Cytokines as determinants of resistance and pathology in human Schistosoma mansoni infection

The role of different cytokines in the peripheral blood mononuclear cell (PBMC) proliferative response and in in vitro granuloma formation was evaluated in a cross-sectional study with patients with the different clinical forms and phases of Schistosoma mansoni infection, as well as a group of individuals "naturally" resistant to infection named normal endemic (NE). The blockage of IL-4 and IL-5 using anti-IL-4 and anti-IL-5 antibodies significantly reduced the PBMC proliferative response to soluble egg (SEA) and adult worm (SWAP) antigens in acute (ACT), chronic intestinal (INT) and hepatosplenic (HS) patients. Similar results were obtained in the in vitro granuloma formation. Blockage of IL-10 had no significant effect on either assay using PBMC from ACT or HS. In contrast, the addition of anti-IL-10 antibodies to PBMC cultures from INT patients significantly increased the proliferative response to SEA and SWAP as well as the in vitro granuloma formation. Interestingly, association of anti-IL-4 and anti-IL-10 antibodies did not increase the PBMC proliferative response of these patients, suggesting that IL-10 may act by modulating IL-4 and IL-5 secretion. Addition of recombinant IL-10 decreased the proliferative response to undetectable levels when PBMC from patients with the different clinical forms were used. Analysis of IFN-g in the supernatants showed that PBMC from INT patients secreted low levels of IFN-g upon antigenic stimulation. In contrast, PBMC from NE secreted high levels of IFN-g. These data suggest that IL-10 is an important cytokine in regulating the immune response and possibly controlling morbidity in human schistosomiasis mansoni, and that the production of IFN-g may be associated with resistance to infection.

Absence of linkage between MHC and a gene involved in susceptibility to human schistosomiasis

Six hundred million people are at risk of infection by Schistosoma mansoni. MHC haplotypes have been reported to segregate with susceptibility to schistosomiasis in murine models. In humans, a major gene related to susceptibility/resistance to infection by S. mansoni (SM1) and displaying the mean fecal egg count as phenotype was detected by segregation analysis. This gene displayed a codominant mode of inheritance with an estimated frequency of 0.20-0.25 for the deleterious allele and accounted for more than 50% of the variance of infection levels. To determine if the SM1 gene segregates with the human MHC chromosomal region, we performed a linkage study by the lod score method. We typed for HLA-A, B, C, DR and DQ antigens in 11 informative families from an endemic area for schistosomiasis in Bahia, Brazil, by the microlymphocytotoxicity technique. HLA-DR typing by the polymerase chain reaction with sequence-specific primers (PCR-SSP) and HLA-DQ were confirmed by PCR-sequence-specific oligonucleotide probes (PCR-SSOP). The lod scores for the different q values obtained clearly indicate that there is no physical linkage between HLA and SM1 genes. Thus, susceptibility or resistance to schistosomiasis, as defined by mean fecal egg count, is not primarily dependent on the host's HLA profile. However, if the HLA molecule plays an important role in specific immune responses to S. mansoni, this may involve the development of the different clinical aspects of the disease such as granuloma formation and development of hepatosplenomegaly.

Antigenic stimulation is more efficient than LPS in inducing nitric oxide production by human mononuclear cells on the in vitro granuloma reaction in schistosomiasis

Nitric oxide (NO) is an extremely important and versatile messenger in biological systems. It has been identified as a cytotoxic factor in the immune system, presenting anti- or pro-inflammatory properties under different circumstances. In murine monocytes and macrophages, stimuli by cytokines or lipopolysaccharide (LPS) are necessary for inducing the immunologic isoform of the enzyme responsible for the high-output production of NO, nitric oxide synthase (iNOS). With respect to human cells, however, LPS seems not to stimulate NO production in the same way. Addressing this issue, we demonstrate here that peripheral blood mononuclear cells (PBMC) obtained from schistosomiasis-infected patients and cultivated with parasite antigens in the in vitro granuloma (IVG) reaction produced more nitrite in the absence of LPS. Thus, LPS-induced nitrite levels are easily detectable, although lower than those detected only with antigenic stimulation. Concomitant addition of LPS and L-N-arginine methyl ester (L-NAME) restored the ability to produce detectable levels of nitrite, which had been lost with L-NAME treatment. In addition, LPS caused a mild decrease of the IVG reaction and its association with L-NAME was responsible for reversal of the L-NAME-exacerbating effect on the IVG reaction. These results show that LPS alone is not as good an NO inducer in human cells as it is in rodent cells or cell lines. Moreover, they provide evidence for interactions between LPS and NO inhibitors that require further investigation.

