Abstract: Glycerol derivatives are a class of compounds, which are easy and inexpensive to produce with potent anti-malarial activities against blood stages of Plasmodium falciparum in vitro. In the present study, one of these compounds, termed 1t, which had the lowest IC(50) values, was assessed in a murine malarial model. Nuclear magnetic resonance imaging and Balb/c mice infected with Plasmodium berghei ANKA strain were treated in a 4-day suppressive test. Mice received a once-daily intraperitoneal administration of 50 mg/Kg of the drug for 4 days. Although no parasitaemia clearance was reached, a slower parasite proliferation and a slightly longer survival time compared with the placebo group were observed.
Abstract: Numerous epidemiological studies have shown an inverse correlation between helminth infections and the manifestation of atopic diseases, yet the immunological mechanisms governing this phenomenon are indistinct. We therefore investigated the effects of infection with the filarial parasite Litomosoides sigmodontis on allergen-induced immune reactions and airway disease in a murine model of asthma. Infection with L. sigmodontis suppressed all aspects of the asthmatic phenotype: Ag-specific Ig production, airway reactivity to inhaled methacholine, and pulmonary eosinophilia. Similarly, Ag-specific recall proliferation and overall Th2 cytokine (IL-4, IL-5, and IL-3) production were significantly reduced after L. sigmodontis infection. Analysis of splenic mononuclear cells and mediastinal lymph nodes revealed a significant increase in the numbers of T cells with a regulatory phenotype in infected and sensitized mice compared with sensitized controls. Additionally, surface and intracellular staining for TGF-beta on splenic CD4(+) T cells as well as Ag-specific TGF-beta secretion by splenic mononuclear cells was increased in infected and sensitized animals. Administration of Abs blocking TGF-beta or depleting regulatory T cells in infected animals before allergen sensitization and challenges reversed the suppressive effect with regard to airway hyperreactivity, but did not affect airway inflammation. Despite the dissociate results of the blocking experiments, these data point toward an induction of regulatory T cells and enhanced secretion of the immunomodulatory cytokine TGF-beta as one principle mechanism. In conclusion, our data support the epidemiological evidence and enhance the immunological understanding concerning the impact of helminth infections on atopic diseases thus providing new insights for the development of future studies.
Abstract: Helminths facilitate their own survival by actively modulating the immune systems of their hosts. We investigated the impacts that different life cycle stages of the rodent filaria Litomosoides sigmodontis have on the inflammatory responses of mice injected with sublethal doses of lipopolysaccharide (LPS). Mice infected with female adult worms from prepatent infections, worms which have not yet started to release microfilariae, developed lower levels of proinflammatory cytokines in the peripheral blood after LPS challenge than sham-treated controls, demonstrating that female adult worms can mitigate the innate immune response. The presence of microfilariae in mice, however, through either direct injection or implantation of microfilaria-releasing adult female worms, turned the LPS challenge fatal. This lethal outcome was characterized by increased plasma levels of gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), interleukin 12 (IL-12), and IL-6, greater numbers of macrophages and granulocytes in the peripheral blood, and decreased body temperatures in microfilaria-infected mice. Microfilaria-infected mice deficient in IFN-gamma receptor and TNF receptor 1 had increased survival rates after LPS challenge compared to immune-competent mice, suggesting that microfilariae worsen LPS-induced sepsis through actions of IFN-gamma and TNF-alpha. In summary, we have demonstrated that infection of mice with L. sigmodontis female adult worms from prepatent infections protects mice injected with LPS whereas microfilariae worsen LPS-induced sepsis through the induction of proinflammatory cytokines and upregulation of granulocytes, NK cells, and monocytes in the peripheral blood.
Abstract: Strategies for the control of human parasitic diseases such as onchocerciasis and lymphatic filariasis require an understanding of how the parasite successfully infects and persists in humans. Despite the fact that the vast majority of infective larvae are eliminated after infection, this 'protection' is far from being an all-or-nothing response. The hypothesis presented here, which is based on epidemiological observations, suggests that the resistance against filarial parasites includes a time-dependent component, probably caused by an early immune response with short-term memory. Validating this hypothesis requires experimental studies with a longitudinal component. Such experiments would help to clarify whether the infection can be controlled by vaccination strategies at all.
