Marta de Almeida Santiago

Characterisation of Lymphocyte Response and Cytokine Patterns in Patients with Dengue Fever

It is believed that the pathogenesis of dengue is generated by a deregulation of the immunological response. Dengue virus-infected monocytes/macrophages are likely to secrete monokines, which play a role in clinical features observed in patients with dengue haemorrhagic fever or dengue shock syndrome. This is a report on a study on 45 individuals presenting clinical and laboratory characteristics of dengue virus infection. During the acute phase of infection, immunophenotyping of peripheral mononuclear leukocytes was carried out in 19 patients and demonstrated a reduced frequency of CD2+ lymphocytes and their CD4+ and CD8+ subsets. Normal ratios were recovered during convalescence. Also, during the acute phase, mononuclear cells proliferated poorly in response to mitogens and dengue antigens as detected by incorporation of radiolabeled thymidine. During convalescence the lymphoproliferative response was re-established. In addition, the presence of circulating cytokines was investigated in the plasma of the same 45 patients. Concentrations of tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-10 (IL-10) and soluble tumor necrosis factor receptor (sTNF-Rp75) were found to be significantly elevated in patients when compared to normal controls. The increase in TNF-alpha was correlated with haemorrhagic manifestations and the increase in IL-10 with platelet decay. The data demonstrate that during the acute phase of dengue infection subsets of T lymphocytes are depressed in terms of both rate and function and provide evidence that circulating pro-inflammatory cytokines, such as TNF-alpha, are important in the pathogenesis and severity of dengue. IL-10 may be downregulating lymphocyte and platelet function.

Immunologic patterns associated with cure in human American cutaneous leishmaniasis

Patients with American cutaneous leishmaniasis were studied before therapy (active lesion) and at the end of therapy (cured patients). Assays of lymphocyte proliferative responses of peripheral blood mononuclear cells induced in vitro by Leishmania braziliensis promastigote antigens (Lb) were performed. Antigen-stimulated cells were harvested for CD4 and CD8 phenotype analysis and the levels of gamma interferon (IFN-gamma) and interleukin 4 (IL-4) produced were also determined in the culture supernatants. Two different patterns of Lb-induced T cell responses were observed: a) predominance of responding CD4+ cells and mixed type 1 and type 2 cytokine production (IFN-gamma and IL-4) during the active disease, and b) similar proportions of responding CD4+ and CD8+ cells, and type 1 cytokine production (presence of IFN-gamma and very low IL-4) at the end of therapy (healed lesions). This last pattern is probably associated with a beneficial T cell response.

Detection of early apoptosis and cell death in T CD4+ and CD8+ cells from lesions of patients with localized cutaneous leishmaniasis

Human localized cutaneous leishmaniasis (LCL), induced by Leishmania braziliensis, ranges from a clinically mild, self-healing disease with localized cutaneous lesions to severe forms which can present secondary metastatic lesions. The T cell-mediated immune response is extremely important to define the outcome of the disease; however, the underlying mechanisms involved are not fully understood. A flow cytometric analysis of incorporation of 7-amino actinomycin D and CD4+ or CD8+ T cell surface phenotyping was used to determine whether different frequencies of early apoptosis or accidental cell death occur at different stages of LCL lesions. When all cells obtained from a biopsy sample were analyzed, larger numbers of early apoptotic and dead cells were observed in lesions from patients with active disease (mean = 39.5 +/- 2.7%) as compared with lesions undergoing spontaneous healing (mean = 17.8 +/- 2.2%). Cells displaying normal viability patterns obtained from active LCL lesions showed higher numbers of early apoptotic events among CD8+ than among CD4+ T cells (mean = 28.5 +/- 3.8 and 15.3 +/- 3.0%, respectively). The higher frequency of cell death events in CD8+ T cells from patients with LCL may be associated with an active form of the disease. In addition, low frequencies of early apoptotic events among the CD8+ T cells were observed in two patients with self-healing lesions. Although the number of patients in the latter group was small, it is possible to speculate that, during the immune response, differences in apoptotic events in CD4+ and CD8+ T cell subsets could be responsible for controlling the CD4/CD8 ratio, thus leading to healing or maintenance of disease.

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.

AN EXPERIMENTAL MODEL OF THE PRODUCTION OF METASTASES IN MURINE CUTANEOUS LEISHMANIASIS

An experimental investigation into the influence of artificially induced trauma in the production of leishmanial metastatic lesions and into the possible role played by Leishmania-reactive T cell populations in the metastatic process was carried out. Trauma was induced by incising a small cut into the shaved rump of Leishmania amazonensis-infected BALB/c mice. Ten days after the trauma, mice were killed to quantify the parasite load in the traumatic lesion or in the equivalent area in nontraumatized mice, by limiting dilution analysis. Results demonstrated that metastatic lesions occurred earlier in traumatized animals and that parasites could be detected sooner in traumatic lesions than in equivalent areas in nontraumatized mice. When lymph node cells from L. amazonensis antigen-immunized BALB/c mice were adoptively transferred intravenously to L. amazonensis-infected syngeneic mice, the parasite load in the metastatic lesions was greater in the animals that received La Ag-reactive T cells than in the controls. When CD4(+)- or CD8(+)-depleted T cell populations from La Ag-immunized mice were adoptively transferred to infected traumatized or nontraumatized animals, we observed that the metastatic lesions in CD4(+)-inoculated animals had a greater number of parasites than the lesions in mice from all other groups. Thus, a new and reliable mouse model for studying the mechanisms involved in leishmanial metastasis is described.