IgE expression on the surface of B1 and B2 lymphocytes in experimental murine schistosomiasis

In a previous study we monitored the distribution and phenotype expression of B1 cells during the evolution of experimental murine schistosomiasis mansoni and we proposed that the B1 cells were heterogeneous: a fraction which originated in the spleen and followed the migratory pathway to mesenteric ganglia, while the other was the resident peritoneal B1-cell pool. In the present study, we have addressed the question of whether these two B1-lymphocyte populations are involved in the production of the late Ig isotype IgE, which is present in high levels in schistosomal infection. Lymphocyte expression of surface markers and immunoglobulins were monitored by immunofluorescence flow cytometry. Both in the spleen and mesenteric ganglia, the B1 and B2 cells were induced to switch from IgM to IgE in the early Th2-dominated phase of the disease, with an increase of IgE in its later phases. Conversely, peritoneal B1-IgM+ switched to the remaining IgE+ present in high numbers in the peritoneal cavity throughout the disease. We correlated the efficient induction of the expression of late Ig isotypes by B1 cells with high levels of inflammatory cytokines due to the intense host response to the presence of worms and their eggs in the abdominal cavity. In conclusion, B1 cells have a different switch behavior from IgM to IgE indicating that these cell sub-populations depend on the microenvironment.

Activity of liver microsomal enzymes during the chronic phase of murine schistosomiasis

The effects of schistosomiasis on microsomal enzymes were studied on post-infection day 90 when accumulated damage and fibrosis are most intense but granulomatous reaction around the eggs harbored in the liver is smaller than during the earlier phases. Swiss Webster (SW) and DBA/2 mice of either sex (N = 12 per sex per group) were infected with 100 Schistosoma mansoni cercariae on postnatal day 10 and killed on post-infection day 90. Cytochrome P-450 (CYP) concentration and alkoxyresorufin-O-dealkylases (EROD, MROD, BROD, and PROD), p-nitrophenol-hydroxylase (PNPH), coumarin-7-hydroxylase (COH), and UDP-glucuronosyltransferase (UGT) activities were measured in hepatic microsomes. Age-matched mice of the same sex and strain were used as controls. In S. mansoni-infected mice, CYP1A- and 2B-mediated activities (control = 100%) were reduced in SW (EROD: male (M) 36%, female (F) 38%; MROD: M 38%, F 39%; BROD: M 46%, F 19%; PROD: M 50%, F 28%) and DBA/2 mice (EROD: M 64%, F 58%; MROD: M 60%; BROD: F 49%; PROD: M 73%) while PNPH (CYP2E1) was decreased in SW (M 31%, F 38%) but not in DBA/2 mice. COH did not differ between infected and control DBA/2 and UGT, a phase-2 enzyme, was not altered by infection. In conclusion, chronic S. mansoni infection reduced total CYP content and all CYP-mediated activities evaluated in SW mice, including those catalyzed by CYP2E1 (PNPH), CYP1A (EROD, MROD) and 2B (BROD, PROD). In DBA/2 mice, however, CYP2A5- and 2E1-mediated activities remained unchanged while total CYP content and activities mediated by other CYP isoforms were depressed during chronic schistosomiasis.