Abstract: River blindness is a seriously debilitating disease caused by the filarial parasite Onchocerca volvulus, which infects millions in Africa as well as in South and Central America. Research has been hampered by a lack of good animal models, as the parasite can only develop fully in humans and some primates. This review highlights the development of two animal model systems that have allowed significant advances in recent years and hold promise for the future. Experimental findings with Litomosoides sigmodontis in mice and Onchocerca ochengi in cattle are placed in the context of how these models can advance our ability to control the human disease.
Abstract: BACKGROUND: MHC-class-II-deficient mice lack T helper cell dependent immune reactions. T cell related immune functions are critical for normal wound healing. We hypothesized that MHC-II-deficiency compromises wound repair by affecting the normal wound immune response. MATERIAL AND METHODS: Groups of 10 male MHC-class II-knockout mice and wild-type controls underwent dorsal skin incision. Polyvinyl alcohol sponges were then inserted subcutaneously. The mice were sacrificed 10 days later to determine wound breaking strength and reparative collagen deposition. Activity of T cells and macrophages isolated from the spleens and from the healing wounds was investigated. Fibroblasts derived from the wounds were tested ex vivo for proliferative activity and collagen synthesis. RESULTS: Wound collagen deposition and wound breaking strength were impaired in MHC-class-II-knockout mice (P < 0.05). Impaired healing was reflected in diminished mitogen-reactivity of splenic T-cells (P < 0.01), and decreased CD4 expression in wounds. In addition, basal and LPS + IFN-gamma-induced synthesis of TNF-alpha and nitric oxide by wound-derived macrophages was impaired. Exvivo, fibroblast proliferation and fibroblast collagen production from MHC-II-deficient mice was decreased. CONCLUSION: MHC-II-deficiency compromises wound healing. This may be a reflection of impaired wound immune cell function and decreased activity of wound fibroblasts.
Abstract: Infection with the cestode Echinococcus multilocularis causes human alveolar echinococcosis (AE), a life-threatening disease affecting primarily the liver. Despite the severity of AE, clinical symptoms often develop only many years after infection, which suggests that E. multilocularis has developed mechanisms which depress anti-parasite immune response, thus favouring immune evasion. In this study we examined the production of cytokines, chemokines and the expression of CD molecules on peripheral blood mononuclear cells (PBMC) from AE patients and healthy controls in response to E. multilocularis metacestode culture supernatant, viable E. multilocularis vesicles and E. multilocularis vesicle fluid antigen in vitro. After 48 h of co-culture, E. multilocularis metacestode culture supernatant and E. multilocularis vesicles depressed the release of the proinflammatory cytokine interleukin (IL)-12 by PBMC. This effect was dose-dependent and a suppression of tumour necrosis factor (TNF)-alpha and IL-12 was observed even when PBMC were activated with lipopolysaccharide (LPS). Comparing proinflammatory cytokine release by AE patients and controls showed that the release of IL-12 and TNF-alpha was reduced in AE patients, which was accompanied by an increased number of CD4+ CD25+ cells and a reduced release of the Th2 type chemokine CCL17 (thymus and activation regulated chemokine, TARC), suggesting an anti-inflammatory response to E. multilocularis metacestode in AE patients. Instead the production of interferon (IFN)-gamma and the expression of CD28 on CD4+ T cells were increased in PBMC from AE patients when compared to controls. This was accompanied by a higher release of the Th2-type chemokine CCL22 (macrophage derived chemokine, MDC) supporting that E. multilocularis also generates proinflammatory immune responses. These results indicate that E. multilocularis antigens modulated both regulatory and inflammatory Th1 and Th2 cytokines and chemokines. Such a mixed profile might be required for limiting parasite growth but also for reducing periparasitic tissue and organ damage in the host.
