点击显示 收起
Institut Pasteur, Antananarivo, Madagascar
Danish Bilharziasis Laboratory, Departments of Infectious Diseases and Clinical Immunology, Rigshospitalet, Department of Pathology, Hvidovre Hospital
Department of Bacteriology, Parasitology and Mycology, Statens Serum Institut, Copenhagen, Denmark
In this study, we investigated the seminal inflammatory response to egg infestation of the urogenital organs in 240 semen-donating men aged 1549 years living in a Schistosoma haematobiumendemic area of Madagascar. In 29 subjects (12%) with excretion of 5 ova/ejaculate, leukocytospermia (>106 leukocytes/mL) and the presence of seminal lymphocytes and eosinophil leukocytes were each significantly more prevalent than in 74 subjects (31%) who were S. haematobium negative (P < .01). In addition, seminal levels of interleukin (IL)4, IL-6, IL-10, and tumor necrosis factor were significantly higher among seminal eggexcreting subjects than among infection-negative subjects (P < .001). Sexually transmitted infection (STI) with Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, and/or Trichomonas vaginalis did not act as a confounding factor for the observed associations. At follow-up, 6 months after systematic antischistosomiasis and STI syndrome treatment at baseline, the prevalence of seminal leukocytes decreased significantly among the previously seminal eggpositive subjects. The same tendency was observed for the posttreatment levels of cytokines. Numerous studies have already shown an association between STI-associated genital inflammation and human immunodeficiency virus (HIV) propagation. Therefore, the results of the present study suggest that male urogenital schistosomiasis may constitute a risk factor for HIV transmission, as a result of egg-induced inflammation in the semen-producing pelvic organs.
Africa, more than any other continent on the globe, is ravaged by the HIV pandemic, which has resulted in an enormous amount of human suffering and socioeconomic consequences [1]. Epidemiological, clinical, and immunological studies have, in addition to the behavioral element, pointed towards several host, viral, and other biological factors that play a role [25].
Sexually transmitted infections (STIs) have, with substantial supporting evidence, been identified as one of the most important risk factors for transmission HIV in sub-Saharan Africa [69]. HIV-1 transmission is reflected by the size of the viral inoculum in different types of body fluid, including semen from HIV-1infected men [4, 1012]. Gonococcal urethritis has, in this context, been found to be associated with increased seminal viral shedding in HIV-1infected men, in whom treatment was followed by a significant reduction in the seminal load of HIV-1 RNA [13]. Furthermore, Neisseria gonorrhoeae induces cytokine production as part of genital inflammation, and cytokine production mediates the transport of potentially virus-hosting cells into the genital tract and enhance viral replication [8, 1416].
Schistosoma haematobium infection has been hypothesized to cause increased viral shedding into the semen of HIV-1infected men, in a way analogous to STIs, as a result of egg-induced inflammation [17], which, in addition to affecting the bladder, also commonly affects the seminal vesicles and the prostate [1820]. Feldmeier et al. have raised the complementary hypothesis that egg-induced inflammatory lesions in the epithelial lining of the lower reproductive tract in women, analogous to venereal lesions, are associated with an increased risk of HIV transmission [21]. To provide data validating the hypothesized association between male genital schistosomiasis and HIV transmission, we conducted a study of men living in an area where urogenital schistosomiasis and STIs coexist.
MATERIALS AND METHODS
Study area, population, and design.
This study was conducted in a rural setting in the Ambilobe region in northern Madagascar. Participants were, as part of a community-based study of urogenital schistosomiasisassociated morbidity, recruited from 4 villages (Tanambao, Ambodimanga, Ambodikataka, and Ankatoka) randomly selected from among the 16 villages accommodating the workers and their families in the Sirama sugarcane plantation. The area is endemic for S. haematobium, which is transmitted through the canals used for irrigation of the sugarcane fields and for household, hygienic, and leisure activities. A presurvey by urine examination revealed an S. haematobium prevalence of 53.5% among 298 men in the 4 Sirama villages (range, 46.2%58.3%). The village of Mataipako was identified outside of the plantation as a study village of lower endemicity, on the basis of an observed S. haematobium prevalence of 28.7% among 172 presurveyed men. Only subjects with confirmed diagnoses of S. haematobium infection in the Sirama villages were enrolled in the baseline study, whereas S. haematobium infection was not a mandatory criterion for enrollment for subjects from Mataipako. The age range criterion for inclusion in the study was 1549 years. The subjects were examined at baseline and then again after 6 months, by use of an identical examination protocol.