Association of MICA gene polymorphisms with liver fibrosis in schistosomiasis patients in the Dongting Lake region

Major histocompatibility complex class I chain-related A (MICA) is a highly polymorphic gene located within the MHC class I region of the human genome. Expressed as a cell surface glycoprotein, MICA modulates immune surveillance by binding to its cognate receptor on natural killer cells, NKG2D, and its genetic polymorphisms have been recently associated with susceptibility to some infectious diseases. We determined whether MICA polymorphisms were associated with the high rate of Schistosoma parasitic worm infection or severity of disease outcome in the Dongting Lake region of Hunan Province, China. Polymerase chain reaction-sequence specific priming (PCR-SSP) and sequencing-based typing (SBT) were applied for high-resolution allele typing of schistosomiasis cases (N = 103, age range = 36.2-80.5 years, 64 males and 39 females) and healthy controls (N = 141, age range = 28.6-73.3 years, 73 males and 68 females). Fourteen MICA alleles and five short-tandem repeat (STR) alleles were identified among the two populations. Three (MICA*012:01/02, MICA*017 and MICA*027) showed a higher frequency in healthy controls than in schistosomiasis patients, but the difference was not significantly correlated with susceptibility to S. japonicum infection (Pc > 0.05). In contrast, higher MICA*A5 allele frequency was significantly correlated with advanced liver fibrosis (Pc < 0.05). Furthermore, the distribution profile of MICA alleles in this Hunan Han population was significantly different from those published for Korean, Thai, American-Caucasian, and Afro-American populations (P < 0.01), but similar to other Han populations within China (P > 0.05). This study provides the initial evidence that MICA genetic polymorphisms may underlie the severity of liver fibrosis occurring in schistosomiasis patients from the Dongting Lake region.

The value of the Widal test in the diagnosis of Prolonged Septicemic Salmonellosis

Twenty patients with prolonged septicemic salmonellosis (Group 1) and 20 with schistosomiasis mansoni (Group 2) were selected for this study. In both groups, the Widal test was done using antigens of the sample Ty 901 (S. typhi). The test was also applied in 6 group 1 patients with antigens prepared from salmonellae isolated from these patients (autoantigens). Titres over 1:200 were considered significant. Ten group 1 patients (50%) were positive for antigen "H" and 5 (25%) were positive for antigen "O". Three patients with negative "H" and "O" reactions became positive with high titres when using autoantigens. Two other cases maintained the same positive titres and one case showed a fourfold increase in titres when the test was done 'with antigens of the Salmonella isolated. The Widal test was positive in most patients infected with group D Salmonellae. Considering titres above 1:200, all cases were negative in Group 2. The authors conclude that the Widal test has low positivity in prolonged septicemic salmonellosis. The test may be valuable in the diagnosis of this disease when using S. paratyphi "A" and "B" antigens and a mixture of Salmonella antigens taken from other groups.

On the finding of biomphalaria tenagophila naturally infected with Schistosoma mansoni in the State of Minas Gerais, Brazil

Natural infection in Biomphalaria tenagophila with Schistosoma mansoni was observed for the first time in a small area, in Jaboticatubas, in the State of Minas Gerais, Brazil. In view of this finding, the Authors decided to carry out some detailed investigations, since B. glabrata has been known as the vector snail of schistosomiasis in that State. B. tenagophila was found naturally infected throughout the investigation period (15 months). Strains of the snail and of the parasite were isolated four times, and then maintained at the laboratory through B. tenagophila — hamster passages. The possibilities of B. tenagophila to play a role in the epidemiology of the human disease in the State of Minas Gerais, in the future, are discussed.

The effect of Schistosoma mansoni infection on child morbidity in the State in Bahia, Brazil: II — Analysis at the individual level

The present investigation was carried out on a sample of 840 children (5 to 16 years old) from ten small towns of the State of Bahia in northeastern Brazil. The objetive was to study, by using a cross sectional methodology, the evolution of schistosomiasis morbidity (hepatic and splenic enlargement) in children, and the role of the intensity of S. mansoni infection in this process. The children were analised in three age groups (5 to 8, to 12 and 13 to 16 years old) and classified as uninfected, mildly infected, moderately infected and heavily infected according to the number of eggs in the stool. In children aged 5 to 8 years, increasing egg counts were not associated with increasing frequencies of hepatic or splenic enlargement. In the 9 to 12 years old group and association was observed with the prevalence of hepatic enlargement, but not with the prevalence of spleen enlargement. In the oldest group, 13 to 16 years old, an association was observed with the prevalence of enlargement of both organs. It was evident that in this population schistosomiasis morbidity develops in the early period of life as a gradual process starting with liver enlargement and followed by spleen enlargement some years later. It was found that the intensity of infection has a fundamental role in this process, although there is a latent period of some years before clinical splenomegaly appears in moderate-heavily infected children. The Authors suggest that the prevalence of splenomegaly in the 13 to 16 years old group is a good measure of the community level of schistosomiasis morbidity and could be used to measure the impact of control programs.

