Cytokine responses to the anti-schistosome vaccine candidate antigen glutathione-S-transferase vary with host age and are boosted by praziquantel treatment.

BACKGROUND: Improved helminth control is required to alleviate the global burden of schistosomiasis and schistosome-associated pathologies. Current control efforts rely on the anti-helminthic drug praziquantel (PZQ), which enhances immune responses to crude schistosome antigens but does not prevent re-infection. An anti-schistosome vaccine based on Schistosoma haematobium glutathione-S-transferase (GST) is currently in Phase III clinical trials, but little is known about the immune responses directed against this antigen in humans naturally exposed to schistosomes or how these responses change following PZQ treatment. METHODOLOGY: Blood samples from inhabitants of a Schistosoma haematobium-endemic area were incubated for 48 hours with or without GST before (n = 195) and six weeks after PZQ treatment (n = 107). Concentrations of cytokines associated with innate inflammatory (TNFα, IL-6, IL-8), type 1 (Th1; IFNγ, IL-2, IL-12p70), type 2 (IL-4, IL-5, IL-13), type 17 (IL-17A, IL-21, IL-23p19) and regulatory (IL-10) responses were quantified in culture supernatants via enzyme-linked immunosorbent assay (ELISA). Factor analysis and multidimensional scaling were used to analyse multiple cytokines simultaneously. PRINCIPAL FINDINGS: A combination of GST-specific type 2 (IL-5 and IL-13) and regulatory (IL-10) cytokines was significantly lower in 10-12 year olds, the age group at which S. haematobium infection intensity and prevalence peak, than in 4-9 or 13+ year olds. Following PZQ treatment there was an increase in the number of participants producing detectable levels of GST-specific cytokines (TNFα, IL-6, IL-8, IFNγ, IL-12p70, IL-13 and IL-23p19) and also a shift in the GST-specific cytokine response towards a more pro-inflammatory phenotype than that observed before treatment. Participant age and pre-treatment infection status significantly influenced post-treatment cytokine profiles. CONCLUSIONS/SIGNIFICANCE: In areas where schistosomiasis is endemic host age, schistosome infection status and PZQ treatment affect the cellular cytokine response to GST. Thus the efficacy of a GST-based vaccine may also be shaped by the demographic and epidemiological characteristics of targeted populations.

Elevated oxidative stress and DNA damage and repair levels in urinary bladder carcinomas associated with schistosomiasis.

To cast light on mechanisms underlying development of urothelial carcinomas (UCs) of the urinary bladder associated with Schistosomiasis, we immunohistochemically analyzed the relationship between oxidative stress markers, DNA single strand breaks (ssDNA) which could also measure the levels of base damage and apoptosis in DNA, and expression of DNA repair genes with levels of nitric oxide synthases in bladder carcinomas of Egyptian patients with or without Schistosoma hematobium infection. Marked elevation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels was found in squamous cell carcinomas and UCs associated with Schistosomiasis when compared with non-Schistosomal carcinomas. This was accompanied by strong over expression of the DNA-repair genes, 8-oxoguanine-DNA-glycosylase and apurinic/apyrimidinic endonuclease, as well as increased formation levels of ssDNA. Expression levels of inducible nitric oxide synthase (iNOS) which is known to be indirectly related to oxidative stress was higher in Schistosomal than in the non-Schistosomal carcinomas. However, expression of endothelial nitric oxide synthase was slightly stronger in non-Schistosomal than in the Schistosomal carcinomas. In conclusion, these findings suggest a strong correlation between Schistosoma haematobium infection and increased levels of oxidative stress accompanied by a continuous DNA damage and repair in UCs, all directly correlating with elevated iNOS.

Effects of Schistosoma haematobium infection on drug-metabolizing enzymes in human bladder cancer tissues.

