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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2/3). The CD4+/CD8+ ratio in men (1.34 ± 0.11) was significantly lower than that of women (1.58 ± 0.15). CD4+ T cells and the ratio of CD4+/CD8+ were significantly decreased both in subjects infected with S. japonicum and those with parenchymal fibrosis. However, levels of total IgA, IgM, and anti-schistosome egg antigen IgG correlated positively with infection intensity and pathologic lesion number. These results suggest an imbalance between cell-mediated and humoral immunity in these fishermen, the precise cause of which remains undetermined. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The human immune response is regulated by a network of cross-regulatory cytokines released by T helper (Th) cells. However, the exact role played by cytokines in regulating susceptibly or resistance remains unclear. Reports on the immune status of individuals with schistosomiasis at different stages of pathology (acute, chronic, and advanced) have shown that the phenomenon of cellular immune suppression exists among people with chronic and advanced schistosomiasis.2–5 However, the majority of these studies mainly focused on patients in hospitals who were classified clinically. Population-based studies of human immune status are lacking, especially in populations at high risk of infection. This study aimed to explore the relationship between immune responses and infection intensity or pathologic changes in the liver among a group of migrant fishermen who live and work on boats in the Dongting Lake in Hunan Province, China; these fishermen have a very high level of water exposure and a high frequency of infection.6 The completed analysis was based on infection studies, morbidity measurements, and estimates of the levels of CD4+ and CD8+ T cells and antibodies in infected individuals compared with controls.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In addition, 10 subjects (men = 5; women = 5) with a mean age of 28 years from a non-endemic area for schistosomiasis were included as normal controls in this study. All 10 controls were Hunan University students and staff who had no previous history of water contact and who were negative for S. japonicum by fecal examination and serology.
Infection assessment. Individual infection with S. japonicum was assessed by both the quantitative Kato-Katz thick smear examination and the miracidium hatching test, a traditional method developed in China.7 Both methods, based on one stool sample, were described in detail in a previous report.6 Infection intensity (eggs per gram of stool [epg]) was determined by the Kato-Katz technique.
Morbidity assessment of liver and spleen. A portable ultrasonograph (EUB 200; Hitachi Medical Corp., Tokyo, Japan) was used in the field to assess hepatosplenic morbidity in each subject. Protocols for measurement, classification, standard scan positions, and views followed the World Health Organization (WHO) standard protocol established in Cairo in 1990.8 To prevent observer measurement bias, only one experienced observer was invited to conduct all ultrasound measurements. The measurements taken were reported in detail in a previous report.6
Measurement of peripheral blood CD4+ and CD8+ T cells. CD4+ and CD8+ peripheral blood T-lymphocyte subset counts were measured by flow cytometry using a FACS-420 at the Tumor Institute of He Bei Medical University, China. Blood samples (0.5 mL for each subject) were collected in heparinized tubes and distilled water was added (4.5 mL/ tube).9,10 The tubes were left at room temperature for 1 minute, and 0.5 mL sodium chloride (0.9% wt/vol) was added. The supernatants were removed after centrifugation at 1,000 rpm for 5 minutes, and the pellets were washed in phosphate-buffered saline (PBS). The supernatants were removed again by centrifugation at 1,000 rpm for 5 minutes. Formaldehyde (4% vol/vol)-PBS solution was added to the remaining pellets and stored at 4°C. Before measuring CD4+ and CD8+ T cells, the pellets were washed by centrifugation, and mouse anti-human CD4+ and CD8+ monoclonal antibodies were added, together with isosulfocyanic acid fluorandiol. To cell counts of 4 x 106/mL, the same volume of methanol was added to the tubes and lightly mixed. The tubes were left at 4°C for 1 hour, and the cells were processed using ribonuclease and stained by iodide pyridine. The percentage of CD4+/CD8+ T lymphocytes was based on the measurement of 10,000 cells.
Detection of total IgA and IgM levels in serum. Diagnostic reagent kits, based on published procedures,11 were purchased from Shanghai Ke Hua Biology Project Corporation (Shanghai, China), and IgA and IgM levels were assessed by immuno-turbidimetry, performed using an auto-biochemical analyzer at the Clinical Laboratory of Xiang Ya Hospital. Measured units are expressed as milligrams per milliliter.
