Cytokine Pattern of T Lymphocytes in Acute Schistosomiasis mansoni Patients following Treated Praziquantel Therapy.

Acute schistosomiasis is associated with a primary exposure and is more commonly seen in nonimmune individuals traveling through endemic regions. In this study, we have focused on the cytokine profile of T lymphocytes evaluated in circulating leukocytes of acute Schistosomiasis mansoni-infected patients (ACT group) before and after praziquantel treatment (ACT-TR group). Our data demonstrated increased values of total leukocytes, eosinophils, and monocytes in both groups. Interestingly, we have observed that patients treated with praziquantel showed increased values of lymphocytes as compared with noninfected group (NI) or ACT groups. Furthermore, a decrease of neutrophils in ACT-TR was observed when compared to ACT group. Analyses of short-term in vitro whole blood stimulation demonstrated that, regardless of the presence of soluble Schistosoma mansoni eggs antigen (SEA), increased synthesis of IFN-γ and IL-4 by T-cells was observed in the ACT group. Analyses of cytokine profile in CD8 T cells demonstrated higher percentage of IFN-γ and IL-4 cells in both ACT and ACT-TR groups apart from increased percentage of IL-10 cells only in the ACT group. This study is the first one to point out the relevance of CD8 T lymphocytes in the immune response induced during the acute phase of schistosomiasis.

Immune response and pathogenesis of neuroschistosomiasis mansoni.

The involvement of the central nervous system (CNS) by schistosomes may or may not determine clinical manifestations. When symptomatic, neuroschistosomiasis (NS) is one of the most severe presentations of schistosome infection. Considering the symptomatic form, Schistosoma mansoni causes almost always spinal cord disease. Cerebral and spinal cord disorders in S. mansoni infections are inflammatory conditions of the CNS that cause mild-moderate impairment of the blood-brain barrier and intrathecal synthesis of antibodies against schistosomal antigens. Little is known about the pathogenesis of NS, but available evidence strongly suggests that it depends basically on the presence of parasite eggs in the nervous tissue and on the host's immune response against the trapped eggs. Numerous eggs surrounded by granulomas lodged together in circumscribed areas of the CNS damage the nervous tissue by both the mass effect and the egg-induced inflammatory reaction. Vasculitis of immune etiology, which causes isquemic lesions, may also play an important role in the genesis of the neurological symptoms. Although the mechanisms involved in the immunophatogenesis of NS are largely unknown, initial investigations on cerebrospinal fluid (CSF) and serum cytokine profiles suggest the occurrence of inflammation as well as a skewed Th2 immune response that probably occur both locally and systemically.

Human schistosomiasis mansoni: immune responses during acute and chronic phases of the infection.

Schistosoma mansoni infection may occur either as an acute infection in individuals who have recently visited an endemic area, with no previous contact with the parasite, or as a lasting chronic disease, if not interrupted by specific chemotherapy. The acute phase is characterized by symptoms such as fever, cough, diarrhea, anorexia, and arthralgias in combination with leukocytosis and eosinophilia, and a high cellular immune response to schistosome antigens especially those from the parasite's eggs. In the chronic phase, most patients living in endemic areas are asymptomatic, and their immune responses to egg antigens are modulated. A few develop periportal fibrosis of the liver, which may result in the hepatosplenic form of the disease. The humoral response (IgG, IgM and IgE) in acute patients to egg and worm antigens does not differ from the chronic phase. However, a high level of IgG and IgM antibodies to KLH were detected in acute patients. Acute patients express a considerably higher in vitro cellular responsiveness than do chronic patients, especially to egg antigens. They present a mixed profile of Th1 and Th2 cytokines. Ultrasound examinations of endemic population reveal a high heterogeneity between the patients as regards the presence and intensity of periportal fibrosis. Most patients are asymptomatic and their immune responses to schistosoma egg antigens (SEA) are modulated. In contrast, a high percentage of patients with incipient fibrosis (early stage of hepatosplenic) responded strongly to SEA. Patients with advanced hepatosplenic disease were likely to be non-responders to SEA. Most of the chronic patients presented a Th2 profile with low production of interferon-gamma (IFN-gamma). The intensity of infection favors the production of interleukin (IL)-10. After adjusting for age, sex, and intensity of infection, a strong correlation was observed between the production of IL-13 and the degree of fibrosis. Chronic asymptomatic patients and those with incipient fibrosis expressed very high levels of heterogeneity of their antibody responses. IgG response to soluble worm antigen preparation (SWAP) was distinct and significantly higher in hepatosplenic patients than in those asymptomatic or with incipient fibrosis. Levels of IgG4 to SEA were significantly higher in sera from patients with incipient fibrosis as compared to uninfected and hepatosplenic groups. Polyclonal idiotypic antibodies and their fragments F(ab')2, directly stimulate in culture T cells of schistosomiasis patients in presence of IL-1. Polyclonal idiotypic antibodies are able to modulate in vitro granuloma formation around SEA-polyacrylamide. The importance of idiotypes for protection or pathology in schistosomiasis is still not clear.