Stability of Schistosoma mansoni progeny to antischistosomal drugs

The susceptibility of the MAP Brazilian strain (F1 to F5 progenies) of S. mansoni to four antischistosomal drugs has been reported in a previous study. In the present investigation, progeny F14 of the same strain, was tested for stability to the same 4 drugs. A new medication, Oltipraz (35,972 RP), was added to the study. Five groups of 12 mice infected with cercariae by tail immersion were treated with hycanthone, oxamniquine, niridazole, praziquantel and Oltipraz. An untreated group was used as control. Schistosomal activity was assessed by the localization of worms in the portal vein system, by oogram changes, and percentage of parasite reduction. The stability of the susceptibility of progeny F14 did not change in relation to generations F1 to F5; the progeny was resistant to hycanthone and oxamniquine; but sensitive to niridazole, praziquantel and Oltipraz. We emphasize the importance of the phenomenon of resistance of the worm in view of the fact that oxamniquine has been widely used in Brazilian areas where mansonic schistosomiasis is endemic.

Schistosoma mansoni: effects of anesthetics and antimonial drugs on worm shift in the mouse

Mice experimentally infected with Schistosoma mansoni were injected with sodium thiopental or sodium antimonyl gluconate (Triostib R), or submitted to halothane inhalation, with or without a previous injection of thiopental. Data obtained showed that halothane and thiopental induce worm shift to the liver (99 and 76%, respectively). Sodium gluconate and antimonium (Triostib R) shifted 52% of worms towards the liver. These results seem to indicate that the use of antimonium would be unnecessary, when surgical removal of schistosomules is carried out through the extracorporeal filtration technique, in patients with portal hypertension.

Avaliação terapêutica duplo-cega baseada na técnica do oograma quantitativo, comparando praziquantel com oxamniquine na esquistossomose mansônica humana

Um total de 54 pacientes adultos, com esquistossomose mansônica crônica, nas formas intestinal ou hepatintestinal, participou de um ensaio clínico duplo-cego, para comparar o praziquantel com a oxamniquine. De acordo com uma distribuição aleatória, 27 casos receberam o praziquantel (65 mg/kg de peso corporal) e 27 a oxamniquine (18 mg/kg), administrados em dose oral única. A incidência, intensidade e duração dos efeitos colaterais foram similares para os dois medicamentos A avaliação da eficácia terapêutica baseou se na técnica do oograma quantitativo por biópsia da mucosa retal, realizada ao final de um, dois, quatro e seis meses depois do tratamento. Nessas mesmas ocasiões foram feitos exames de fezes pelos métodos de HOFFMAN, PONS e JANER e de KATO-KATZ, com a finalidade de confrontar seu resultados com os achados do oograma. Para averiguar o efeito imediato do tratamento sobre a atividade ovipositora do parasito, um número restrito de pacientes foi submetido a biópsias retáis no 6,° e 18.° dias subsequentes a administração da medicação. Ambas as drogas provaram ser ativas contra o esquistossoma, vez que os respectivos coeficientes de variação, determinados a partir de oogramas efetuados imediatamente após o tratamento, foram superiores a 60%. Ademais, dentre os 27 pacientes de cada grupo, 24 tratados com praziquantel e 22 com oxamniquine completaram o período de seis meses, requerido para controle parasitológico. Os índices de cura, segundo os achados do oograma e dos exames de fezes pelos métodos de HPJ e KK, foram, respectivamente, 29%,— 50% e 92% com o praziquantel; 23%, 50% e 86% com a oxamniquine. Apesar do baixo percentual de cura, observou-se nos oogramas pós tratamento, uma pronunciada queda no número de ovos vivos por grama de tecido. Esses resultados revelam que ambas as drogas foram semelhantemente eficazes, embora já se tenha comprovado que a susceptibilidade do S. mansoni não seja sempre igual para cada um desses medicamentos, pois linhagens resistentes à oxamniquine evidenciaram ser 100% sensíveis ao praziquantel. Por outro lado, constatou-se uma nítida diferença nos percentuais de cura em função do método utilizado para controle parasitológico. O oograma foi o mais preciso, seguido pelo HPJ e, finalmente, pelo K-K. Tendo ocorrido uma correlação direta entre o número de ovos vivos no oograma e a positividade dos exames de fezes, a percentagem de resultados falso-negativos aumentou acentuadamente após o tratamento, alcançando 47,3% com o HPJ e 92,9% com o K-K. Antes da medicação esses índices eram, respectivamente, 0% e 64,8%. Os autores depreendem que a diferença de precisão da metodologia aplicada para avaliar a eficácia terapêutica pode explicar a divergência encontrada entre o índice de cura obtido neste - ensaio clínico e os relatados por outros investigadores, tanto com o praziquantel quanto com a oxamniquine.

