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.

The sample processing time interval as an influential factor in flow cytometry analysis of lymphocyte subsets

The objective of this paper is to propose a protocol to analyze blood samples in yellow fever 17DD vaccinated which developed serious adverse events. We investigated whether or not the time between sample collection and sample processing could interfere in lymphocyte subset percentage, for it is often impossible to analyze blood samples immediately after collection due to transport delay from collection places to the flow cytometry facility. CD4+CD38+ T, CD8+CD38+ T, CD3+ T, CD19+ B lymphocyte subsets were analyzed by flow cytometry in nine healthy volunteers immediately after blood collection and after intervals of 24 and 48 h. The whole blood lysis method and gradient sedimentation by Histopaque were applied to isolate peripheral blood mononuclear cells for flow cytometry analyses. With the lysis method, there was no significant change in lymphocyte subset percentage between the two time intervals (24 and 48 h). In contrast, when blood samples were processed by Histopaque gradient sedimentation, time intervals for sample processing influenced the percentage in T lymphocyte subsets but not in B cells. From the results obtained, we could conclude that the whole blood lysis method is more appropriate than gradient sedimentation by Histopaque for immunophenotyping of blood samples collected after serious adverse events, due to less variation in the lymphocyte subset levels with respect to the time factor.

Single and concomitant experimental infectionsby Endotrypanum spp. and Leishmania (Viannia) guyanensis (Kinetoplastida: Trypanosomatidae) in the Neotropical sand fly Lutzomyia longipalpis (Diptera: Psychodidae)

Lutzomyia longipalpis females received single and mixed infections with Endotrypanum and Leishmania. Two biological parameters were analyzed: the percentage of infected females and the distribution of flagellates in the gut of the females. The principal comparisons were performed between (1) two strains of Endotrypanum, (2) cloned versus primary sample of one strain of Endotrypanum, (3) Endotrypanum versus Leishmania guyanensis, and (4) the pattern of flagellates behaviour by optical microscopy in females with single or mixed infection versus the identification of parasites isolated from digestive tracts by isoenzyme electrophoresis. Flagellates of Endotrypanum showed distinct patterns of infection suggesting that there is variation between and within strains. The distribution of Endotrypanum and L. guyanensis differed significantly in relation to the colonization of the stomodeal valve. In co-infection with L. guyanensis, a large number of flagellates were seen to be plentifully infecting the stomodeal valve in significantly more specimens than in females infected by Endotrypanum only. However, the electrophoretic profiles of isoenzymes of parasites recovered from all co-infected specimens corresponded to Endotrypanum. This suggests that the mere correlation sand fly infection-biochemical analysis of isolates may induce parasitological incorrect consideration.

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

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 percent (SE ± 4 percent) to 61.15 percent (SE ± 4.2 percent), CD4+ T cells from 22.4 percent (SE ± 3.6 percent) to 39.17 percent (SE ± 2 percent) with 43 percent of these cells corresponding to CD4+CD45RO+ T cells, CD8+ T cells from 15.2 percent (SE ± 2.9 percent) to 27 percent (SE ± 3 percent) with 70 percent corresponding to CD8+CD45RO+ T cells in first-time vaccinees. In re-vaccinees, the CD3+ T cells increased from 50.7 percent (SE ± 3 percent) to 80 percent (SE ± 2.3 percent), CD4+ T cells from 24.9 percent (SE ± 1.4 percent) to 40 percent (SE ± 3 percent) presenting a percentage of 95 percent CD4+CD45RO+ T cells, CD8+ T cells from 19.7 percent (SE ± 1.8 percent) to 25 percent (SE ± 2 percent). Among CD8+CD38+ T cells there could be observed an increase from 15 to 41.6 percent in first-time vaccinees and 20.7 to 62.6 percent 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.

Comparison of dengue infection in human mononuclear leukocytes with mosquito C6/36 and mammalian Vero cells using flow cytometry to detect virus antigen

Fluorescent activated cell sorter (FACS) analysis is useful for the detection of cellular surface antigens and intracellular proteins. We used this methodology in order to detect and quantify dengue antigens in highly susceptible cells such as clone C6/36 (Aedes albopictus) and Vero cells (green monkey kidney). Additionally, we analyzed the infection in vitro of human peripheral blood mononuclear leukocytes (PBML). FACS analysis turned out to be a reliable technique to quantify virus growth in traditional cell cultures of C6/36 as well as Vero cells. High rates of infection were achieved with a good statistical correlation between the virus amount used in infection and the percentage of dengue antigen containing cells detected in infected cultures. We also showed that human monocytes (CD14+) are preferred target cells for in vitro dengue infection among PBML. Monocytes were much less susceptible to virus infection than cell lines but they displayed dengue antigens detected by FACS five days after infection. In contrast, lymphocytes showed no differences in their profile for dengue specific immunofluorescence. Without an animal model to reproduce dengue disease, alternative assays have been sought to correlate viral virulence with clinical manifestations and disease severity. Study of in vitro interaction of virus and host cells may highlight this relationship.