The mixed function oxidase system includes the phase I drug oxidation proteins e.g. aryl hydrocarbon hydroxylase (AHH), N-nitrosodimethylamine-N-demethylase I (NDMA-dI) and cytochrome b5 which metabolize most carcinogens and xenobiotics into less and/or more active intermediates. These were determined in human bladder tissues diagnosed as bladder cancer only (10 samples) and bladder cancer associated with Schistosoma haematobium (12 samples) and normal bladder tissues (12 samples). In addition to the above enzymes, agents involved in Phase II drug metabolism e.g. glutathione and glutathione S-transferase as well as free radicals (detected as thiobarbituric acid-reactive substances, TBARS) were also determined in these tissues samples. AAH and NDMA-dI, cytochrome b5, and glutathione S-transferase activity decreased by 42, 28, 47 and 32%, respectively, in human bladder cancer tissues. In bladder cancer tissues associated with S. haematobium infection NDMA-dI and GST activity decreased further by 65 and 56%, respectively, whereas AHH activity increased by 50% and levels of reduced glutathione also increased by 43% in cancer tissue and by 29% in schistocome infected bladder cancer tissue. The level of free radicals also increased significantly (by 57%) in infected bladder cancer tissue but not at all in non-infected cancer tissue. Alterations in the activity of phase I and II of drug-metabolizing enzymes in human bladder tissues as a result of S. haematobium infection may therefore change the bladder's capacity to detoxify many endogenous compounds and may also potentiate the deleterious effects of bladder carcinogens, (e.g. N-nitrosamines) which are known to be present in relatively large quantities in the bladder of patients with schistosomiasis. The present study thus provides new insights into mechanisms for the genesis of bladder cancer initiated in association with schistosomiasis.

Changes in expression and activity of glutathione S-transferase in different organs of schistosoma haematobium-infected hamster.

Schistosomiasis is a major health problem in many subtropical developing countries, causing a number of serious pathologies, including bladder cancer. Most of the toxic compounds formed as a result of these infestations are derived either exogenously or formed endogenously and can be conjugated with glutathione (GSH) via glutathione S-transferase (GST). The present study investigates the effect of Schistosma haematobium infection on the activity of GST and glutathione reductase (GR) and levels of glutathione and free radicals (measured as thiobarbituric acid reactive substances) in different organs of the male hamster. The total activity of GST was increased in several organs; in kidney by 50 and 46% at 6 and 10 weeks postinfection, respectively, and in bladder tissues by 169, 23, and 130% at 2, 4, and 6 weeks postinfection, respectively. In support of this, the expression of GST isozymes was also induced in kidney and bladder tissues at early stages (2, 4, and 6 weeks) and reduced at the later stages of infection (8 and 10 weeks). In contrast, the expression of these isozymes was decreased in the spleen and liver at 2, 4, 6, 8, and 10 weeks postinfection. Also, such activity was decreased in lungs by 74 and 78% and in bladders by 65 and 72% at 8 and 10 weeks postinfection, respectively. GSH levels increased in lungs by 95, 40, and 56% at 2, 4, and 6 weeks and in spleen by 26 and 74% at 4 and 6 weeks, respectively, but decreased at later stages of S. haematobium infection in these organs. The depletion of GSH levels also occurred in bladders by 72 and 54% at 8 and 10 weeks postinfection, respectively. The activity of GR was increased in the livers, lungs, and kidneys of the S. haematobium-infected hamster. TBARS also increased in the lung by 14, 65, 53, 828, and 624% and in the kidney by 64, 29, 87, 190, and 111%, and in the bladder by 216, 23, 1468, 528, and 1025% at 2, 4, 6, 8, and 10 weeks postinfection, respectively. This study indicates that low GST expression and high levels of free radicals could provide new evidence for damage to the bladder and other organs as a result of S. haematobium infection.

Infection induces antibodies against the cercarial secretions, but not against the cercarial elastases of Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum and Trichobilharzia ocellata.