The present study was approved by the Committee of Ethics in the Ministry of Health in Madagascar. Subjects were enrolled in the study only after signed consent was received. The subjects were informed verbally and in writing that participation in the study was on a voluntary basis and that they could withdraw from the study at any given time.
S. haematobium infection and STIs.
The mean egg count in 10 mL of urine was determined by filtration of urine samples collected on 2 different days, by use of a Nucleopore membrane (Costar). Total egg excretion levels in ejaculates were measured by a filtration technique, using a Whatman membrane (Whatman International).
Two urethral swabs were obtained from each subject, for culture of N. gonorrhoeae and Trichomonas vaginalis. One swab was kept in liquid nitrogen at -193°C until culture for gonococci could be performed in a reference laboratory (Statens Serum Institute, Copenhagen, Denmark). The second swab was examined on location for the presence of T. vaginalis, by use of the InPouch TV culture system (BioMed Diagnostics). Chlamydia trachomatis and Mycoplasma genitalium were diagnosed in first-voided urine samples, by use of in-house polymerase chain reaction assays in a reference laboratory (Statens Serum Institute). As part of the community-based study of urogenital schistosomiasisassociated morbidity, 643 men and women were tested for HIV antibodies by use of ELISA (Genscreen HIV 1/2, version 2; Bio-Rad) and Western blotting (New Lav Blot 1 and New Lav Blot 2; Bio-Rad); of these, 6 (1%) tested positive.
Seminal leukocyte count.
After 3 days of ejaculatory abstinence, an ejaculate was collected in a dry plastic container or in a nonspermicidal condom (Seminal Collection Device; HDC Corporation). Sperm count was performed upon receipt of the sample, by use of a counting chamber (Markler; Sefi-Medical Instruments), as described elsewhere [22]. Total and differential leukocyte counts were performed in 20 microscopy fields (×40) in a Papinocolaou-stained seminal smear from each sample. The concentration of leukocytes was expressed as millions of cells per milliliter of seminal fluid, calculated from their incidence relative to the average number of observed spermatozoa in the microscopy fields and with reference to the sperm count value.
Seminal cytokines.
For cytokine analysis, 200 L of seminal fluid from each sample was stored in a container with liquid nitrogen at -193°C until the specimens could be processed. Seminal plasma was centrifuged from seminal fluid at 700 g for 10 min. The concentrations of interferon (IFN), tumor necrosis factor (TNF), interleukin (IL)10, IL-6, IL-4, and IL-2 were measured in the seminal plasma by use of the Cytometric Bead Array (CBA) kit (BD Biosciences) [23].
To validate the method on seminal plasma, samples were spiked with cytokines from the CBA kit, reaching final concentrations of 650, 312, 156, 40, 20, 10, 5, 2.5, 1.25, 0.6, and 0.3 pg/mL. Furthermore. seminal plasma was spiked to a concentration of 666 pg/mL and was subsequently diluted 2-fold to reach a concentration of 0.3 pg/mL (data not shown). Both the spiking assay and the dilution assay showed good discrimination in both the upper and the lower concentration range. The cytokine-specific detection limits were calculated from the average fluorescence of 15 negative samples from the standard curve, multiplied by 3 times the SD, and were as follows: IFN-, 8.0 pg/mL; TNF-, 0.4 pg/mL; IL-10, 2.0 pg/mL; IL-6, 1.2 pg/mL; IL-4, 2.8 pg/mL; and IL-2, 0.8 pg/mL. Cytokine concentrations >5 pg/mL were calculated using CBA software (BD Biosciences). Cytokine concentrations <5 pg/mL were calculated using values between concentrations of 0 and 5 pg/mL on the linear curve of the plot of concentration against fluorescence.
Specificity was tested by the addition of antiTNF- and antiIL-6 to spiked (400 pg/mL) and nonspiked seminal plasma. This procedure completely inhibited the relevant fluorescence signal of all of the samples (data not shown). To test for the influence of the sample collection procedure on the outcome of the seminal cytokine level measurement under the given field-study conditions, spiked (400 pg/mL) and nonspiked samples were left for 8 h at room temperature. This storage had only minimal effect on the cytokines (data not shown).
Treatment.