Abstract: Macrophage-derived nitric oxide is a critical mediator in wound healing. Its regulation in vivo, however, remains unclear. We hypothesized that interferon (IFN)-gamma plays an important role in the regulation of nitric oxide in wounds. Groups of 12 male IFN-gamma -knockout mice and wild-type controls underwent dorsal skin incision and polyvinyl alcohol sponges were inserted subcutaneously. Mice were sacrificed 10 days later to determine wound breaking strength and reparative collagen deposition. Synthesis of nitric oxide (NO), tumor necrosis factor (TNF)-alpha, and IFN-gamma was measured in the wound. Wound-derived macrophages were tested for NO synthesis in the presence or absence of IFN-gamma, TNF-alpha, and anti-TNF-alpha antibody. In a separate experiment, IFN-gamma -knockout mice and wild-type controls were treated with molsidomine, a nitric oxide donor. It was found that wound collagen deposition and wound breaking strength were impaired in IFN-gamma-knockout mice (p < .05). Impaired healing was reflected in diminished synthesis of TNF-alpha and NO in wounds (p < .05). In vivo treatment with molsidomine reversed impaired healing in IFN-gamma-deficient mice. Ex vivo, addition of IFN-gamma stimulated the synthesis of TNF-alpha and NO in wound-derived macrophages. IFN-gamma -induced NO synthesis by wound-derived macrophages was abolished by anti-TNF-alpha-antibody-treatment, which could be fully reversed by exogenous TNF-alpha. Thus we conclude that IFN-gamma-deficiency impairs wound healing and diminishes NO synthesis in wound-derived macrophages. The stimulatory effect of IFN-gamma on macrophage NO production depends on endogenous TNF-alpha synthesis.
Abstract: BALB/c and C57BL/6 mice were experimentally infected with Angiostrongylus costaricensis and the parasitic parameters and antibody response during the acute and chronic phases of infection were analyzed. Following administration of six third-stage larvae (L3), there was no significant difference in the mean worm recovery or mean larval output. Coinciding with the maturation of worms in infected animals and with the egg output in mesenteric arteries, a strong increase in the humoral immune response was observed in both mouse strains. This response was characterized by a hypergammaglobulinemia, with a predominance of IgA and IgG1 during the acute phase of infection, and IgG1 and total IgE during the patent and post-patent periods. Significantly higher levels of IgM, IgG and IgG1 were found in BALB/c mice compared with C57BL/6 mice. On the other hand, a significantly higher concentration of IgA was detected at 6 and 7 weeks post-infection in C57BL/6 mice compared with BALB/c mice. Specific IgE could not be detected in any of the mouse strains. Our results suggest that immunoglobulins, mainly IgG1, contribute to the outcome of a primary A. costaricensis infection with respect to the period of patency and to mortality during the chronic phase.
Abstract: Passage of parasites and their antigens across the placenta occurs with metazoan as well as protozoan parasites, and this study addressed to which extent exposure to and infection of mothers with Plasmodium spp. and Entamoeba histolytica/dispar has sensitized their offspring for parasite-specific immune responses. While at delivery none of the mothers presented with an acute malaria attack, 42% were seropositive for P. falciparum. In half of the mothers cysts of E. histolytica/dispar were detected in stool specimen, 51% of them were found seropositive for E. histolytica, and E. histolytica-specific immunoglobulin A (IgA) responses were detected in neonates of seropositive mothers as well. Umbilical cord blood cells (UCBC) from neonates, when activated with the mitogen phytohaemagglutinine (PHA) and bacterial streptolysin O (SL-O), released significantly less interferon (IFN)-gamma, interleukin (IL)-10 and tumor necrosis factor (TNF)-alpha into cell culture supernatants than peripheral blood cells (PBMC) of mothers. In response to Plasmodium- and Entamoeba-specific antigens UCBC and PBMC produced equal amounts of IL-1beta, TNF-alpha, IFN-gamma and IL-5, but PBMC from mothers secreted significantly more IL-10. Parasite-specific production of inflammatory and Th(1)- and Th(2)-type cytokines was similar in newborns of Plasmodium and Entamoeba seropositive and seronegative mothers. In summary, repeated exposure and subclinical infection of mothers with E. histolytica or P. falciparum will suffice to prime in utero their children for inflammatory and both Th(1)- and Th(2)-type cytokine responses, and such broad and mixed cytokine spectrum may be of advantage upon secondary parasite challenge in later life.
