Memórias do Instituto Oswaldo Cruz Fundação Oswaldo Cruz, Fiocruz ISSN: 1678-8060 EISSN: 1678-8060 Vol. 102, Num. 2, 2007, pp. 225-228

Memórias do Instituto Oswaldo Cruz, Vol. 102, No.2, March 2007, pp. 225-228

Treatment of human acute schistosomiasis with oxamniquine induces an increase in interferon-γ response to Schistosoma mansoni antigens

Joelma R de Souza*/⁺⁺, Clarice NL Morais, Marcílio L Aroucha**, Paulo JC Miranda****, Constança S Barbosa/⁺⁺⁺, Ana Lúcia C Domingues**, Luiz B Carvalho Júnior*/***/⁺⁺⁺, Frederico GC Abath /⁺⁺⁺, Silvia ML Montenegro/⁺/⁺⁺⁺

Centro de Pesquisas Aggeu Magalhães-Fiocruz, Av. Prof. Moraes Rego s/n^o, Cidade Universitária, 52020-020 Recife, PE, Brasil *Departamento de Bioquímica **Hospital das Clínicas ***Departamento de Patologia ****Laboratório de Imunopatologia Keizo Asami, UFPE, Recife, PE, Brasil
⁺Corresponding author: silvia@cpqam.fiocruz.br, ⁺⁺Capes fellow, ⁺⁺⁺CNPq research scholarships

Financial support: CNPq, Capes, Fiocruz

Received 17 August 2006
Accepted 23 January 2007

Code Number: oc07039

Patients with acute schistosomiasis were studied before and after oxamniquine treatment. They had been exposed to cercariae 5 to 9 weeks before, and presented compatible clinical manifestations, eosinophilia, and high levels of total IgE. Interferon-g (IFN-γ) and interleukin-4 were measured by ELISA in whole blood samples under soluble egg antigen or soluble adult worm preparation stimulation. After treatment, the reduction of leukocytosis and eosinophilia were not significant, but total IgE levels decreased significantly, in contrast to IFN-γ levels that were significantly increased. The oxamniquine treatment of acute schistosomiasis patients is followed by an improvement of a Th1 response in vitro. If this response has a protective aspect is unknown, and some investigations need to be realized.

Key words: Schistosoma mansoni - acute schistosomiasis - oxamniquine - cytokines

In Brazil, schistosomiasis is historically considered to be a rural endemic disease. However, it is gradually expanding into some coastal regions of the state of Pernambuco, due to the exodus of rural workers to the urban areas, resulting in unusual outbreaks of acute schistosomiasis (Barbosa et al. 1998, 2000, 2001). Although immunological responses have been extensively examined in chronic patients, relatively few studies have been carried out with human acute schistosomiasis (Montenegro et al. 1999, Ribeiro de Jesus et al. 2002). In the present paper, patients selected from one of the rare outbreaks of acute schistosomiasis occurred in Brazil, were studied before and after oxamniquine treatment and the immunological findings reported.