Each subject was offered the following treatment with oral agents against schistosomiasis and STIs: praziquantel (40 mg/kg) as a single dose and 100 mg of doxycycline twice per day for 7 days (or, alternatively, 1 g of azithromycine as a single dose), in combination with 500 mg of ciprofloxacin and 2 g of metronidazole (both as single doses). The partner(s) of each subject was also offered STI syndrome treatment in accordance with existing recommendations.
Statistics.
Data were analyzed using EpiInfo (version 6.02; Centers for Disease Control and Prevention). Differences in proportions were tested by 2 or Fisher's exact test. The means from different groups at baseline were compared by Kruskall-Wallis test (Mann-Whitney U test), and means from the same group at baseline and at follow-up were compared by Wilcoxon's signed rank test. Correlation between seminal cytokine levels was tested by nonparametric Spearman's test.
RESULTS
A complete data set was obtained from 240 subjects at baseline: 126 subjects in the Sirama villages and 114 in Mataipako. The median age of the study subjects was 31 years. At the 6-month follow-up, 160 subjects (67%) from the baseline study sample were reinvestigated.
S. haematobium infection.
At baseline, urogenital schistosomiasis was diagnosed in 166 (69%) of 240 subjects (table 1). S. haematobium ova were detected in urine from 160 subjects (67%) and in semen from 67 subjects (28%). Moderate to high levels of egg excretion, defined as 50 ova/10 mL in urine and as 5 ova/ejaculate, were observed in 28 subjects (12%) and in 29 subjects (12%), respectively. In the Sirama villages, moderate to high egg levels of excretion in urine and in semen were significantly more prevalent in subjects aged 1539 years than in subjects aged 4049 years: 22%38% versus 5%, respectively, for urine (P = .03) and 26%38% versus 5%, respectively, for semen (P = .02) (table 2). In Mataipako, ova were detected in urine from 34 subjects (30%) and in semen from 21 subjects (18%), and the prevalences of moderate to high levels of egg excretion were 6% and 0%, respectively.
At follow-up, the overall S. haematobium infection prevalence had declined significantly, to 18% (P < 10-6) (table 1). None of the 16 subjects with ova detected in urine at follow-up had moderate to high levels of egg excretion, compared with 3 (23%) of the 13 subjects with seminal egg excretion. Of 29 subjects who received diagnoses of S. haematobium infection at follow-up, 11 (38%) were found to be egg positive in semen only.
STIs.
STIs were diagnosed at baseline in 43 subjects (18%) (table 1). The overall STI prevalences in the Sirama villages and in Mataipako were similar (17% and 19%, respectively), and the age-specific prevalences also did not differ significantly (table 2). At follow-up, the STI prevalence had declined to 7% (P < .001).
Seminal leukocytes and cytokines.
Leukocytospermia, defined as >106 leukocytes/mL, was detected at baseline in 42 subjects (18%)27 (21%) in the Sirama villages and 15 (13%) in Mataipakobut the difference did not reach a significant level (table 2). Lymphocytes in semen were more frequently observed in the Sirama villages than in Mataipako (30% vs. 18%, respectively; P < .02), whereas the prevalence of eosinophil leukocytes was the same in the 2 locations. With regard to age-dependent differences in S. haematobium infection intensity in the Sirama villages, the prevalence of leukocytospermia and seminal lymphocytes was significantly higher in subjects aged 1539 years than in those aged 4049 years: for leukocytospermia, 21%38% versus 8%, respectively (P < .05), and for presence of lymphocytes, 34%38% versus 11%, respectively (P < .01).
In the comparison between the seminal leukocyte findings at baseline and those for the 160 subjects at follow-up, the prevalence of leukocytospermia did not change significantly, whereas the prevalence of lymphocyte-positive smears decreased from 24% at baseline to 4% at follow-up (P < 10-5). A significant decrease was also observed for the eosinophil leukocytepositive smears, from 13% to 6% (P < .03).
Complete baseline and follow-up seminal cytokine data were obtained from 116 subjects (47%) in the study group. Of the 6 cytokines being investigated, IL-6 was detected most often, in 115 (99%) of the baseline ejaculates, followed by TNF- in 89 (77%), IFN- in 31 (27%), IL-10 in 25 (22%), IL-2 in 8 (7%), and IL-4 in 5 (4%). In the correlation analysis between the 6 cytokines, TNF-, IL-10, and IL-6 were each correlated with one another: IL-6/TNF-, r = 0.67; IL-6/IL-10, r = 0.52; and TNF-/IL-10, r = 0.50 (P < .001). Compared with baseline values, the overall prevalence of detectable seminal cytokines remained unaltered at follow-up, whereas there was a nonsignificant tendency toward a decrease in the geometric mean levels.