Detection of specific anti-SEA IgG in serum. Indirect ELISA was used for measuring anti-SEA IgG antibodies. Coating concentration with SEA was 5 µg/mL. Three controls on the ELISA plates included a blank PBS and negative and positive reference sera. Primary human serum samples were diluted at 1:800, and the secondary antibody used was horse-radish peroxidase-staphylococcal protein A (HRP-SPA). OD values (596 mm) were measured using an E960 microplate reader.
Statistical analysis.
SPSS10.0 statistical software was used for data entry and analysis. Student t test was used to compare the means of two groups. The averages among multi-groups were compared by analysis of variance and others by standard 2 x 2 tables (
2). Differences with P < 0.05 were considered statistically significant.
Ethical considerations. An information sheet describing the aims of the study was issued to each participant. Signed consent was collected from the participants or their parents. All patients who were stool-egg positive, except pregnant women, were offered a single dose (40 mg/kg body weight) of praziquantel. All the examination outcomes of the study were reported to local physicians who visited the fishermen’s village. Ethical clearance for this study was obtained from the Medical Ethics Committee of Hunan Province and The Queensland Institute of Medical Research.
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RESULTS |
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Thirty-seven of 94 (39.4%) subjects were diagnosed with parenchymal fibrosis (stage 2 or 3) and 17.7% (17/94) of subjects had periportal fibrosis assessed by ultrasound. Of the 37 patients with parenchymal fibrosis stage 2 or 3, 70.3% (N = 26) were shown to be excreting S. japonicum eggs, and fibrosis appeared to increase with infection intensity. Of the subjects with no parenchymal fibrosis, 52.9% (18/34) also had S. japonicum infection.
CD4+T cells, CD4+/CD8+ T-cell count ratios and antibody levels in fishermen compared with naïve controls. In the 10 control subjects from non-endemic areas, the percentage of CD4+ T cells in the measured 10,000 cells was 49.8% and the ratio of CD4+/CD8+ was 1.62. These values were 39.5% CD4+ T cells and a ratio of 1.37 for CD4+/CD8+ T cells measurable in the migrant fishermen. CD4+ T cell numbers and the ratio of CD4+/CD8+ T cells in the migrant fishermen were significantly lower than those of controls from non-schistosomiasis endemic areas. In general, the levels of serum IgA, IgM, and specific anti-SEA-IgG in the fishermen were higher than those of the controls. There was no significant difference in the levels of serum IgA and IgM between the controls and the 34 egg-negative persons, but the levels of specific anti-SEA-IgG showed a statistically significant difference (P < 0.01) between the controls and the egg-negative individuals. In the migrant fishermen, the CD4+/CD8+ T-cell ratio (1.34 ± 0.11) in men was significantly lower (P < 0.01) than that (1.58 ± 0.15) of women. There was no significant difference in the ratios of CD4+/CD8+ T cells among the age groups (age 6–17 years, 1.37 ± 0.33 years; age 18–44 years, 1.37 ± 0.11; age > 44 years, 1.38 ± 0.10).
Implications of CD4+, CD4+/CD8+ T-cell ratios, and antibodies in infection and intensity of infection with S. japonicum.