Eleven patients infected with acute schistosomiasis mansoni were selected in 2000, during an outbreak occurred in Porto de Galinhas beach, located in the southern coastal area of the state of Pernambuco, Brazil. They had been exposed to cercariae 5 to 9 weeks before, and presented fever, eosinophilia, cough, and, gastrointestinal symptoms. Informed consent was obtained from all patients, in line with the Brazilian ethical guidelines. The 11 subjects studied were 7 males and 4 females with ages ranging from 8 to 34 years (mean = 14 years). Physical, hematological, and stool examinations (according to Hoffman et al. (1934) and Katz et al. (1972) were performed in all patients. Detection of Schistosoma mansoni eggs was achieved in all acute patients studied. No other parasites were detected by stool examination. The parasite burden before treatment ranged from 24 to 1512 eggs/g of stool (mean = 220; SEM = 131). Blood samples (5 ml) were collected into heparin (10 U/ml) for leukocyte counts and cellular culture, before and 6 months after specific treatment with oxamniquine (15 mg/kg of body weight, single oral dose). Total blood leukocytes were stained with Turk's solution and counted in a Neubauer chamber. The relative and absolute eosinophil levels were determined in Giemsa stained blood smears. Whole blood without separation of peripheral blood mononuclear cells, were diluted in RPMI 1640 medium (1:3) plus penicilin (100 U/ml) and streptomicyn (100 μg/ml), and 0.7 ml were placed per well, as described previously (Montenegro et al. 1999). The cells were stimulated with soluble egg antigens (SEA, 20 μg/ml), soluble worm antigen preparations (SWAP, 50 μg/ml), and phorbol myristate acetate (PMA, 50 ng/ml) plus ionomycin (IONO, 1 μg/ml) as mitogens in a 48 well flat-bottom plates and incubated at 37^oC in a 5% CO₂ humidified atmosphere. Then, supernatants were collected after 96 h, quickly frozen, and stored at 70^oC until use. Interferon-g (IFN-γ) and interleukin-4 (IL-4) were quantified by capture ELISA (enzyme linked immunosorbent assay) as described previously (Montenegro et al. 1999). Eleven healthy Brazilian individuals, living in non-endemic areas of Pernambuco, with negative stool examinations and no previous history of exposure to schistosoma infection were included as controls. Total IgE (E immunoglobulin) antibodies were measured in plasma by the ACCESS (Beckman Coulter) immunoassay, as described elsewhere (Addison et al. 1972). Differences between paired groups were assessed by the Wilcoxon signed rank test and correlation between variables was determined by calculating the Spearman's rank correlation coefficients. Statistical significance was considered at P < 0.05.

Most of the reinfection studies indicate that schistosome-specific Th2 responses are associated with protective immunity after treatment of chronically infected patients (Butterworth 1998). In this context, it has been previously suggested that drug-dependent killing of worms in situ might boost protective immunity against reinfection by immunizing with antigens released from dying worms (Dunne et al. 1992, Webster et al. 1997, Woolhouse & Hagan 1999). More recently, it was shown that the immune responses induced by chemotherapy is heterogeneous, depending on the type of antigen used and time of analysis after treatment (Fitzsimmons et al. 2004, Joseph et al. 2004). However, most of the immunological studies after schistosomiasis treatment were undertaken in chronically infected patients living in endemic areas. We studied the effects of oxamniquine treatment in acutely infected patients that did not live in endemic areas and were selected during an outbreak of acute schistosomiasis in a coastal area of Brazil. Although the cytokine assays were not performed in a large sample, the present paper contributes to a better understanding of the immune responses after treatment, because acute schistosomiasis is rare in endemic areas. Our results demonstrated that after treatment the IFN-γ levels increased significantly in response to SEA (P = 0.0076), SWAP (P = 0.0076), and mitogens (P = 0.0099, Figure). Before treatment, the production of IFN-γ was similar to controls (data not shown). This is in line with studies that demonstrated increased IFN-γ production by T cell clone populations derived from treated acute patients (Contigli et al. 1999). Similarly, it was reported that patients with chronic disease living in endemic area showed increased levels of IFN-γ after treatment, in response to parasite antigens (Correa-Oliveira et al. 2000). Probably, the increase in T cell reactivity after chemotherapy can be explained by exposure of released antigens to the immune system following destruction of worms by chemotherapy (Araújo et al. 1996, Grogan et al. 1996, Brito et al. 2000, Correa-Oliveira et al. 2000). Nonetheless, the production of IFN-γ post-treatment did not correlate significantly with the parasite burden before treatment, possibly because of the small number of patients analyzed. It has been suggested that schistosome infection downmodulates the immune responses (Colley et al. 1986, Zwingenberger et al. 1989). Thus, alternatively, the increase in T cell reactivity after the treatment of infection, could be explained by termination of modulation and release from the repression of the immune response (Joseph et al. 2004). Although there are evidence for a role of IL-10 in the suppression of the Th1 response in late stages of the acute phase (16 weeks after exposure) of human schistosomiasis (Montenegro et al. 1999, Abath et al. 2006), it is not clear if this would occur at earlier stages of acute human schistosomiasis. On the other hand, there are reports that levels of IFN-γ induced by SEA and SWAP were not significantly changed by treatment in chronic patients from endemic areas 7 weeks after treatment with praziquantel (Joseph et al. 2004).