Bivariate analyses.
In the comparison of the 74 (31%) S. haematobium infectionnegative subjects with the 137 subjects (57%) with low levels of seminal egg excretion (04 ova/ejaculate) and with the 29 subjects (12%) with moderate to high levels of seminal egg excretion (5 ova/ejaculate), it was observed that leukocytospermia was significantly less prevalent in the infection-negative group: 8%, versus 18% in the group with low levels of egg excretion (P < .05) and 38% in the group with moderate to high levels of egg excretion (P < .01) (table 3). Lymphocytes and eosinophil leukocytes were also detected in significantly higher proportions in the group with moderate to high levels of egg excretion than in the infectionnegative group (P < .001 and P < .01, respectively). STIs were diagnosed in the same proportions among all 3 groups.
Of the 116 subjects with complete cytokine data, 45 (39%) were from among the 74 S. haematobium infectionnegative subjects, 59 (51%) were from among the 137 subjects with low levels of seminal egg excretion, and 12 (10%) were from among the 29 subjects with moderate to high levels of seminal egg excretion (table 4). As was observed for the seminal leukocytes, the difference was most significant in the comparison between infection-negative subjects and subjects with moderate to high levels of egg excretion. The prevalence of detectable IL-10 was 75% in the egg-positive group, compared with 13% in the infection-negative group (P < .0001). Of the 5 subjects with seminal IL-4 detected, all belonged to the group of the 12 subjects with egg excretion (P < .0001). Geometric mean levels of IFN-, TNF-, and IL-6 were also significantly higher in the group with moderate to high levels of egg excretion. In the same group of subjects with complete cytokine data, no significant differences were found in geometric mean cytokine levels or in the prevalence of detectable seminal cytokines, leukocytospermia, lymphocytes, and eosinophil leukocytes in the comparison between subjects with diagnoses of STIs and those without diagnoses of STIs (data not shown). In the correlation analysis of seminal egg count, leukocyte count, and cytokine level, the following correlations were observed: egg count/lymphocytes, r = 0.53 (P < .001); egg count/IFN- level, r = 0.37 (P < .05); egg count/IL-10 level, r = 0.59 (P < 0.01), egg count/IL-4 level, r = 0.63 (P < .001), leukocytes/IFN- level, r = 0.65 (P < .001); leukocytes/TNF- level, r = 0.56 (P < .001); leukocytes/IL-10 level, r = 0.57 (P < .001); and leukocytes/IL-6 level, r = 0.68 (P < .001).
Of the 29 subjects with moderate to high levels of seminal egg excretion observed at baseline, 22 (76%) were reexamined at follow-up. The number of subjects with leukocytospermia and differential leukocytes in this group decreased from baseline to follow-up: leukocytospermia, from 9 subjects to 3 subjects (P = .10); lymphocytes, from 12 subjects to 1 subject (P < .001); and eosinophil leukocytes, from 9 subjects to 1 subject (P < .02). IL-4 was not detected in any of the 12 ejaculates collected at follow-up (P < .02), and the geometric mean levels of IFN- and IL-6 decreased significantly, to 16.9 pg/mL (P < .02) and 106.3 pg/mL (P < .05), respectively. The same tendency, although nonsignificant, was observed for TNF- (11.9 pg/mL) and IL-10 (6.3 pg/mL) at follow-up.
DISCUSSION
It seems plausible that urogenital schistosomiasis may be a contributing factor in HIV transmission from a dually infected man to his partner. This study has shown that S. haematobium egg excretion in semen is associated with the presence of neutrophils, lymphocytes, and eosinophil leukocytes. In addition, seminal egg count was correlated with seminal lymphocyte count. Elevated numbers of polymorphonuclear leukocytes in semen are invariably accompanied by increased levels of macrophages and CD4+ lymphocytes, which are the primary HIV host cells [24], and, consequently, HIV is more readily cultured from the semen of seropositive men with leukocytospermia [13]. Eosinophil leukocytes can also express CD4 protein and CD4 receptors for HIV-1 [25, 26], since they may act as host cells for the virus [2729]. As part of the host's immune response to schistosomiasis, activated eosinophil leukocytes secrete, among different mediators, eosinophilic cationic protein (ECP) [30]. Measurable seminal ECP is a reliable indicator of egg-induced eosinophil leukocyterich genital inflammation, since it is found in high concentrations in the presence of seminal S. haematobium ova [17].