CD4+ T-cell numbers and CD4+/CD8+ ratios in the fishermen who were stool-egg positive were significantly lower than in those subjects who were stool-egg negative. However, the numbers of CD8+ T cells were not significantly different in the two groups. The numbers of CD4+ T cells and the CD4+/CD8+ ratios declined with infection intensity (P < 0.05; Table 1
). The levels of total IgM and anti-SEA-IgG, but not IgA, were significantly higher in the stool-egg positive subjects than in the stool-egg negative (Table 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our study, focusing on migrant fishermen at high risk of schistosome infection, measured peripheral blood CD4+ T cells, CD8+ T cells, CD4+/CD8+ T-cell ratios, and antibodies in patients with different infection intensities and with different liver pathologic changes assessed by ultrasound. The results showed that CD4+ T cells and CD4+/CD8 + T-cell ratios in the migrant fishermen were significantly lower than those in control subjects from non-schistosomiasis areas. In the patients infected with S. japonicum, the percent of CD4+ cells in the measured 10,000 cells was markedly lower than those in subjects who were stool-egg negative. Liver parenchymal fibrosis increased with infection intensity. This study showed that the decline in immune response was caused by infection intensity and liver pathology. Others have shown that immunosuppression in schistosome infection may be caused by the activation of CD8+ T-cell function suppressing the maturation of CD4+ T cell subsets or T-cell production, which could account for the decline in CD4 + T cells and the decrease in the CD4+/CD8+ T-cell ratio.16 The relationship between pathogenesis and cellular immunoreactivity in hosts infected with S. japonicum has been linked to the release of components from the worms as they develop inside the host.17,18 In this study, those patients with heavy infections or with stage 2/3 liver fibrosis had reduced CD4+ T cells and a lower CD4+/CD8+ T-cell ratio. A previous study in China also showed that CD4+/CD8+ T-cell ratios were normal or higher in the acute or early stage of schistosomiasis, but in the chronic or advanced stages, the ratios declined.19,20 A possible explanation is that those patients with heavy infections had increased release of antigens from developing worms.21
The immune response to schistosomes at the early stage of infection is determined by CD4+ helper Th1 T-cell and CD8+ T-cell increases, leading to pathologic liver changes. When pathologic changes develop in the liver, the host immune response is modulated by CD4+ helper Th2 T cells and suppressive CD8+ T cells. This modulation of the immune response ensures pathologic changes are kept in check with the onset of the fibrosis that is promoted by Th2 cell-excreted interleukin (IL)-13.22
The status of the humoral immune responses was measured by the levels of total IgA, IgM, and anti-SEA IgG antibodies. These assays showed that the levels of IgM and anti-SEA-IgG antibodies were increased with infection intensity and that IgA and anti-SEA-IgG antibodies, but not IgM, were increased with the staging of parenchymal fibrosis assessed by ultrasonography. It thus seems that the humoral immune responses to S. japonicum infection, especially in heavy infections, and with liver morbidity caused by S. japonicum infection, are increased, which contrasts with the cellular immune responses measured in the same subjects.
This study has shown that there is an imbalance between humoral and cellular immune function in this group of migrant fishermen from the Dongting Lake in China, reflected by a weakened level of cell-mediated immunity concomitant with an increase in humoral immunity. The imbalance is more pronounced with increased infection intensity and aggravation of liver morbidity, but the precise cause of this imbalance remains to be determined.
Received January 31, 2006. Accepted for publication June 23, 2006.
Acknowledgments: The authors thank the fishermen who participated in this survey. We sincerely thank the staff at Hunan Institute of Parasitic Diseases, China for assistance in carrying out this project.
Financial support: This study was supported financially by the National Health and Medical Research Council of Australia and the UK Wellcome Trust.
* Address correspondence to Magda K. Ellis, Molecular Parasitology Laboratory, Queensland Institute of Medical Research, 300 Herston Road, Herston, Brisbane, Queensland 4029, Australia. E-mail: magda.ellis{at}qimr.edu.au 
Authors’ addresses: Yuesheng Li, HHMI International Scholar, Molecular Parasitology Laboratory, Queensland Institute of Medical Research, 300 Herston Road, Herston, Brisbane, Queensland 4029, Australia, Telephone: 61-7-33620405, Fax: 61-7-33620104, E-mail: Yuesheng.Li{at}qimr.edu.au. Qinren Zeng, Xiangya School of Medicine, Central South University, Xiangya Road, Changsha, Hunan, People’s Republic of China. Magda Ellis, Julie Balen (Northcote Graduate Scholar), and Don McManus, Molecular Parasitology Laboratory, Queensland Institute of Medical Research, 300 Herston Road, Herston, Brisbane, Queensland 4029, Australia, Telephone: 61-7-33620405, Fax: 61-7-33620104. Tie Xiong, Hunan Institute of Parasitic Diseases, Huabian Qiao Road, Yueyang, Hunan Province, People’s Republic of China;
Reprint requests: Yuesheng Li, Molecular Parasitology Laboratory, Queensland Institute of Medical Research, 300 Herston Road, Herston, Brisbane, Queensland 4029, Australia.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