Insucesso na indução de resistência a drogas esquistossomicidas em uma cepa brasileira humana de Schistosoma mansoni

No presente estudo, realizou-se uma tentativa de indução de resistência a 3 drogas esquistossomicidas em uma cepa brasileira de S. mansoni, segundo o esquema de indução de resistência tipo II preconizado por JANSMA et al. em 1977. Houve insucesso nas 3 tentativas realizadas. A geração parental tratada com a droga durante o estágio imaturo do verme mostrou-se menos suscetível aos quimioterápicos do que as gerações F1 e F2 do verme. Uma hipótese é levantada para a explicação do fato.

Splenomegaly in relation to Schistosoma mansoni egg counts: a population based study

A cross: sectional survey on schistosomiasis was done in Comercinho (Minas Gerais State, Brazil), a town with 1474 inhabitants. Stool (Kato-Katz method) and physical examinations were done on 90% of the population and on 84% of the individuals over 2 years of age, respectively. The ecological and individual (case-control) analysis were used to investigate the relation between splenomegaly and S. mansoni egg counts in different age groups. In the ecological analysis there was a clearly correspondence between higher geometric mean of eggs and higher percentage of splenomegaly in the age groups 5-9 and 10-12 years. In the individual analysis it was found that only in the youngest individuals (5-8 or 5-9 years old) the splenomegaly was related with higher mean egg counts in the feces, having been a tendency to the decrease of excretion of eggs in patients with splenomegaly as the age increased. These results strongly suggest that the ecological data are' better indicator of the severity of schistosomiasis in endemic areas, as the decrease of the egg excretion in patients with splenomegaly may be a confounding variable for the individual analysis.

Water-contact patterns and schistosoma mansoni infection in a rural community in northeast Brazil

We evaluated the influence of water-related human activities, contaminative behaviour, house location, education and socio-economic status on endemic Schistosoma mansoni infection. The study was conducted in a hilry non-irrigated area of rural northeast Brazil amongst a defined population of subsistence farmers, of whom 93% were infected by age 20. The area was mapped, water bodies were surveyed, and a detailed questionnaire was performed on each household. Infection was assessed by duplicate stool examinations using the sensitive Bell technique to quantify egg excretion. For each household, and index of intensity of infection was computed by grouping individual log-transformed egg counts as an age-sex adjusted Z score. Few households had a sanitary installation or a domestic water supply. However, neither water-contact nor contaminative behavior were indiscriminate. The people made considerable effort to defaecate far from a water source, to obtain household drinking water from the cleanest source, and to bathe only at certain sites where privacy is assured. Land ownership and literacy correlated poorly with the household index of intensity of infection. The key influence on infection status was the relative location of the house and snail-free or snail colonized water sources. In this area, a safe domestic water supply is the critical input needed to achieve definitive control of endemic Schistosomiasis.