A postmortem study of schistosomiasis-infected individuals showed that S. haematobium ova were deposited as frequently in the seminal vesicles as in the bladder wall [18]. However, the egg counts per field were highest in the bladder. The prostate was less frequently affected and had lower egg counts per field than the other pelvic organs. The same study also demonstrated that inflammatory lesions were directly associated with the presence of S. haematobium ova in the seminal vesicles and, to a lesser extent, in prostate tissue. However, it was also observed that the presence of even a few ova provokes marked inflammation, although the reverse situation, in which dense ova deposition is associated with limited inflammation, may occur as well. The variation in the level of egg-induced inflammation from person to person could be explained by many factors, including patient history of infection and treatment, the level of immune status, and genetic susceptibility [3133].
The presence of S. haematobium ova in semen was also associated with an increased prevalence of detectable levels of IL-4 and IL-10 in semen and with significantly higher levels of IFN-, TNF-, and IL-6. Furthermore, both seminal egg count and leukocyte count were correlated with cytokine level.
The retained ova in the host tissue evokes a delayed-hypersensitivity granulomatous T cellmediated immune response mediated by soluble egg antigens, resulting in an accumulation of eosinophils, macrophages, and lymphocytes and thus forming the classic Schistosoma granuloma [34]. The cytokines involved in the formation of granuloma in schistosomiasis infection include TNF-, IL-2, IL-4, and IL-5, whereas IFN-, IL-10, and IL-12 are known for their immune modulating role [3538].
The results we obtained at the 6-months-posttreatment follow-up have added support to the conclusion that semen containing S. haematobium ova is associated with the presence of inflammatory cells and immunological mediators. After the therapeutic clearance of urogenital schistosomiasis, the prevalence of leukocytospermia declined significantly, as did the prevalence of detectable IL-4 in semen. Moreover, a clearand, in some cases, significanttendency for a decrease in cytokine levels was observed at follow-up.
Experimental studies indicate that various cytokines, including IFN-, TNF-, and IL-4, produced by inflammatory and immunocompetent cells attracted to genital infection amplify the local immune response in the epithelial and mucosal cells in the urethra, prostate, and seminal vesicles [24, 39]. Of the 6 cytokines investigated in the present study, TNF- and IL-6 are associated with a stimulatory effect on HIV-1 replication in monocyte-derived macrophages and T cells, and IL-10 is associated with an inhibitory effect [40, 41]. These inflammatory cytokines, along with other soluble mediators [42], may be expected to increase the transport of HIV-1harboring cells into the genital tract and to enhance the rate of viral replication and viral release into the semen. Cytokines, which are characteristically associated with most inflammatory lesions, have been shown to induce latently infected T cells and macrophages to produce large amounts of HIV-1 [43]. Alternatively, urethral inflammation itself could be responsible for an increased migration of cell-free HIV-1 from the blood to the seminal plasma [13].
STIs did not act as confounding variables for the observed associations between seminal egg excretion and leukocytospermia, and STIs were not found to be associated with elevated levels of seminal cytokines. In this regard, previous studies of N. gonorrhoeae immunopathology have demonstrated that this organism induces an increased secretion of TNF-, IL-4, IL-6, and IL-10 in the genital tract [8, 1416]. Our study sample of STI-infected subjects may have been too small to demonstrate a similar association.
Recently, interest in the detrimental influence of parasitic diseases on the natural history and transmission of HIV infection has increased [4446]. So far, no studies have been able to demonstrate an exacerbating effect of schistosomiasis on the course of HIV-1 infection [47, 48], whereas it has been established that the latter condition affects the immune response patterns of patients with schistosomiasis [4951]. From a therapeutic perspective, it has been shown that the effect of praziquantel treatment on schistosomiasis infection is similar among patients with HIV infection and those without HIV infection [52].