Cytokines, chemokine receptors, CD4+CD25HIGH+ T-cells and clinical forms of human schistosomiasis.

Previous studies have demonstrated that distinct immune response profiles can be correlated with the development/maintenance of different clinical forms of human schistosomiasis. We have previously shown that individuals with the more severe clinical forms of the disease such as those presenting different levels of fibrosis or with the hepatosplenic (HS) clinical form of the disease show significantly different immune response when compared with those with the intestinal clinical form (INT). To better understand the immune mechanisms associated with the clinical form of the schistosomiasis, in this study, we present the results of the evaluation, at a single cell level, of the cytokine patterns as well as the chemokine receptors expression by T-cell subsets after in vitro short-term stimulation with soluble egg antigens as well as the ex vivo frequency analysis of putative regulatory CD4+CD25HIGH+ T-cell subset in the peripheral blood mononuclear cells. We observed an increase on IL-4+, IL-5+ and IL-10+ cells both in the CD4+ and CD8+ lymphocytes in INT and a significant decrease on the number of IL-4+, IL-5+ and IL-10+ T-lymphocytes for HS. However, patients with detectable fibrosis presented decrease on IL-10+ (both CD4+ and CD8+ lymphocytes) and basal levels of IL-4 and IL-5. These data suggested that although INT group is under the influence of an effective immunoregulated immune response, mainly due to the high percentage of IL-10+ cells, it presents a mixed type (Type1/Type-2) immune profile. Moreover, the chemokine receptors expression demonstrated that CXCR3 and CXCR4 by CD4+ T-cells in INT may dictate the selective profile of IL-10 associated with the immunomodulatory events in human schistosomiasis. Additionally, the ex vivo analysis also suggests that higher levels of CD4+CD25HIGH+ T-cells may play a role in controlling morbidity in chronic human schistosomiasis. Taken together, these data suggest a major role of IL-10-producing CXCR4+ CD4+ T-cell subset for the asymptomatic outcome of the disease.

In vitro cultured peripheral blood mononuclear cells from patients with chronic schistosomiasis mansoni show immunomodulation of cyclin D1,2,3 in the presence of soluble egg antigens.

Infection with Schistosoma mansoni induces a wide range of effects on the immune responses of the host. In the present study we investigated the influence of soluble egg antigens (SEA) on the cell cycle of peripheral blood mononuclear cells (PBMC) from infected and non-infected individuals with S. mansoni resident in an endemic area and blood donors from non-endemic area. The cell cycle, the expression of activation markers and cyclin D(+)(1,2,3) CD3(+) frequency was assessed by flow cytometry. Stimulation of PBMC from infected patients with SEA resulted in a lower frequency of CD3(+) T cells in S phase when compared with the non-infected group. In addition, infected patients presented a decrease of activation marker expression (CD69(+), HLA-DR(+) and CD28(-) on CD4(+) cells and CD25(+), HLA-DR(+) on CD8(+) cells). A reduced frequency was observed of cyclin D(1,2,3) expression in SEA-stimulated T cells from infected individuals when compared with those from the non-infected group. The decreased expression of activation markers and frequency of cyclin D(1,2,3) in T cells may result in arrest of T cells in the G(0)/G(1) phase of the cell cycle, thus explaining the down-regulation observed in chronic schistosomiasis.

