The spleen is an important site of T cell activation during human hepatosplenic schistosomiasis

We have undertaken a comparative immunephenotypic study of spleen cells from hepatosplenic patients (HS) and uninfected individuals (NOR) using flow cytometry. Our data did not show any significant differences in the mean percentage of T-cells and B-cells between the two groups. Analysis of activated T-cells demonstrated that HS present an increased percentage of CD3+HLA-DR+ splenocytes in comparison to NOR. Analysis of T-cell subsets demonstrated a significant increase on the percentage of both activated CD4+ T-splenocytes and CD8+ cells in HS. We did not find any difference in the mean percentage of CD28+ T-cells. Analysis of the B-cell compartment did not show any difference on the percentage of B1-splenocytes. However, the spleen seems to be an important reservoir/source for B1 lymphocytes during hepatosplenic disease, since after splenectomy we found a decreased the percentage of circulating B1-lymphocytes. We observed an increase on the percentage of CD2+CD3- lymphocytes in the spleen of HS suggesting that the loss of CD3 by activated T-cells or the expansion of NK-cells might play a role in the development/maintenance of splenomegaly.

Immunological evaluation of human immunedeficiency virus infected individuals by flow cytometry

Human immunodeficiency virus (HIV) infection heavily compromises the immune system. The decrease of the T cell CD4+ subset along the evolution to acquired immunodeficiency syndrome has been considered as a hallmark of HIV infection. In this paper we review some aspects of the immunopathology of HIV infection and discuss the importance of the flow cytometry for the evaluation of the T lymphocyte subsets in the follow-up of HIV infected children and adults, and for the monitoring of the immune reconstitution upon antiretroviral therapy.

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.

Protective CD8+ T cell responses against the pre-erythrocytic stages of malaria parasites: an overview

CD8+ T cells have been implicated as critical effector cells in protection against the pre-erythrocytic stage of malaria in mice and humans following irradiated sporozoite immunization. Immunization experiments in animal models by several investigators have suggested different strategies for vaccination against malaria and many of the targets from liver stage malaria antigens have been shown to be immunogenic and to protect mice from the sporozoite challenge. Several prime/boost protocols with replicating vectors, such as vaccinia/influenza, with non-replicating vectors, such as recombinant particles derived from yeast transposon (Ty-particles) and modified vaccinia virus Ankara, and DNA, significantly enhanced CD8+ T cell immunogenicity and also the protective efficacy against the circumsporosoite protein of Plasmodium berghei and P. yeti. Based on these experimental results the development of a CD8+ T cell inducing vaccine has moved forward from epitope identification to planning stages of safety and immunogenicity trials of candidate vaccines.

The identification and characterization of new immunogenic egg components: implications for evaluation and control of the immunopathogenic T cell response in schistosomiasis

In schistosomiasis, granuloma formation to parasite eggs signals the beginning of a chronic and potentially life-threatening disease. Granulomas are strictly mediated by CD4+ T helper (Th) cells specific for egg antigens; however, the number and identity of these T cell-sensitizing molecules are largely unknown. We have used monoclonal T cell reagents derived from egg-sensitized individuals as probes to track down, isolate and positively identify several egg antigens; this approach implicitly assures that the molecules of interest are T cell immunogens and, hence, potentially pathogenic. The best studied and most abundant egg component is the Sm-p40 antigen. Sm-p40 and its peptide 234-246 elicit a strikingly immunodominant Th-1-polarized response in C3H and CBA mice, which are H-2k strains characterized by severe egg-induced immunopathology. Two additional recently described T cell-sensitizing egg antigens are Schistosoma mansoni phosphoenolpyruvate carboxykinase (Sm-PEPCK) and thioredoxin peroxidase-1 (Sm-TPx-1). In contrast to Sm-p40, both of these molecules induce a more balanced Th-1/Th-2 response, and are relatively stronger antigens in C57BL/6 mice, which develop smaller egg granulomas. Importantly, Sm-p40 and Sm-PEPCK have demonstrated immunogenicity in humans. The findings in the murine model introduce the important notion that egg antigens can vary significantly in immunogenicity according to the host's genetic background. A better knowledge of the principal immunogenic egg components is necessary to determine whether the immune responses to certain antigens can serve as indicators or predictors of the form and severity of clinical disease, and to ascertain whether such responses can be manipulated for the purpose of reducing pathology.