Abstract: This study analysed the impact and the extent by which parental Onchocerca volvulus infection, intensity of transmission of O. volvulus infective 3rd-stage larvae (L3) and anthropometric factors may influence the acquisition, development and persistence of O. volvulus infection in offspring. A total of 15290 individuals in 3939 families with 9640 children were surveyed for microfilariae of O. volvulus, and prevalence and level of O. volvulus infection in children aged 0 to 20 years from infected and non-infected parents were followed longitudinally for 18 years. Children from O. volvulus-infected mothers had not only a substantially higher risk to become infected; they also acquired infection earlier in life and developed higher infection levels. Multiple logistic regression analysis showed that maternal O. volvulus infection and children's age are the predominant predictors for patent O. volvulus infection, while the intensity of transmission, measured by the annual transmission potential (ATP) of O. volvulus L3, was less decisive. Longitudinal follow up of children showed that during vector control activities by the Onchocerciasis Control Programme (OCP) and in low-level transmission areas, infection persisted at higher levels in children from O. volvulus-positive mothers. In summary, the dominant risk factor for children to become infected is maternal onchocerciasis, and also age-associated factors will strongly impact on the development of patent O. volvulus infection in offspring.
Abstract: During experimental Angiostrongylus costaricensis infections in several inbred mouse strains, genetic factors as well as different cytokine secretion patterns have recently been shown to play a role in the outcome of infection in terms of morbidity and mortality, e.g. BALB/c mice show a high and C57BL/6 mice a low mortality during the acute phase of infection. In this study, C57BL/6 MHC-II knockout mice infected with A. costaricensis did not show increased mortality during the acute phase of infection when compared with wild-type mice. Furthermore, MHC-II knockout mice showed a strongly diminished parasite-specific humoral and cellular immune response, which can be explained by the nearly complete lack of CD4+ T cells in the periphery. This defect in MHC-II genes, the lack of CD4+ T cells, and the resulting cellular and humoral unresponsiveness resulted in a three times higher output of first-stage larvae in feces compared with wild-type animals. The results indicate that during experimental A. costaricensis infection a parasite-specific immune response, directed via MHC-II molecules and CD4+ T cells, is not essential for the survival of C57BL/6 mice during the acute phase of infection, whereas the elimination of first-stage larvae seems to be regulated by a MHC-II- and CD4+ T-cell-dependent mechanism.
Abstract: In the present study, the cytokines interleukin (IL)-12 and IL-18 were evaluated for their capacity to modulate and to re-direct in vitro parasite antigen-specific cellular responsiveness in patients exposed to Onchocerca volvulus and Entamoeba histolytica infection. We found that IL-18 was highly capable of reducing parasite antigen-induced IL-10 production by PBMC. In contrast, addition or neutralization of IL-12, also in combination with IL-18 and the interferon-gamma-inducible chemokine IP-10 did not affect IL-10 production. Interestingly, the highest IL-10 levels were measured when IL-18 and IP-10 were both neutralized. Although having no effect on IL-10, IL-12 strongly promoted spontaneous and parasite antigen-driven IFN-gamma production by PBMC, whereas IL-18 was only moderately affecting IFN-gamma release by PBMC re-stimulated with E. histolytica- or O. volvulus-specific antigens. Both IL-12 and IL-18 diminished the cellular production of IL-13, and a synergistic effect was observed when the cytokines were combined. Likewise, neutralization of IL-12 enhanced Entamoeba and Onchocerca antigen-driven IL-13 production, but no further increase of IL-13 was observed, when anti-IL-12 and anti-IL-18 were used together. This study disclosed that IL-18 will significantly down-regulate parasite-specific IL-10 production, whereas IL-12 induced IFN-gamma and inhibited IL-13 production by PBMC from humans exposed to O. volvulus and E. histolytica. Such selective immune-regulatory capacity of IL-12 and IL-18 may comprise an important tool to re-direct polarized cytokine responses towards a balanced Th1/Th2 cytokine profile, which may prevent pathology and promote immunity against helminth and protozoan parasite infections.