Cercarial secretions from different species of the parasite Schistosoma and from Trichobilharzia ocellata contain a proteolytic activity, cercarial elastase, which was demonstrated by a 30 kDa band in gelatin gels. Sera of patients infected with Schistosoma mansoni, Schistosoma haematobium or Schistosoma japonicum contain immunoglobulin G which react in ELISA with cercarial secretions from all schistosomes and cross-react among the different parasite species. In Western blots, however, infection sera from patients, as well as heavily infected mice or rabbits, did not react with a 30-kDa protein. Moreover, when sections from infected snails (Biomphalaria, Bulinus and Lymnaea) were analysed by immunofluorescence using the same infection sera, only the tegument of the developing cercariae was recognized, but not the acetabular glands. In contrast, when antisera against purified cercarial elastase from either S. mansoni or S. haematobium were tested with sections of infected Biomphalaria or Bulinus, fluorescence was strong in the preacetabular glands of the cercariae of either species, but undetectable with the tegument. Cross-reactivity of both antisera extended to T. ocellata-infected Lymnaea, but not to S. japonicum-infected Oncomelania. In conclusion, although immunization with purified cercarial elastase results in antibody production, the enzyme does not induce an apparent antibody response following natural infection.

Cyclooxygenase-2: a possible target in schistosoma-associated bladder cancer.

OBJECTIVE: To analyse and compare the expression of cyclooxygenase (COX) enzymes in schistosoma-associated bladder cancer, and to determine any association with tumour grade or stage. MATERIALS AND METHODS: Sixty paired samples of tumour and adjacent nonmalignant urothelium were identified. There were 25 squamous and 28 transitional cell carcinomas, and seven adenocarcinomas. Serial sections were obtained and a standard three-layer immunohistochemistry protocol, using COX-1- and COX-2-specific mouse monoclonal antibodies, applied. RESULTS: COX-1 was expressed mostly in nonvascular smooth muscle with weak reactivity in malignant and nonmalignant urothelium. Nonmalignant urothelium expressed COX-2 weakly, notably in areas of dysplasia and squamous metaplasia whereas there was a significant increase in COX-2 (P < 0.001) with moderate to strong granular cytoplasmic expression in all three malignant histological types. The COX-2 reactivity was higher in transitional and adenocarcinomas than in squamous cell carcinoma (P < 0.001). Areas of carcinoma in situ showed COX-2 reactivity comparable with that in invasive areas and more intense than that detected in dysplastic or metaplastic urothelium (P < 0.001). There was a statistically significant positive correlation between COX-2 expression and tumour grade (P = 0.0052). CONCLUSION: COX-2 is over-expressed in schistosoma-associated bladder cancer, consistent with a potential role for COX-2 inhibitors in the prevention and management of this disease.

Some urinary enzymes in bilharziasis.

Urinary alkaline phosphatase (ALP), acid phosphatase (ACP), aryl sulphatase (Ar. sulph.), beta-glucuronidase (beta-gluc.) and galactosidase were assayed in a group of Bilharzia haematobium patients and another group of healthy subjects (control group). The results for most of the determined enzymes revealed high activities as compared to the controls. The activity of acid phosphatase in male urine samples increased also, though not significantly. These elevated enzyme activities could be used to establish the diagnosis of schistosomiasis in patients whose urine contains no ova or when it is difficult to detect them. The results are discussed in the light of localization of each enzyme in the urinary tract as well as in other organs like the liver.

A preliminary report on the prognostic value of selected diagnostic enzymes among certain malignant and schistosomal malignant patients.

The study is a trial to test certain biochemical parameters as differential diagnostic markers between some pathological malignant cases. The first part of the present article was carried out in order to investigate the effect of both cancerous infestation and schistosomal infection on Lactate dehydrogenase (LDH), two transaminases (ALT and AST) activities and total proteins in both serum and tumor tissue isolated from bladder carcinoma patients. The activities were measured in neighboring mucosa to carcinoma tissues together with bladded tissues excised because of malignant lesions and malignant tissues excised because of urinary schistosomiasis, in Egyptian human patients. The second part was design in order to estimate the effect of cancerous disorders on the previous parameters in serum and isolated tumors among colonic carcinoma patients. In addition, the study was extended to explore the changes that might occurred in serum LDH isoenzymatic pattern among some selected cases from these patients.