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% (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.

Studies in a co-infection murine model of Plasmodium chabaudi chabaudi and Leishmania infantum: interferon-g and interleukin-4 mRNA expression

This work aimed to study the T helper type 1/2 (Th1/Th2) cytokine profile in a co-infection murine model of Plasmodium chabaudi chabaudi and Leishmania infantum. Expression of interferon-gamma (IFNT FACE=Symbol>-gT>) and interleukin-4 (IL-4) was analyzed, in spleen and liver of C57BL/6 mice, by reverse transcriptase-polymerase chain reaction. High levels of IFNT FACE=Symbol>-gT> expression did not prevent the progression of Leishmania in co-infected mice and Leishmania infection did not interfere with the Th1/Th2 switch necessary for Plasmodium control. The presence of IL-4 at day 28 in co-infected mice, essential for Plasmodium elimination, was probably a key factor on the exacerbation of the Leishmania infection.

Activated peripheral lymphocytes with increased expression of cell adhesion molecules and cytotoxic markers are associated with dengue fever disease

The immune mechanisms involved in dengue fever and dengue hemorrhagic/dengue shock syndrome are not well understood. The ex vivo activation status of immune cells during the dengue disease in patients was examined. CD4and CD8 T cells were reduced during the acute phase. Interestingly, CD8 T cells co-expressing activation marker HLA-DR, Q, P, and cytolytic granule protein-Tia-1 were significantly higher in dengue patients than in controls. Detection of adhesion molecules indicated that in dengue patients the majority of T cells (CD4 and CD8) express the activation/memory phenotype, characterized as CD44HIGH and lack the expression of the naïve cell marker, CD62L LOW. Also, the levels of T cells co-expressing ICAM-1 (CD54), VLA-4, and LFA-1 (CD11a) were significantly increased. CD8 T lymphocytes expressed predominantly low levels of anti-apoptotic molecule Bcl-2 in the acute phase, possibly leading to the exhibition of a phenotype of activated/effector cells. Circulating levels of IL-18, TGF-b1 and sICAM-1 were significantly elevated in dengue patients. Early activation events occur during acute dengue infection which might contribute to viral clearance. Differences in expression of adhesion molecules among CD4 and CD8 T cells might underlie the selective extravasation of these subsets from blood circulation into lymphoid organs and/or tissues. In addition, activated CD8 T cells would be more susceptible to apoptosis as shown by the alteration in Bcl-2 expression. Cytokines such as IL-18, TGF-b1, and sICAM-1 may be contributing by either stimulating or suppressing the adaptative immune response, during dengue infection, thereby perhaps establishing a relationship with disease severity.

T-cell responses associated with resistance to Leishmania infection in individuals from endemic areas for Leishmania (Viannia) braziliensis

Subclinical or asymptomatic infection is documented in individuals living in endemic areas for leishmaniasis suggesting that the development of an appropriate immune response can control parasite replication and maintain tissue integrity. A low morbidity indicates that intrinsic factors could favor resistance to Leishmania infection. Herein, leishmanial T-cell responses induced in subjects with low susceptibility to leishmaniasis as asymptomatic subjects were compared to those observed in cured cutaneous leishmaniasis (CCL) patients, who controlled the disease after antimonial therapy. All of them have shown maintenance of specific long-term immune responses characterized by expansion of higher proportions of CD4+ as compared to CD8+ Leishmania reactive T-lymphocytes. Asymptomatic subjects had lower indexes of in vitro Leishmania induced lymphoproliferative responses and interferon-gamma (IFN-gamma) production in comparison to CCL patients. On the other hand, interleukin (IL-10) production was much higher in asymptomatics than in CCL, while no differences in IL-5 levels were found. In conclusion, long lived T-cell responses achieved by asymptomatic individuals differed from those who had developed symptomatic leishmaniasis in terms of intensity of lymphocyte activation (proliferation or IFN-gamma) and regulatory mechanisms (IL-10). The absence of the disease in asymptomatics could be explained by their intrinsic ability to create a balance between immunoregulatory (IL-10) and effector cytokines (IFN-gamma), leading to parasite destruction without producing skin tissue damage. The establishment of profiles of cell-mediated immune responses associated with resistance against Leishmania infection is likely to make new inroads into understanding the long-lived immune protection against the disease.