Abstract: During chronic filariasis, parasite-specific cellular responsiveness is profoundly down-regulated. Cystatins, a group of cysteine protease inhibitors, have been implicated in this suppressive activity. In an attempt to investigate the effects of cystatins in vivo, we isolated and expressed a 14 kDa protein of the rodent filaria Litomosoides sigmodontis with substantial homologies to cystatins from human pathogenic filariae. Cystatin was detected in antigen preparations of several developmental stages of L. sigmodontis, as well as in the supernatants of in vitro cultured adult worms. On closer examination, L. sigmodontis cystatin (Ls-Cystatin) migrated as two separate bands at 14 and 15 kDa. When cystatin was introduced into the peritoneal cavity of C57BL/6 mice via micro-osmotic pumps, the production of nitric oxide was profoundly reduced upon microfilarial challenge and, at the same time, synthesis of TNF-alpha mRNA became up-regulated. Furthermore, antigen-specific proliferative response of spleen cells to circulating L. sigmodontis microfilariae was significantly diminished in the presence of cystatin, whereas the antibody production was not suppressed. In vaccination trials, using the L. sigmodontis/BALB/c mouse model of filariasis, L. sigmodontis cystatin did not generate protective effects in terms of adult worm recovery, however, lower numbers of patent infections, i.e. less infections with microfilaraemia were observed in vaccinated animals. These results suggested that cystatin acts as an immunomodulatory molecule during the course of a filarial infection, and its neutralisation might contribute to generate protective immune responses.
Abstract: Filarial infections of humans are chronic diseases. Despite an ongoing immune response, adult filariae continuously produce their offspring, the microfilariae (Mf), which are able to persist in sufficient numbers to ensure transmission. In this study, host- and parasite-derived factors, which contribute to persistence of Mf, were investigated using the filariasis model of Litomosoides sigmodontis in mice. Different strains of mice were found to differ widely in their capability to eliminate circulating Mf. Studies of congenic mouse strains showed that early and rapid clearance of Mf was mediated by activation pathways relevant to innate immunity, whereas late or delayed clearance of Mf was pre-determined by MHC-related factors. Genetic knock-out of genes for the MHC class-II molecules totally abrogated resistance. Most interestingly, the presence of only I adult female, but not male worms, renders all mice susceptible, irrespective of the genetic background, enabling Mf to circulate for extended periods of time. Such prolonged microfilaraemia was also observed in L. sigmodontis-infected animals challenged with heterologous Mf of Acanthocheilonema viteae. The use of cytokine gene knock-out mice showed that persistence of L. sigmodontis Mf was facilitated by IL-10, but not by IL-4 or IFN-gamma. In conclusion, irrespective of a resistant or susceptible host genetic background, survival of Mf of L. sigmodontis in mice is decisively regulated by the presence of adult female L. sigmodontis which will skew and exploit immune responses to facilitate the survival and persistence of their offspring in the infected host.
Abstract: In our experimental study we were able to show that the contrasting outcome of Angiostrongylus costaricensis infection in C57BL/6 and BALB/c mice, in respect of morbidity and mortality, can be explained by divergent cellular immune responses and a different cytokine pattern in each strain. In BALB/c mice (i.e. those with high mortality), the initial high proliferation of ConA or LPS stimulated spleen cells dropped to very low levels after 2 weeks post-infection (p.i.), whereas in C57BL/6 mice (i.e. those with low mortality), only a minor reduction in lymphoproliferative responses after mitogenic stimulation was observed. The specific proliferation of spleen cells after stimulation with A. costaricensis adult worm antigen remained low in BALB/c mice throughout the experiment, but showed an augmented proliferation in C57BL/6 mice, especially from 2 weeks p.i. onwards. The mitogen-induced production of Th2-type cytokines (IL-4, IL-5, IL-6, IL-10) in spleen cell cultures remained low in BALB/c mice until 4 weeks p.i., but production of Th1-type cytokines (IL-2, IFN-gamma) was highly elevated at 14 and 28 days p.i. In C57BL/6 mice, an upregulated and balanced production of both Th1- and Th2-type cytokines was measured during the course of infection. In summary, a polarization of the immune response towards cellular hyporesponsiveness and a predominantly Th1 cytokine profile was observed in A. costaricensis infected BALB/c mice, which may contribute to pathogenesis and increased morbidity.