Detection of intracytoplasmic cytokines by flow cytometry

Flow cytometry has been used as a powerful technique for studying cell surface antigen expression as well as intracellular molecules. Its capability of analyzing multiple parameters simultaneously on a single cell has allowed identification and studies of functional cell subsets within heterogeneous populations. In this respect, several techniques have been developed during the past few years to study cytokine-producing cells by flow cytometry in humans and several animal models.

Flow cytometry in the study of cell death

In this report we present a concise review concerning the use of flow cytometric methods to characterize and differentiate between two different mechanisms of cell death, apoptosis and necrosis. The applications of these techniques to clinical and basic research are also considered. The following cell features are useful to characterize the mode of cell death: (1) activation of an endonuclease in apoptotic cells results in extraction of the low molecular weight DNA following cell permeabilization, which, in turn, leads to their decreased stainability with DNA-specific fluorochromes. Measurements of DNA content make it possible to identify apoptotic cells and to recognize the cell cycle phase specificity of apoptotic process; (2) plasma membrane integrity, which is lost in necrotic but not in apoptotic cells; (3) the decrease in forward light scatter, paralleled either by no change or an increase in side scatter, represent early changes during apoptosis. The data presented indicate that flow cytometry can be applied to basic research of the molecular and biochemical mechanisms of apoptosis, as well as in the clinical situations, where the ability to monitor early signs of apoptosis in some systems may be predictive for the outcome of some treatment protocols.

Conteúdo de ADN em nódulos de tireóide / DNA contents in thyroid nodules

A importância prognóstica negativa de um conteúdo anormal de ADN em carcinomas diferenciados de tireóide tem sido estabelecida por vários autores. avaliamos o conteúdo de ADN em 19 pacientes com nódulos de tireóide, submetidos à punçao aspirativa por agulha fina. As suspensoes celulares obtidas foram marcadas com iodeto de propídio e analisadas pela citometria de fluxo. Houveram sete instâncias de aneuploidia nas seguintes situaçoes clínicas: um adenoma folicular, um bócio colóide, uma tireodite autoimune, um possível bócio colóide e uma possível tireodite autoimune; em dois casos o material nao foi adequado para exame citopatológico. Os casos restantes apresentaram pico diplóide de ADN e, quando foi possível estabelecer um diagnóstico citológico, foram benignos, à exceçao de um caso de carcinoma papilífero. A análise da atividade proliferativa pelo percentual de células em fase S mostrou grande dispersao, com valores variando de 5 a 42 por cento. Todos os casos de aneuploidia tiveram percentuais acima de 20 por cento. A ausência de correlaçao de ploidia com doença benigna ou maligna concorda com os achados relatados na literatura. Um acompanhamento mais rigoroso dos casos com aneuploidia, ainda que benignos, é sugerida.

Characterization of CD4 and CD8 bearing cells in intestines of rotavirus susceptible (suckling) and resistant (adult/weanling) mice by flow cytometry

Suckling mice are susceptible to several virus infections and develop diarrhea after rotavirus inoculation whereas 3 week-old and older mice are resistant. Since young mice have a n immature immune system, we investigated the status of CD4 and CD8 bearing T-lymphocytes in intestines of 1, 3-4 and 8-10 week-old mice. Unicellular suspensions of the total small intestine were prepared. Cells were stained with monoclonal antibodies reactive to CD4 and CD8 molecules and were analyzed by flow cystometry. Percentages of CD8+ and CD4+CD8+ cells were markedly increased in intestines of suckling mice when compared to adults. CD4+ cells were apparently not altered. Rotavirus SA-11 infected diarrheic suckling mice presented a decrease of all three studied lymphocyte subpopulations, whereas no changes were observed in virus inoculated weanling mice. We suggest that higher proportions of CD4+CD8+ and CD8+ cells in intestines of suckling mice may play a role in the susceptibility to rotavirus, which would disable the animals to develop a rapid and efficient immune response resulting in resistance.