Capillaria spp. eggs in Patagonian archaeological sites: statistical analysis of morphometric data

Discriminant analysis was used to identify eggs of Capillaria spp. at specific level found in organic remains from an archaeological site in Patagonia, Argentina, dated of 6,540 ± 110 years before present. In order to distinguish eggshell morphology 149 eggs were measured and grouped into four arbitrary subsets. The analysis used on egg width and length discriminated them into different morphotypes (Wilks' lambda = 0.381, p < 0.05). The correlation analysis suggests that width was the most important variable to discriminate among the Capillaria spp. egg morphotypes (Pearson coefficient = 0.950, p < 0.05). The study of eggshell patterns, the relative frequency in the sample, and the morphometric data allowed us to correlate the four morphotypes with Capillaria species.

Circulating natural killer and γδ T cells decrease soon after infection of rhesus macaques with lymphocytic choriomeningitis virus

Rhesus macaques infected with the WE strain of lymphocytic choriomeningitis virus (LCMV-WE) serve as a model for human infection with Lassa fever virus. To identify the earliest events of acute infection, rhesus macaques were monitored immediately after lethal infection for changes in peripheral blood mononuclear cells (PBMCs). Changes in CD3, CD4, CD8 and CD20 subsets did not vary outside the normal fluctuations of these blood cell populations; however, natural killer (NK) and γδ T cells increased slightly on day 1 and then decreased significantly after two days. The NK subsets responsible for the decrease were primarily CD3-CD8+ or CD3-CD16+ and not the NKT (primarily CD3+CD56+) subset. Macaques infected with a non-virulent arenavirus, LCMV-Armstrong, showed a similar drop in circulating NK and γδ T cells, indicating that this is not a pathogenic event. V³9 T cells, representing the majority of circulating γδ T cells in rhesus macaques, displayed significant apoptosis when incubated with LCMV in cell culture; however, the low amount of cell death for virus-co-cultured NK cells was insufficient to account for the observed disappearance of this subset. Our observations in primates are similar to those seen in LCMV-infected mice, where decreased circulating NK cells were attributed to margination and cell death. Thus, the disappearance of these cells during acute hemorrhagic fever in rhesus macaques may be a cytokine-induced lymphopenia common to many virus infections.

Regulatory elements involved in the post-transcriptional control of stage-specific gene expression in Trypanosoma cruzi: a review

Trypanosoma cruzi, a protozoan parasite that causes Chagas disease, exhibits unique mechanisms for gene expression such as constitutive polycistronic transcription of protein-coding genes, RNA editing and trans-splicing. In the absence of mechanism controlling transcription initiation, organized subsets of T. cruzi genes must be post-transcriptionally co-regulated in response to extracellular signals. The mechanisms that regulate stage-specific gene expression in this parasite have become much clearer through sequencing its whole genome as well as performing various proteomic and microarray analyses, which have demonstrated that at least half of the T. cruzi genes are differentially regulated during its life cycle. In this review, we attempt to highlight the recent advances in characterising cis and trans-acting elements in the T. cruzi genome that are involved in its post-transcriptional regulatory machinery.