Differential impact of metacyclic and blood trypomastigotes on parasitological, serological and phenotypic features triggered during acute Trypanosoma cruzi infection in dogs.

A detailed follow-up investigation of the major parasitological, serological and phenotypic features in dogs experimentally infected with metacyclic (MT) and blood (BT) trypomastigotes of Trypanosoma cruzi strain Berenice-78, typifying vectorial and transfusional transmission of human Chagas disease, has been conducted. Although there were no changes with respect to the window of patent-parasitaemia, significant differences between MT- and BT-infected dogs in both the prepatent period (days 23 and 19, respectively) and the day of maximum parasitaemia (days 26 and 22, respectively) were recorded. A progressive enhancement in the level of T. cruzi-specific antibodies accompanied infection by both MT and BT forms, although higher IgG titres developed on days 14 and 21 following infection with MT forms. Higher Thy-1(+)/CD21(+) and lower CD4(+)/CD8(+) cell ratios, occasioned by increased levels of Thy-1(+) and CD8(+) T-cells and reduced frequencies of CD4(+) T-cells and CD21(+) B-lymphocytes, were observed in both MT- and BT-infected animals. The reduced frequency of CD14(+) leukocytes was revealed as the most relevant phenotypic feature intrinsic to T. cruzi infection independent of inoculum source. BT-specific phenotypic features included an early reduction in the percentage of circulating CD21(+) and CD14(+) leukocytes, together with a higher Thy-1(+)/CD21(+) cell ratio on day 42. On the other hand, higher levels of CD8(+) T-cells, together with a lower CD4(+)/CD8(+) cell ratio on day 28, were characteristic of MT infection. These findings emphasise the importance of inoculum source and suggest that vectorial or transfusional routes of T. cruzi infection may trigger distinct parasite-host interactions during acute Chagas disease.

Intrathecal cytokines in spinal cord schistosomiasis.

We investigate the cytokine profile in the cerebrospinal fluid (CSF) and serum of patients with spinal cord schistosomiasis (SCS). Increased levels of IL-1beta, IL-4, IL-6 and IL-10 and low concentrations of TNF-alpha and IFN-gamma were observed in both CSF and serum. CSF showed higher levels of IL-4 and IL-6 when compared to the paired serum samples. A negative correlation between the concentrations of IL-10 and IFN-gamma was observed in the CSF. These findings suggest an inflammatory as well as a skewed type-2 immune response that probably occur both locally and systemically and may be involved in the pathogenesis of SCS.

Comparative clinical and ultrasound study of egg-negative and egg-positive individuals from Schistosoma mansoni low morbidity endemic areas, and hospitalized patients with hepatosplenic disease.

Two hundred and twenty three subjects from a Schistosoma mansoni low morbidity endemic area and nine hospitalized hepatosplenic patients were submitted to stool test and clinical examination and abdomen ultrasound assessments. According to stool examination and ultrasound results, they were grouped as follows: G1 -- 63 Schistosoma mansoni egg-negative individuals; G2 -- 141 egg-positive patients and without evidence of periportal fibrosis; G3 -- 19 egg-positive patients with periportal echogenicity (3-6 mm); and G4 -- 9 hepatosplenic patients with periportal echogenicity (> 6 mm). Hepatomegaly detected by physical examination of the abdomen evaluated in the midclavicular line was verified in G1, G2 and G3, respectively, in 11.1, 12.1 and 26.3%. In G1, G2 and G3, periportal thickening occurred only in schistosomal patients (8.5%). Mild pathological alterations in patients that cannot yet be detected by clinical examination were detectable in the liver by ultrasound and can be due to fibrosis. The degree of mild periportal fibrosis was diminished in 57.9% of patients 12 months after treatment of schistosomiasis with oxamniquine. At ultrasonography, the mean liver left lobe measurement of G3 was larger than that of G1, and that of G4 larger than that of G1 and G2. The mean size of the spleen of G4 was significantly larger than that of the other three groups, and that of G3 larger than that of G1 and G2.