Epidemiological studies of schistosomiasis in endemic communities have demonstrated an age-associated distribution, with the highest prevalence and intensity of infection found among children and young adults [53]. The same tendency toward age dependence was observed in the present study, in which egg excretion in urine, as well as that in semen, was higher in subjects aged 1539 years than in older subjects. This observation leads to the speculation that young men coinfected with urogenital schistosomiasis and HIV pose a potential additional risk burden in the transmission and propagation of HIV in S. haematobiumendemic areas, since young men in general have already been identified as a particular target group in global HIV/STI control strategies, because of their sexual risk behavior. Accordingly, urogenital schistosomiasis, because of egg-induced inflammatory lesions in the reproductive tract, may also be considered to be an additional risk factor for contracting HIV infection in young women living in settings where S. haematobium and HIV/STIs coexist [21]. If this hypothesis regarding urogenital schistosomiasis and its alleged associations with HIV-1 transmission are proven correct in clinical studies, antischistosomiasis treatment could constitute another important preventive tool in the control of HIV-1 transmission in the major part of sub-Saharan Africa.
Acknowledgments
Benjamin Scheel Jrgensen is acknowledged for excellent technical assistance, and Dr. Charles Laubscher is acknowledged for his careful review of the manuscript.
References
1. Quinn TC. Global burden of the HIV pandemic. Lancet 1996; 348:99106. First citation in article
2. Clerici M, Giorgi JV, Chou C-C, et al. Cell-mediated immune response to human immunodefiency virus (HIV) type 1 in seronegative homosexual men with recent sexual exposure to HIV-1. J Infect Dis 1992; 165:10129. First citation in article
3. Huang Y, Paxton WA, Wolinsky SM, et al. The role of a mutant CCR5 allele in HIV-1 transmission and disease progression. Nat Med 1996; 2:12403. First citation in article
4. Dyer JR, Kazembe P, Vernazza PL, et al. High levels of human immunodeficiency virus type 1 in blood and semen in seropositive men in sub-Saharan Africa. J Infect Dis 1998; 177:17426. First citation in article
5. Rizzardini G, Piconi R, Ruzzante S, et al. Immunological activation markers in the serum of African and European HIV-seropositive and seronegative individuals. AIDS 1996; 10:153542. First citation in article
6. Wasserheit JN. Epidemiological synergy: interrelations between human deficiency virus infection and other sexually transmitted diseases. Sex Transm Dis 1992; 19:6177. First citation in article
7. Dickerson MC, Johnston J, Delea TE, White A, Andrews E. The causal role of genital ulcer diseases as a risk factor for transmission of human immunodeficiency virus: an application of the Bradford Hill Criteria. Sex Transm Dis 1996; 23:42940. First citation in article
8. Anzala AO, Simonsen JN, Kimani J, et al. Acute sexually transmitted infections increase human immunodeficiency virus type 1 plasma viremia, increase plasma type 2 cytokines, and decrease CD4 cell counts. J Infect Dis 2000; 182:45966. First citation in article
9. Nkengasong JN, Kestens L, Ghys PD, et al. Human immunodefiency virus type 1 (HIV-1) plasma virus load and markers of immune activation among HIV-infected female sex workers with sexually transmitted diseases in Adidjan, Cote d'Ivoire. J Infect Dis 2001; 183:14058. First citation in article
10. Fang G, Burger H, Grimson R, et al. Maternal plasma human immunodefiency virus type 1 RNA level: a determinant and projected threshold for mother-to-child transmission. Proc Natl Acad Sci USA 1995; 92:121004. First citation in article
11. Busch MP, Operskalski EA, Mosley JW, et al. Factors influencing human immunodefiency virus type 1 transmission by blood transfusion. J Infect Dis 1996; 174:2633. First citation in article
12. Lee T-H, Skahara N, Fiebig E, Busch MP, O'Brien TR, Herman SA. Correlation of HIV-1 RNA levels in plasma and heterosexual transmission of HIV-1 from infected transfusion recipients. J Acquir Immune Defic Syndr Hum Retrovirol 1996; 12:4278. First citation in article