Dengue-2 and yellow fever 17DD viruses infect human dendritic cells, resulting in an induction of activation markers, cytokines and chemokines and secretion of different TNF-α and IFN-α profiles

Flaviviruses cause severe acute febrile and haemorrhagic infections, including dengue and yellow fever and the pathogenesis of these infections is caused by an exacerbated immune response. Dendritic cells (DCs) are targets for dengue virus (DENV) and yellow fever virus (YF) replication and are the first cell population to interact with these viruses during a natural infection, which leads to an induction of protective immunity in humans. We studied the infectivity of DENV2 (strain 16681), a YF vaccine (YF17DD) and a chimeric YF17D/DENV2 vaccine in monocyte-derived DCs in vitro with regard to cell maturation, activation and cytokine production. Higher viral antigen positive cell frequencies were observed for DENV2 when compared with both vaccine viruses. Flavivirus-infected cultures exhibited dendritic cell activation and maturation molecules. CD38 expression on DCs was enhanced for both DENV2 and YF17DD, whereas OX40L expression was decreased as compared to mock-stimulated cells, suggesting that a T helper 1 profile is favoured. Tumor necrosis factor (TNF)-α production in cell cultures was significantly higher in DENV2-infected cultures than in cultures infected with YF17DD or YF17D/DENV. In contrast, the vaccines induced higher IFN-α levels than DENV2. The differential cytokine production indicates that DENV2 results in TNF induction, which discriminates it from vaccine viruses that preferentially stimulate interferon expression. These differential response profiles may influence the pathogenic infection outcome.

The presence of Helicobacter pylori in the liver depends on the Th1, Th17 and Treg cytokine profile of the patient

The hypothesis that Helicobactermight be a risk factor for human liver diseases has arisen after the detection of Helicobacter DNA in hepatic tissue of patients with hepatobiliary diseases. Nevertheless, no explanation that justifies the presence of the bacterium in the human liver has been proposed. We evaluated the presence of Helicobacterin the liver of patients with hepatic diseases of different aetiologies. We prospectively evaluated 147 patients (106 with primary hepatic diseases and 41 with hepatic metastatic tumours) and 20 liver donors as controls. Helicobacter species were investigated in the liver by culture and specific 16S rDNA nested-polymerase chain reaction followed by sequencing. Serum and hepatic levels of representative cytokines of T regulatory cell, T helper (Th)1 and Th17 cell lineages were determined using enzyme linked immunosorbent assay. The data were evaluated using logistic models. Detection of Helicobacter pylori DNA in the liver was independently associated with hepatitis B virus/hepatitis C virus, pancreatic carcinoma and a cytokine pattern characterised by high interleukin (IL)-10, low/absent interferon-γ and decreased IL-17A concentrations (p < 10-3). The bacterial DNA was never detected in the liver of patients with alcoholic cirrhosis and autoimmune hepatitis that are associated with Th1/Th17 polarisation. H. pylori may be observed in the liver of patients with certain hepatic and pancreatic diseases, but this might depend on the patient cytokine profile.

Chemokines and chemokine receptors expression in the lesions of patients with American cutaneous leishmaniasis

American cutaneous leishmaniasis (ACL) presents distinct active clinical forms with different grades of severity, known as localised (LCL), intermediate (ICL) and diffuse (DCL) cutaneous leishmaniasis. LCL and DCL are associated with a polarised T-helper (Th)1 and Th2 immune response, respectively, whereas ICL, or chronic cutaneous leishmaniasis, is associated with an exacerbated immune response and a mixed cytokine expression profile. Chemokines and chemokine receptors are involved in cellular migration and are critical in the inflammatory response. Therefore, we evaluated the expression of the chemokines CXCL10, CCL4, CCL8, CCL11 and CXCL8 and the chemokine receptors CCR3, CXCR3, CCR5 and CCR7 in the lesions of patients with different clinical forms of ACL using immunohistochemistry. LCL patients exhibited a high density of CXCL10+, CCL4+ and CCL8+ cells, indicating an important role for these chemokines in the local Th1 immune response and the migration of CXCR3+ cells. LCL patients showed a higher density of CCR7+ cells than ICL or DCL patients, suggesting major dendritic cell (DC) migration to lymph nodes. Furthermore, DCL was associated with low expression levels of Th1-associated chemokines and CCL11+ epidermal DCs, which contribute to the recruitment of CCR3+ cells. Our findings also suggest an important role for epidermal cells in the induction of skin immune responses through the production of chemokines, such as CXCL10, by keratinocytes.