Comparative clinical and ultrasound study of egg-negative and egg-positive individuals from Schistosoma mansoni low morbidity endemic areas, and hospitalized patients with hepatosplenic disease

Duzentos e vinte e três indivíduos de área endêmica de baixa morbidade para esquistossomose e nove pacientes hospitalizados com a forma hepatoesplênica foram submetidos ao exame de fezes e clínico e à ultra-sonografia do abdômen. De acordo com os resultado dos exames de fezes e do ultra-som eles foram agrupados do seguinte modo: G1 - 63 indivíduos sem ovos de Schistosoma mansoni nas fezes; G2 - 141 indivíduos apresentando ovos de Schistosoma mansoni nas fezes, sem ecogenicidade periportal. G3 — 19 indivíduos com ovos de Schistosoma mansoni nas fezes e ecogenicidade periportal entre 3-6mm.; G4 — 9 pacientes hepatesplênicos com ecogenicidade periportal > 6mm. Pelo exame físico do abdômen, a hepatomegalia na linha hemiclavicular direita foi constatada em G1, G2 E G3, respectivamente, em 11,1, 12,1 e 26,3%. Nos grupos G1, G2 e G3, houve espessamento periportal somente em esquistossomáticos (8,5%). Alterações patológicas leves em pacientes, as quais não puderam ser detectadas pelo exame clínico, foram evidenciadas no fígado pelo ultra-som e podem ser devidas à fibrose. O grau de fibrose periportal leve foi diminuído em 57,9% dos pacientes 12 meses após tratamento da esquistossomose com oxamniquine. Na ultra-sonografia, a média da medida do lobo esquerdo do fígado dos indivíduos de G3 foi maior que a de G1 e, a de G4 maior que a de G1 e G2. O tamanho médio do baço de G4 foi significativamente maior que o dos outros grupos e o de G3 foi maior que o de G1 e G2.

Human schistosomiasis mansoni: intensity of infection differentially affects the production of interleukin-10, interferon-gamma and interleukin-13 by soluble egg antigen or adult worm antigen stimulated cultures.

The effect of the intensity of infection (eggs per gram faeces, epg) on the production of interferon-gamma (INF-gamma), interleukin (IL)-10 and IL-13 by peripheral blood mononuclear cells (PBMCs) from individuals living in a Schistosoma mansoni-endemic area was evaluated. In vitro stimulation of PBMCs with soluble egg antigen (SEA) resulted in significantly higher secretion levels of IFN-gamma in egg-negative individuals compared with those with an intensity of infection of more than 100 epg. In contrast, the egg-positive group produced significantly higher amounts of IL-10. Levels of IL-13 did not differ significantly between egg-positive and egg-negative groups. These findings suggest that IL-10 is an important cytokine in the control of the T helper cell (Th) type 1 responses during human S. mansoni infection, shifting the immune response from Th0 in egg-negative individuals from an endemic area to a Th2 polarization in chronic infected individuals.

In vitro activity of labdane diterpene from Alomia myriadenia (Asteraceae): immunosuppression via induction of apoptosis in monocytes.

A screening program in Brazilian flora was carried out to detect the presence of immunosuppressive compounds by using the in vitro phytohemagglutinin A (PHA)-induced human peripheral blood mononuclear cell (PBMC) proliferation assay. In this screening, we isolated from Alomia myriadenia Schultz-Bip. ex. Baker (Asteraceae), a labdane-type diterpene named myriadenolide. Incubation of human PBMC with this compound reduced significantly the percentage of CD14(+) cells, but it has no effect on the relative amount of CD3(+)CD4(-)CD8(+) and CD3(+)CD4(+)CD8(-) T lymphocyte subpopulations. Neither viability nor proliferative competence of T lymphocytes was significantly affected by myriadenolide. The toxic effect on monocytes (CD14(+) cells) may explain the inhibitory effect observed on PHA-induced lymphocyte proliferation. The cytotoxic effect of myriadenolide on monocytes was determined by measuring the percentage of hypodiploid nuclei content by propidium iodide staining, electron microscopy and simultaneous detection of CD14 and annexin V binding by flow cytometry. The results showed that myriadenolide induces a dose-dependent apoptosis in monocytes and thus explain the immunosuppressive effect observed.