Abstract: Onchocerciasis and lymphatic filariasis (LF) are major causes of severe morbidity and considerable socio-economic problems throughout the tropics. Vector control and mass chemotherapy have helped to control these infections in some regions, but the temporary success of such measures argues strongly for the development of vaccines. Success in such a venture will require detailed knowledge of protective immune responses in conjunction with the identification of target antigens. By comparison with other important parasitic infections, such as schistosomiasis and leishmaniasis, work on the development of vaccines for onchocerciasis and LF has been constrained because of the difficulties of producing cyclical and patent filarial infection in laboratory mice. Wolfgang Hoffmann and colleagues here outline the opportunities presented by the rodent filaria Litomosoides sigmodontis for filarial research.
Abstract: Litomosoides sigmodontis in the BALB/c mouse is the only model of filariasis which allows the observation of the complete development in an immunocompetent mouse. In this study, we injected microfilariae (mf) intravenously, as well as into the pleural cavity, the site of natural release of mf from adult female worms, and followed the kinetics of elimination within the host. In susceptible BALB/c mice, mf circulated at high levels in the blood. In contrast, in C57BL/6 mice, which are refractory to full development, mf were eliminated rapidly from the peripheral blood. However, 6 days after intrapleural injection, viable larvae could be found in the pleural cavity and lung capillaries of both susceptible and resistant strains. The numbers of mf in the pleural cavity and lung capillaries in individual mice were significantly correlated, but not dependent on strain or peripheral microfilaraemia. Thus, although C57BL/6 mice showed enhanced production of nitric oxide by pleural exudate cells and a faster change in the numbers of circulating leukocytes after injection, rapid killing of mf by cell or nitric oxide-mediated mechanisms were not the reason for the different outcome. Furthermore, 3 h after iv injection, only a small percentage of mf could be recovered from the peripheral circulation, indicating the presence of a reservoir for mf containment. In conclusion, injected mf showed disparate dynamics of persistence within susceptible and resistant hosts, which is similar to the disparate outcome of natural infections with L. sigmodontis. This difference became obvious within 1 day after injection. The lung capillary system plays obviously a crucial part in regulation of microfilaremia. Our model also provides a possible means to explain frequent cases of occult infections in human filariasis.
Abstract: Nitric oxide (NO) has been shown to be an important effector mechanism in the defence against various pathogens, including filariae. The production of NO, as well as H2O2, is induced by the Th1 cytokine IFN-gamma. Therefore, the microfilariae (mf) of filarial nematodes, which are known to elicit the release of IFN-gamma, may be a target of NO release. In this study, we found that mf of the filarial species Litomosoides sigmodontis were resistant to the attack of H2O2, but vulnerable to NO exposure in vitro by a chemical NO donor, as well as activated macrophages. Adult worms were considerably less affected by exposure to NO. In-vivo production of NO following injection of mf, in this and previous studies, suggested a central role in the defence to filariae. However, neither pharmaceutical inhibition of nitric oxide synthesis, nor genetic knockout of the gene for inducible nitric oxide synthase (iNOS), abrogated resistance to circulating mf in mice. Interestingly, however, iNOS-KO mice showed higher interleukin (IL)-2 responses and lower IL-10 production, compared to their wild-type counterparts. In conclusion, despite its effectiveness in vitro and the observed production of NO by ex vivo cells following infection, nitric oxide seems not to be an important factor in elimination of mf of L. sigmodontis in vivo. However, it may have a regulatory role in the immune response.