Circulating enzyme activities of collagen turnover and undulin in patients with various degrees of schistosomiasis and alcoholic liver cirrhosis.

In contrast to alcoholic liver disease, schistosomiasis leads to presinusoidal hepatic fibrosis, which determines the prognosis of the disease. Since conventional liver function tests and liver biopsy provide little information about the dynamics of the fibrotic process, we measured the activities of two circulating enzymes of collagen turnover, namely serum galactosylhydroxylysyl-glucosyl-transferase and plasma prolidase activity, together with undulin, a novel extracellular matrix glycoprotein. The study encompassed 15 healthy control subjects. 69 patients with various stages of Schistosoma mansoni/hematobium infection [28 with early active infection and no organ involvement, 27 with hepatosplenic involvement, and 14 with complications of portal hypertension] and 16 patients with alcoholic liver cirrhosis. Liver biopsies were obtained from 30 schistosomal patients for histopathological grading. Serum galactosylhydroxylysyl-glucosyl-transferase was significantly increased in all clinical stages of schistosomiasis (p < 0.05), but normal in alcoholic cirrhosis. In contrast, plasma prolidase activity showed a significant increase only in early schistosomiasis (p < 0.01), but dropped to subnormal levels in advanced stages (p < 0.001). Undulin was highly elevated both in alcoholic patients and in all schistosomal groups (p < 0.001), and was capable of distinguishing between early and advanced schistosomal stages. We conclude that serum undulin may be a valuable non-invasive parameter for monitoring the course of schistosomal and alcoholic liver disease.

O6-alkylguanine-DNA alkyltransferase activity in schistosomiasis-associated human bladder cancer.

O6-Alkylguanine-DNA-alkyltransferase (ATase) activity was measured in extracts of 55 bladder tissue samples (46 tumour and nine uninvolved mucosal tissue) from Egyptian patients with schistosome-associated bladder carcinoma. Activity varied from 2.0 to 16.2 fmole ATase/microgram DNA (mean +/- S.D.; 5.6 +/- 4.0) or from 28 to 351 fmole ATase/mg (117 +/- 71). ATase levels in schistosome-associated bladder cancer tissues (5.6 +/- 4.0 fmole ATase/microgram DNA) tended to be lower than those observed in normal human bladder mucosal tissue (8.5 +/- 4.4 fmole ATase/microgram DNA). In a previous study (Badawi et al., Carcinogenesis, 1992, 13, 877-881) DNA-alkylation damage (O6-methyldeoxyguanosine) was found in 44/46 of these schistosome-associated bladder cancer samples at levels ranging from 0.012 to 0.485 mumole O6-MedG/mole deoxyguanosine. We now report an inverse correlation between the levels of methylation damage and ATase activity (r = -0.67; P < 0.001). These observations encourage further investigations of the possible role of environmental alkylating agents in the aetiology of early bladder cancer associated with schistosomiasis.

Possible mechanisms of alteration in the capacities of carcinogen metabolizing enzymes during schistosomiasis and their role in bladder cancer induction.

Carcinoma of the urinary bladder is the most common malignancy in many tropical and subtropical countries. There is a well documented association with chronic urinary schistosomal infection, and bladder cancer associated with schistosomiasis is a major cause of morbidity and mortality in the endemic areas. Many factors have been suggested as possible causative agents in schistosome-associated bladder carcinogenesis but theories concerning the possible role of schistosomal infection in altering host metabolism of chemical carcinogens have received most attention. In experimental schistosomiasis there is a common pattern of changes in the activities of several hepatic Phase I and Phase II enzymes. Phase I enzymes show increased activities in the early stages of infection but these activities are reduced to below their preinfection levels in the intermediate and late chronic stages of the disease. The activities of Phase II enzymes are altered in favour of the deconjugation pathways in the later stages of the disease. The possible basic mechanisms that might be involved in such changes during parasitism and their potential role in the induction of bladder neoplasia are discussed.