Chronically infected patients treated with pra-ziquantel produced significantly more IL-4 and IL-5 in response to SWAP, although this was not observed in response to SEA (Joseph et al. 2004). In contrast, we could not demonstrate a significant increase in the production of IL-4 after treatment of acute schistosomiasis, as production of IL-4 in response to SEA or SWAP were similar to controls before and after chemotherapy (data not shown). The majority of studies on human immune responses highlighted the importance of humoral immunity based on IgE responses, having eosinophils as effector cells (Hagan et al. 1991). Herein we showed that acutely infected patients presented leukocytosis with eosinophilia and high levels of total IgE. Plasma IgE levels (Table) and eosinophil counts were significantly decreased after treatment (P = 0.0164 and 0.0208, respectively). Differently, studies in chronic patients demonstrated an increase in the levels of IgE antibodies against adult worm antigens after three months of treatment (Correa-Oliveira et al. 2000).

In summary, treatment of acute schistosomiasis with oxamniquine is followed by an improvement of a Th1 response in vitro. If this response has a protective character is unknown and demands further investigations.

ACKNOWLEDGEMENTS

To Wlademir G de Melo for his technical assistance

REFERENCES

• Abath FGC, Morais CNL, Montenegro CEL, Wynn TA, Montenegro SML 2006. Immunophatogenic mechanisms in schistosomiasis: what can be learnt from human studies? Trends Parasitol 22: 85-91.
• Addison GM, Beamish MR, Hales CN, Hodgkins M, Jacobs A, Llewellin P 1972. An immunoradiometric assay for ferritin in the serum of normal subjects and patients with iron deficiency and iron overload. J Clin Pathol 25: 326-329.
• Araújo MI, Jesus AR, Bacellar O, Sabin E, Pearce EJ, Carvalho EM 1996. Evidence of a T helper type 2 activation in human schistosomiasis. Eur J Immunol 26: 1399-1403.
• Barbosa CS, Domingues AL, Abath FGC, Montenegro SML, Guida U, Carneiro J, Tabosa B, Morais CNL, Spinelli V 2001. Epidemia de esquistossomose aguda na Praia de Porto de Galinhas, Pernambuco, Brasil. Cad Saú Públ 17: 725-728.
• Barbosa CS, Gonçalves JE, Albuquerque Y, Barbosa FS 1998. Urban schistosomiasis in Itamaracá Island, Pernambuco, Brazil: epidemiological factors involved in the recent endemic process. Mem Ist Oswaldo Cruz 93: 265-266.
• Barbosa CS, Pieri OS, Silva CB, Barbosa FS 2000. Ecoepidemiologia da esquistossomose urbana na Ilha de Itamara-cá, Pernambuco. Rev Saú Públ 34: 337-341.
• Brito CFA, Caldas IR, Coura-Filho P, Correa-Oliveira R, Oliveira SC 2000. CD4⁺ T cells of schistosomiasis naturally resistant individuals living in endemic area produce interferon-g and tumour necrosis factor-alpha in response to the recombinant 14 kDa Schistosoma mansoni fatty acid-binding protein. Scand J Immunol 51: 595-601.
• Butterworth AE 1998. Immunological aspects of human schistosomiasis. Br Med Bull 54: 357-368.
• Colley DG, Barsoum IS, Dahawi HS, Gamil F, Habib M, El alamy MA 1986. Immune responses and immunoregulation in relation to human schistosomiasis in Egypt. III Immunity and longitudinal studies of in vitro responsiveness after treatment. Trans R Soc Trop Med Hyg 80: 952-957.
• Contigli C, Silva-Teixeira DN, Prete GD, D'elios MM, De Carli M, Manghetti M, Amedei A, Almerigogn F, Lambertucci JR, Goes AM 1999. Phenotype and cytokine profile of Schistosoma mansoni specific T cell lines and clones derived from schistosomiasis patients with distinct clinical forms. Clin Immunol 91: 338-344.