Abstract: Several density gradients were tested for the isolation of parasitic nematode, Angiostrongylus costaricensis, first-stage larvae from rodent feces. With a 45/72% Percoll gradient, 83-99% (89.56+/-6.57%) of the larvae were recovered in a clean preparation.
Abstract: Comparisons were made between the immune responses evoked during the course of chronic and patient infections of Litomosoides sigmodontis in susceptible BALB/c mice and non-patent infections in resistant B10.D2 mice. Early antigen specific responses of spleen cells were weak in both mouse strains. However, by day 58 post infection a strong Th2 response, as determined by production of IL-4, IL-5 and IL-10, was observed in BALB/c mice but not in B10.D2 mice. Antibody responses seemed to appear sooner in B10.D2 than in BALB/c mice, and these differentially recognised two antigens of 15 kD and 80 kD.
Abstract: Onchocerca volvulus polypeptides in the molecular mass range of 2.2 to 12.5 kD were separated by Tricine-SDS-PAGE and the serological recognition of these very low molecular weight antigens (VLMW-OvAg) was then investigated by immuno-blotting. Sera from 21 onchocerciasis patients as well as from 53 individuals with other filariases were used to determine the sensitivity and specificity of detection of individual VLMW-OvAg. In onchocerciasis patients, up to 16 VLMW-OvAg were recognized predominantly by IgG1 and IgG4, while only few antigens were recognized by IgG2 and IgG3. The antigen recognition pattern varied individually, but 4 VLMW-OvAg of 8.6, 6.2, 5.4, and 5.1 kD, respectively, were bound by IgG4 from more than 90% of the onchocerciasis patients. Six VLMW-OvAg of 7.3, 5.8, 5.4, 4.0, 3.8, and 3.6 kD were recognized exclusively by IgG1 from onchocerciasis patients. In amicrofilaraemic filariasis patients with lymphatic pathology, a strong reactivity of IgG3 to an OvAg of 2.2 kD was observed, indicating a possible contribution of this antigen to the pathogenesis. In the molecular mass range below 13 kD, no specific carbohydrate residues or phosphorylcholine-containing (PC) determinants could be identified by lectin-blotting or PC-specific immunoblotting, respectively. Two-dimensional separation and immunoblotting distinctly resolved more than 40 antigenic polypeptides, the majority focusing at acidic isoelectric points. In O. volvulus-infected chimpanzees the IgG1- and IgG4-reactivity against OvAg below 13 kD appeared concurrently with onset of patent infection. These data suggest that some of these VLMW-OvAg might be associated with the production and release of microfilariae from gravid female worms as well as be involved in immune-mediated pathogenesis during filarial infections.
Abstract: The filaria Litomosoides sigmodontis, which develops a patent infection in BALB/c mice, was used to determine the fate of a challenge inoculum following immunization of mice with irradiation attenuated infective larvae (3 subcutaneous inoculations at weekly intervals with 25 L3 irradiated at 60 krad, and challenge with 25 L3 two weeks after the final immunization). The adult worm burden of vaccinated mice was reduced to 50% of that of controls although the pattern of larval migration and microfilaraemia were not affected. Necropsies showed that the increased killing of the filariae of the challenge inoculum occurred at the L3 stage within the first 2 days of challenge. This result draws attention on the protective mechanisms operating very early and probably in the subcutaneous region.