A randomized double-blind placebo-controlled trial to evaluate the immunogenicity of a candidate vaccine against American tegumentary leishmaniasis

This study was aimed at evaluating the immunogenicity of a vaccine composed of killed Leishmania amazonensis promastigotes using several different protocols in a randomized, double-blind and controlled trial design in order to select one of them for further efficacy trials. One hundred and fourteen leishmanin skin test (LST)-negative healthy volunteers were allocated into eight groups that received either two or three deep intramuscular injections of vaccine at doses of 180, 360 and 540 microg or similar injections of placebo. Cell-mediated immune responses were evaluated before and after vaccination by means of LST as well as proliferative responses and cytokine production in Leishmania antigen-stimulated peripheral blood mononuclear cell cultures. The majority of the subjects who actually received vaccine converted to positive LST (89.5%). On the other hand, none of the subjects who received placebo converted to positive LST. Proliferative responses and production of interferon-gamma and interleukin-2 were significantly higher after vaccination than before vaccination in all groups, including those that received placebo. The dose of 360 microg provided the highest LST conversion rate (100%), as well as the greatest increase in interferon-gamma and interleukin-2 production after vaccination.

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.

Randomized, double-blind, placebo-controlled study on the immunogenicity of the leishmanin skin test

A positive reaction to the leishmanin skin test (LST) indicates previous contact with Leishmania antigens and is a useful criterion for the diagnosis of cutaneous leishmaniasis. In leishmaniasis vaccine trials, selection of volunteers has always been based on skin testing. During 1999 we performed a randomized controlled study in order to evaluate the immunogenicity of the LST. Fifty-nine (29 male and 30 female) healthy volunteer undergraduate students from the Medical School of Volta Redonda, Rio de Janeiro State, Brazil, with no evidence of previous infection with Leishmania, were randomly assigned into 2 groups: 29 subjects received LST and 30 received a placebo (merthiolate-phosphate-buffered saline). All volunteers received LST 41 d after the first injection of LST or placebo. Blood samples were taken immediately before the applications of LST or placebo for the assessment of Leishmania antigen-induced proliferation and cytokine production in peripheral blood mononuclear cell cultures. A significant increase in proliferative responses to L. braziliensis (P < 0.005) and L. amazonensis (P = 0.01) antigens as well as in L. braziliensis antigen-induced interferon-gamma production (P < 0.01) followed the application of LST but not the administration of the placebo. A single LST application is therefore able to induce Leishmania-specific cell-mediated immune responses. This observation should be considered in human trials of candidate vaccines against leishmaniasis.

Severe feline sporotrichosis associated with an increased population of CD8low cells and a decrease in CD4⁺ cells.

Sporotrichosis is a subcutaneous mycosis with worldwide distribution, especially in tropical and subtropical areas. Zoonotic transmission is described with cats being the main animal species involved. The occurrence of severe feline sporotrichosis with high fungal levels demonstrates the susceptibility of cats to this disease and the importance of studying its pathogenesis. This study describes the leukocytes profile in blood of cats with sporotrichosis by flow cytometry and its correlation with histopathology and fungal load. The cats with sporotrichosis were separated into groups L1, L2, and L3 (lesions at one, two, and three or more noncontiguous skin locations, respectively) and were classified as good, fair, or poor general conditions. The highest percentage of CD4+ cells was associated to L1 (P = .04) and to good general condition (P = .03). The percentage of CD8+ cells was greater in L2 and L3 (P = .01). CD8(low) expression occurred in 20 animals with sporotrichosis, mainly in L3 (P = .01) and was not observed in healthy controls. This expression was related to macrophage granulomas (P = .01) and predominated in cases with high fungal load. Altogether, the results indicated that control over feline sporotrichosis, with maintenance of a good general condition, fixed lesions, well-organized response and lower fungal load, is associated with increased CD4+ cells percentages. In contrast, a poor general condition, disseminated lesions and high fungal load were related to increased CD8+ cell percentages and increased expression of CD8(low). As conclusion these results point to an important role of the CD4:CD8 balance in determining the clinical outcome in feline sporotrichosis.

Differentiation between canine cutaneous and visceral leishmaniasis by the detection of immunoglobulin G specific for Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi antigens using flow cytometry

Flow cytometry employing Leishmania (L.) chagasi (Lc) and L. (Viannia) braziliensis (Lb) antigen was used to establish the differential diagnosis between visceral (VL) and cutaneous leishmaniasis (CL) in dogs. Flow cytometry permitted the detection of Leishmania-specific immunoglobulin G in sera from 19 dogs: nine with CL and 10 with VL. A significant difference in the percentage of positive staining was observed in sera from dogs with CL between the homologous antigen (69% for Lb) and the heterologous antigen (42% for Lc). However, this difference was not significant in sera from dogs with VL (61% for Lb and 73% for Lc). No significant staining was observed in control sera (0.6% for Lb and 0.4% for Lc) consisting of samples from healthy dogs, or in the group with sporotrichosis (1.8% for Lb and 1.5% for Lc), a differential diagnosis of CL. The results suggest that flow cytometry might be useful for the differentiation between CL and VL in dogs, with practical applications in areas where the two infections overlap.