13. Cohen MS, Hoffman IF, Royce RA, et al. Reduction of concentration of HIV-1 in semen after treatment of urethritis: implications for prevention of sexual transmission of HIV-1. AIDSCAP Malawi Research Group. Lancet 1997; 349:186873. First citation in article
14. Ramsey KH, Schneider H, Cross AS, et al. Inflammatory cytokines produced in response to experimental human gonorrhea. J Infect Dis 1995; 172:18691. First citation in article
15. Fichorova RN, Desai PJ, Gibson FC 3rd, Genco CA. Distinct pro-inflammatory host responses to Neisseria gonorrhoeae infection in immortalized human cervical and vaginal epithelial cells. Infect Immun 2001; 69:58408. First citation in article
16. Maisey K, Nardocci G, Imarai M, et al. Expression of pro-inflammatory cytokines and receptors by human fallopian tubes in organ culture following challenge with Neisseria gonnorrhoeae. Infect Immun 2003; 71:52732. First citation in article
17. Leutscher P, Ramarokoto CE, Reimert C, Feldmeier H, Esterre P, Vennervald BJ. Community-based study of genital schistosomiasis in men from Madagascar. Lancet 2000; 355:1178. First citation in article
18. Gelfand M, Ross CM, Blair DM, Castle WM, Weber MC. Schistosomiasis of the male pelvic organs: severity of infection as determined by digestion of tissue and histologic methods in 300 cadavers. Am J Trop Med Hyg 1970; 19:77984. First citation in article
19. Smith JH, Kamel IA, Elwi A, Von Lichtenberg FA. Quantitative post mortem analysis of urinary schistosomiasis in Egypt. I. Pathology and pathogenesis. Am J Trop Med Hyg 1974; 23:105471. First citation in article
20. Patil PS, Elem B. Schistosomiasis of the prostate and the seminal vesicles: observations in Zambia. J Trop Med Hyg 1988; 91:2458. First citation in article
21. Feldmeier H, Poggensee G, Krantz I, Helling-Giese G. Female schistosomiasis as a risk factor for HIV transmission. Int J STD AIDS 1994; 5:36872. First citation in article
22. Makler A. A new chamber for rapid sperm count and motility estimation. Fertil Steril 1978; 30:3138. First citation in article
23. Chen R, Lowe L, Wilson JD, et al. Simultaneous quantification of six human cytokines in a single sample using microparticle-based flow cytometric technology. Clin Chem 1999; 45:16934. First citation in article
24. Wolff H, Anderson DJ. Male genital tract inflammation is associated with increased numbers of potential human immunodeficiency virus host cells in semen. Andrologia 1988; 20:40410. First citation in article
25. Lucey DR, Nicholson-Weller A, Weller PF. Human eosinophils express CD4 protein and bind human immunodeficiency virus 1 gp 120. J Exp Med 1989; 169:32732. First citation in article
26. Rubbert A, Combadiere C, Ostrowski M, et al. Dendritic cells express multiple chemokine receptors used as co-receptors for HIV entry. J Immunol 1998; 160:393341. First citation in article
27. Conway B, Baskar P, Bechtel LJ, et al. Eosinophils as host cells for HIV-1. Arch Virol 1992; 127:3737. First citation in article
28. Freedman AR, Gibson FM, Flemming SC, Spry CJ, Griffin GE. Human immunodeficiency virus infection of eosinophil leucocytes in human bone marrow cultures. J Exp Med 1991; 174:16614. First citation in article
29. Weller PF, Marshall WL, Lucey DR, Rand TH, Dvorak AM, Finberg RW. Infection, apoptosis, killing of mature human eosinophiles by human immunodeficiency virus-1. Am J Respir Cell Mol Biol 1995; 13:61020. First citation in article
30. Reimert CM, Ouma JH, Mwanja MT, et al. Indirect assessment of eosinophiluria in urinary schistosomiasis using eosinophil cationic protein (ECP) and eosinophil protein X (EPX). Acta Trop 1993; 54:112. First citation in article
31. Yazdanbakhsh M. Common features of T cell reactivity in persistent helminth infections: lymphatic filariasis and schistosomiais. Immunol Lett 1999; 65:10915. First citation in article
32. Dessein AJ, Hillaire D, Elwali NE, et al. Severe hepatic fibrosis in Schistosoma mansoni infection is controlled by a major locus that is closely linked to the interferon- receptor gene. Am J Hum Genet 1999; 65:70921. First citation in article
33. Mduluza T, Ndhlovu PD, Midzi N, et al. T cell clones from Schistosoma haematobium infected and exposed individuals lacking distinct profiles for Th1/Th2 polarisation. Mem Inst Oswaldo Cruz 2001; 96(Suppl):89101. First citation in article