Abstract: The present study examined the quantitative and qualitative changes registered in the parasite-specific antibody response, cellular reactivity and cytokine production profile in onchocerciasis patients repeatedly treated with ivermectin over a period of 8 years. The densities of Onchocerca volvulus microfilariae (mf) in treated patients remained significantly reduced, whereas the number of permanently amicrofilaridermic patients (subclinical infection) increased with repeated treatments. In vitro cellular responses to O. volvulus antigen (OvAg) were highest (P < 0.01) in untreated control individuals exposed to infection, but negative for mf of O. volvulus (endemic normals). Cellular reactivity in repeatedly treated patients was higher at 84 than at 36 months post initial treatment (p.i.t); furthermore, the proliferative responses to OvAg, mycobacterial purified protein derivative (PPD) and streptococcal SL-O were greater (P < 0.05) at 84 months p.i.t. in amicrofilaridermic than in microfilaria-positive onchocerciasis patients. In amicrofilaridermic patients such reactivity approached the magnitude observed in endemic normals. Peripheral blood mononuclear cells (PBMC) from patients and endemic normals produced equivalent amounts of IL-2, IL-4 and interferon-gamma (IFN-gamma) in response to mitogenic stimulation with phytohaemagglutinin (PHA); in response to OvAg, however, significantly more IL-2 and IFN-gamma were produced by PBMC from subclinical amicrofilaridermic patients or endemic normals than by mf-positive patients. OvAg-specific production of IL-4 by PBMC from treated patients was lower at 84 than at 36 months p.i.t. At three months p.i.t. the titres of circulating OvAg-specific IgG1-3 had increased (P < 0.05), but they then continuously declined with repeated treatments. Only IgG1 and IgG4 bound to OvAg of mol. wt 2-12 kD at 1 month p.i.t., while recognition of OvAg of mol. wt 10-200 kD by IgG1, IgG2 and IgG4 reached a maximum intensity at 3-6 months p.i.t., with the overall intensity of binding to OvAg gradually weakening thereafter. These results suggest that onchocerciasis-associated immunosuppression is reversible following ivermectin-induced permanent clearance of microfilariae from the skin; and that a vigorous parasite-specific cellular reactivity and a sustained production of IL-2 and IFN-gamma in amicrofilaridermic individuals may contribute to controlling O. volvulus infection.
Abstract: A longitudinal investigation has been conducted into the cell-mediated immune responses of onchocerciasis patients after a single-dose treatment with ivermectin. Untreated patients tested for delayed cutaneous hypersensitivity (DCH) to seven recall antigens showed lower responses than infection-free control individuals (P less than 0.01), but 6 and 14 months after treatment DCH reactions increased to similar levels to those seen in the controls. The in vitro cellular reactivity to Onchocerca volvulus-derived antigen (OvAg) was reduced in untreated patients as compared with controls, and the lymphocyte blastogenic responses to OvAg and streptolysin-O clearly improved up to 14 months after treatment. Peripheral blood mononuclear cells (PBMC) from untreated patients produced IL-1 beta, tumour necrosis factor-alpha (TNF-alpha) and IL-6 in response to mitogenic stimulation with phytohaemagglutinin (PHA), only low levels of IL-1 beta, IL-2 and TNF-alpha in response to OvAg, but higher amounts of IL-4 and interferon-gamma (IFN-gamma) in response to OvAg than control individuals. After ivermectin treatment, the OvAg-induced production of IL-1 beta and TNF-alpha increased significantly 1 and 14 months after treatment. The PHA-induced production of IL-2 and IL-4 increased 1 month after treatment and remained significantly elevated until 14 months after treatment, whereas the OvAg-specific secretion of IL-2, IL-4 and IFN-gamma did not change after ivermectin treatment. Flow cytometric analysis of lymphocyte-subsets in the peripheral blood of untreated patients revealed a relative and absolute (P less than 0.01) diminution of CD4+ cells and a significantly smaller CD4+/CD8+ cell ratio as compared with controls. By 4 weeks after treatment and thereafter, CD4+ T cells increased relatively and absolutely (P less than 0.01); likewise there was an absolute increase in T-helper-inducer cells (CD4+CD45RO+) and a temporarily improved CD4+/CD8+ cell ratio (P = 0.001). The expression of the low-affinity receptor for IgE (CD23) on total lymphocytes decreased from 14% to 7% by 14 months after treatment. The CD8+ cells and CD3+TCR gamma delta + cells were higher in patients than in controls and both remained elevated until 14 months after treatment. These results suggest a distinctly improved cellular immunity in human onchocerciasis that was facilitated by ivermectin therapy.