• Correa-Oliveira R, Caldas I, Martins-Filho A, Queiroz C, Lambertucci J, Cunha-Melo J, Silveira A, Prata A, Wilson R, Gazzinelli G 2000. Analysis of the effects of treatment of human Schistosoma mansoni infection on the immune response of patients from endemic areas. Acta Trop 77: 141-146.
• Dunne D, Butterworth AE, Fulford AJC, Kariluki HC, Langley JG, Ouma JH, Capron A, Pierce RJ, Sturrock RF 1992. Immunity after treatmen of human schistosomiasis: association between IgE antibodies to adult worm antigens and resistance to reinfection. Eur J Immunol 22: 1483-1494.
• Fitzsimmons CM, Joseph S, Jones FM, Reimert CM, Hoffmann KF, Kazibwe F, Kimani G, Mwatha JK, Ouma JH, Tukahebwa EM, Kariuki HC, Vennervald BJ, Kabatereine NB, Dunne DW 2004. Chemotherapy for schistosomiasis in Ugandan fishermen: treatment can cause a rapid increase in interleukin-5 levels in plasma but decreased levels of eosinophilia and worm-specific immunoglobulin E. Infect Immun 72: 4023-4030.
• Grogan JL, Kremsner PG, Deelder AM, Yazdanbakhsh M 1996. Elevated proliferation and interleukin-4 release from CD4+ cells after chemotherapy in human Schistosoma of interferon-g and interleukin-10 in the responses of peripheral blood mononuclear cells and splenocytes to parasite antigens. J Infect Dis 179: 1502-1514.
• Hagan P, Blumenthal U, Dunne D, Simpson A, Wilkins H 1991. Human IgE, IgG4 and resistance to reinfection with Schistosoma haematobium. Nature 349: 243-245.
• Hoffman VA, Pons JS, Janer JL 1934. Sedimentation concentration method in schistosomiasis masoni. Puerto Rico J Publ Health Trop Med 9: 283-289.
• Joseph S, Jones FM, Walter K, Fulford AJC, Kimani G, Mwatha JK, Kamau T, Kariuki HC, Kazibwe F, Tukahebwa EM, Kabatereine NB, Ouma JH, Vennervald BJ, Dunne D 2004. Increases in human T helper 2 cytokine responses to Schistosoma mansoni worm and worm-tegument antigens are induced by treatment with praziquantel. J Infect Dis 190: 835-842.
• Katz N, Chaves A, Pellegrino J 1972. A simple device for quantatitive stool thick-smear technique in schistosomiasis mansoni. Rev Inst Med Trop São Paulo 14: 397-400.
• Montenegro SML, Miranda P, Mahanty S, Abath FGC, Teixeira K, Coutinho E, Brinkman J, Golçalves I, Domingues LA, Domingues AL, Sher A, Wynn T 1999. Cytokine production in acute versus chronic human schistosmiasis mansoni: the cross-regulatory role of interferon-g and interleukin-10 in the responses of peripheral blood mononuclear cells and splenocytes to parasite antigens. J Infect Dis 179: 1502-1514.
• Ribeiro de Jesus A, Silva A, Santana LB, Magalhaes A, de Jesus AA, de Almeida RP, Rego MA, Burattini MN, Pearce EJ, Carvalho EM 2002. Clinical and immunologic evaluation of 31 patients with acute schistosomiasis mansoni. J Infect Dis 185: 98-105.
• Webster M, Fallon PG, Fulford AJ, Butterworth AE, Ouma JH, Kimani G, Dunne D 1997. Effect of praziquantel and oxamniquine treatment on human isotype responses to Schistosoma mansoni: elevated IgE to adult worm. Parasite Immunol 19: 333-335.
• Woolhouse ME, Hagan P 1999. Seeking the ghost of worms past. Nat Med 5: 1225-1227.
• Zwingenberger K, Irschink E, Siqueira Vergetti JG, correia Dacal AR, Janssen-Rosseck R, Bienzle U, Huber C, Feldmeier H 1989. Relese of interleukin-2 and interferon-g by peripheral mononuclear cells in human Schistosoma mansoni infection normalizes after chemotherapy. Scand J Immunol 30: 463-471.

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