PE conjugates.

<p>A: Representative histograms of PE-fluorescence intensity (X axis) vs. count (Y axis). 0.5 μg/mL = white peak and 5 μg/mL = black peak. B: Coefficient of variation of 0.5 μg/mL (white bars) and 5 μg/mL (black bars). Data expressed as the means of CVs from of three tests ± standard deviation. T test was performed for group comparison. An asterisk indicates a significantly different value assumed at p≤0.05.</p