Minha convivência com Zigman Brener: fatos e circunstâncias / Working with Zigman Brener: facts and circumstances

Depoimento do autor sobre seu convívio com Zigman Brener, mais intenso na década de 70, quando organizaram um curso sobre a técnica de produzir linhagens de células produtoras de anticorpos e quando participaram, no Rio de Janeiro, de uma reuniäo organizada para discutir problemas relacionados com o Trypanosoma cruzi, e que resultou na idéia de se estabelecer um programa denominado "Programa Integrado para o estudo de Doenças Endêmicas - Pide". (MAM)

Screening and fractionation of plant extracts with antiproliferative activity on human peripheral blood mononuclear cells

Three hundred and thirteen extracts from 136 Brazilian plant species belonging to 36 families were tested for their suppressive activity on phytohemaglutinin (PHA) stimulated proliferation of human peripheral blood mononuclear cells (PBMC). The proliferation was evaluated by the amount of [ H]-thymidine incorporated by the cells. Twenty extracts inhibited or strongly reduced the proliferation in a dose-dependent manner at doses between 10 and 100 æg/ml. Three of these extracts appeared to be non-toxic to lymphocytes, according to the trypan blue permeability assay and visual inspection using optical microscopy. Bioassay-guided fractionation of Alomia myriadenia extract showed that myriadenolide, a labdane diterpene known to occur in this species, could account for the observed activity of the crude extract. Using a similar protocol, an active fraction of the extract from Gaylussacia brasiliensis was obtained. Analysis of the H and13C NMR spectra of this fraction indicates the presence of an acetylated triterpene whose characterization is underway. The extract of Himatanthus obovatus is currently under investigation

High levels of IgG4 to Schistosoma mansoni egg antigens in individuals with periportal fibrosis.

Specific IgG4 and IgE responses to adult worm antigen and soluble egg antigen (SEA) were examined in 267 individuals from an area in which schistosomiasis mansoni is endemic. Based on information obtained from clinical and sonographic examinations of this sample, the individuals were divided in three groups: 1) 204 individuals without periportal fibrosis, and liver and spleen enlargements; 2) 41 individuals without periportal fibrosis, but presenting with organopathy, with or without organomegaly; and 3) 22 individuals with periportal fibrosis, regardless of their status as having hepatomegaly and/or splenomegaly. Levels of IgG4 to SEA were significantly higher in sera from patients with fibrosis compared with the patients from the other two groups. We also found significantly higher levels of IgG4 against SEA in egg-negative patients with fibrosis compared with egg-negative patients from the other two groups. This report demonstrates a specific humoral response in patients presenting with initial fibrosis, a form of schistosomiasis transient between intestinal and severe hepatosplenic.

Infection with Schistosoma mansoni correlates with altered immune responses to Ascaris lumbricoides and hookworm.

Studies were performed on humoral and cellular immune responses of patients from areas in Brazil endemic for hookworm and Ascaris lumbricoides, and either endemic or non-endemic for Schistosoma mansoni. Humoral and cellular responses were evaluated by enzyme-linked immunosorbant assay (ELISA) and peripheral blood mononuclear cell (PBMC) proliferation assays against larval hookworm antigens, A. lumbricoides egg antigens, and soluble egg antigens (SEA) or soluble whole adult antigenic preparation (SWAP) from S. mansoni. Patients from S. mansoni-endemic areas, who currently had only hookworm or Ascaris infections, expressed lower humoral and cellular responses to hookworm or Ascaris antigens, respectively, than did their counterparts from areas not endemic for S. mansoni. Individuals from S. mansoni endemic area, although without detectable S. mansoni infection, do mount humoral and cellular responses to SEA and SWAP. This group of individuals has been probably in contact with S. mansoni antigens, since the groups harboring A. lumbricoides or hookworm infections from non-S. mansoni endemic areas do not have detectable anti-S. mansoni responses. PBMC proliferative responses discriminated well between patients with active hookworm infections versus ascariasis, if they were from areas not endemic for S. mansoni.