34. El Ridi R, Ismail S, Gaafar T, El Demellawy M. Differential responsiveness of humans with early-stage schistosomiasis haematobium to Schistosoma haematobium soluble adult-worm and egg antigens. Parasitol Res 1997; 83:4717. First citation in article
35. Amiri P, Locksley RM, Parslow TG, et al. Tumour necrosis factor restores granulomas and induces parasite egg-laying in schistosome SCID mice. Nature 1992; 356:6047. First citation in article
36. Wynn TA, Cheever AW. Cytokine regulation of granuloma formation in schistosomiasis. Curr Opin Immunol 1995; 7:50511. First citation in article
37. King CL, Malhotra I, Mungai P, et al. Schistosoma haematobiuminduced urinary tract morbidity correlates with increased tumor necrosis factor and diminished interleukin-10 production. J Infect Dis 2001; 184:117682. First citation in article
38. Henri S, Chevillard C, Mergani A, et al. Cytokine regulation of periportal fibrosis in humans infected with Schistosoma mansoni: IFN- is associated with protection against fibrosis and TNF- with aggravation of disease. J Immunol 2002; 169:92936. First citation in article
39. McGhee JR, Mestecky J, Elson CO, Kiyono H. Regulation of IgA synthesis and immune response by T cells and interleukins. J Clin Immunol 1989; 9:17599. First citation in article
40. Kedzierska K, Crowe SM. Cytokines and HIV-1: interactions and clinical implications. Antivir Chem Chemother 2001; 12:13350. First citation in article
41. Kedzierska K, Crowe SM, Turville S, Cunningham AL. The influence of cytokines, chemokines and their receptors on HIV-1 replication in monocytes and macrophages. Rev Med Virol 2003; 13:3956. First citation in article
42. Ho JL, He S, Hu A, et al. Neutrophils from human immunodeficiency virus (HIV)-seronegative donors induce HIV replication from HIV-infected patients' mononuclear cells and cell lines: an in vitro model of HIV transmission facilitated by Chlamydia trachomatis. J Exp Med 1995; 181:1493505. First citation in article
43. Rosenberg ZF, Fauci AS. Immunopathogenic mechanisms of HIV infection: cytokine induction of HIV expression. Immunol Today 1990; 11:17680. First citation in article
44. Bentwitch Z, Kalinkowich A, Weisman Z. Immune activation is a dominant factor in the pathogenesis of African AIDS. Immunol Today 1995; 16:18791. First citation in article
45. Wolday D, Berhe N, Akuffo H, Britton S. Leishmania-HIV interaction: immunopathogenic mechanisms. Parasitol Today 1999; 15:1827. First citation in article
46. Harms G, Feldmeier H. HIV infection and tropical parasitic diseasesdeleterious interactions in both directions Trop Med Int Health 2002; 7:47988. First citation in article
47. Lawn SD, Karanja DM, Mwinzia P, et al. The effect of treatment of schistosomiasis on blood plasma HIV-1 RNA concentration in co-infected individuals. AIDS 2000; 14:243743. First citation in article
48. Elliott AM, Mawa PW, Joseph S, et al. Associations between helminth infection and CD4+ T cell count, viral load and cytokine responses in HIV-1-infected Uganda adults. Trans R Soc Trop Med Hyg 2003; 97:1038. First citation in article
49. Fontanet AL, Woldemichael T, Sahlu T, et al. Epidemiology of HIV and Schistosoma mansoni infections among sugar-estate residents in Ethiopia. Ann Trop Med Parasitol 2000; 94:14555. First citation in article
50. Mwinzi PN, Karanja DM, Colley DG, et al. Cellular immune response of schistosomiasis patients are altered by human immunodefiency virus type 1 coinfection. J Infect Dis 2001; 184:48896. First citation in article
51. Karanja DM, Hightower AW, Colley DG, et al. Resistance to reinfection with Schistosoma mansoni in occupationally exposed adults and effect of HIV-1 co-infection on susceptibility to schistosomiasis: a longitudinal study. Lancet 2002; 360:5926. First citation in article
52. Karanja DM, Boyer AE, Strand M, et al. Studies on schistosomiasis in western Kenya. II. Efficacy of praziquantel for treatment of schistosomiaisis in persons coinfected with human immunodeficiency virus-1. Am J Trop Med Hyg 1998; 59:30711. First citation in article
53. Serieye J, Boisier P, Ravaolimalala VE, et al. Schistosoma haematobium infection in western Madagascar: morbidity determined by ultrasonography. Trans R Soc Trop Med Hyg 1996; 90:398401. First citation in article