Screening and fractionation of plant extracts with antiproliferative activity on human peripheral blood mononuclear cells.

Three hundred and thirteen extracts from 136 Brazilian plant species belonging to 36 families were tested for their suppressive activity on phytohemaglutinin (PHA) stimulated proliferation of human peripheral blood mononuclear cells (PBMC). The proliferation was evaluated by the amount of [3H]-thymidine incorporated by the cells. Twenty extracts inhibited or strongly reduced the proliferation in a dose-dependent manner at doses between 10 and 100 micro g/ml. Three of these extracts appeared to be non-toxic to lymphocytes, according to the trypan blue permeability assay and visual inspection using optical microscopy. Bioassay-guided fractionation of Alomia myriadenia extract showed that myriadenolide, a labdane diterpene known to occur in this species, could account for the observed activity of the crude extract. Using a similar protocol, an active fraction of the extract from Gaylussacia brasiliensis was obtained. Analysis of the 1H and 13C NMR spectra of this fraction indicates the presence of an acetylated triterpene whose characterization is underway. The extract of Himatanthus obovatus is currently under investigation.

Some aspects of idiopatyc regulation in Chagas disease and human schistosomiasis

We developed an approach for the examination of antiidiotypic cell-mediated reactivity during chronic human infections. Antibodies against Schistosoma mansoni egg antigens (anti-SEA) and against Trypanosoma cruzi epimastigotes (anti-EPI) prepared from pooled and individual sera from patients with either schistosomiasis mansoni or Chagas disease were immunoaffinity purified on appropriate columns and used to stimulate patients' T cells. These antibodies preparations and their F (ab)2 fragments stimulated in a dose-dependent assay the proliferations of peripheral blood mononuclear cells (PBMC) and T lymphocytes from some, but not all actively infected individuals. Anti-idiotypic T cells can recognize and respond to anti-SEA or anti-EPI idiotypes directly. We contend that the most likely explanation of this stimulations is that the idiotype expressed on these anti-SEA or anti-EPI antibodies are acting as immunogens to stimulate antiidiotypic T lymphocytes that develop during the course of the infection. Immunization of rabbits with these antibodies (anti-SEA or anti-EPI) preparations, followed by absorption of the rabbit antisera on absorbants of normal Ig, produced specific anti-id reagents. Use of these reagents in competitive ELISA distinguished the idiotypic expression on anti-SEA or anti-EPI preparations obtained from patients' sera with different clinical forms of schistosomiasis or Chagas disease, respectively. The anti-SEA Id-reactive T cells from S. mansoni infected patients were capable of regulating autologous in vitro granuloma formation.

Human immune responses during schistosomiasis mansoni

O estudo da resposta imune de pacientes com esquistossomose progride em funçäo do avanço tecnológico, apesar das dificuldades de manipulaçäo in vivo deste complexo sistema parasita-hospedeiro. A ênfase nos estudos tem sido mais freqüentemente dirigida para comparaçöes entre grupos característicos de indivíduos, tais como infectado/näo infectado, reinfectado/näo reinfectado, assintomático/hepato-esplênico, com intensidade de infecçäo alta/baixa, etc. Baseado nessas comparaçöes razoáveis com relaçäo à regulaçäo imunológica, susceptibilidade e resistência e até mesmo consideraçöes sôbre mecanismos, que têm sido mais apropriadamente analisados, entretanto, em sistemas experimentais. Tais estudos têm como finalidade a compreensäo das causas e eventos que levam à morbidade e ao desenvolvimento de uma vacina humana anti-esquistosoma