Development and application of three-dimensional skin equivalents for the investigation of percutaneous worm invasion.

Investigation of percutaneous helminth infection is generally based on animal models or excised skin. As desirable replacement of animal experiments, tissue-engineered skin equivalents have recently been applied in microbial and viral in vitro infection models. In the present study, the applicability of tissue-engineered skin equivalents for the investigation of percutaneous helminth invasion was evaluated. Epidermal and a full-thickness skin equivalents that suit the requirements for helminth invasion studies were developed. Quantitative invasion assays were performed with the skin-invading larvae of the helminths Strongyloides ratti and Schistosoma mansoni. Both skin equivalents provided a physical barrier to larval invasion of the nematode S. ratti, while these larvae could invade and permeate a cell-free collagen scaffold and ex vivo epidermis. In contrast, the epidermal and full-thickness skin equivalents exhibited a human host-specific susceptibility to larvae of trematode S. mansoni, which could well penetrate. Invasion of S. mansoni in cell-free collagen scaffold was lowest for all experimental conditions. Thus, reconstructed epidermis and full-thickness skin equivalents confirmed a high degree of accordance to native tissue. Additionally, not only tailless schistosomula but also cercariae could permeate the skin equivalents, and thus, delayed tail loss hypothesis was supported. The present study indicates that the limitations in predictive infection test systems for human-pathogenic invading helminths can be overcome by tissue-engineered in vitro skin equivalents allowing a substitution of the human skin for analysis of the interaction between parasites and their hosts' tissues. This novel tissue-engineered technology accomplishes the endeavor to save animal lives.

Immune recognition of Onchocerca volvulus proteins in the human host and animal models of onchocerciasis.

Onchocerca volvulus is a tissue-dwelling, vector-borne nematode parasite of humans and is the causative agent of onchocerciasis or river blindness. Natural infections of BALB/c mice with Litomosoides sigmodontis and of cattle with Onchocerca ochengi were used as models to study the immune responses to O. volvulus-derived recombinant proteins (OvALT-2, OvNLT-1, Ov103 and Ov7). The humoral immune response of O. volvulus-infected humans against OvALT-2, OvNLT-1 and Ov7 revealed pronounced immunoglobulin G (IgG) titres which were, however, significantly lower than against the lysate of O. volvulus adult female worms. Sera derived from patients displaying the hyperreactive form of onchocerciasis showed a uniform trend of higher IgG reactivity both to the single proteins and the O. volvulus lysate. Sera derived from L. sigmodontis-infected mice and from calves exposed to O. ochengi transmission in a hyperendemic area also contained IgM and IgG1 specific for O. volvulus-derived recombinant proteins. These results strongly suggest that L. sigmodontis-specific and O. ochengi-specific immunoglobulins elicited during natural infection of mice and cattle cross-reacted with O. volvulus-derived recombinant antigens. Monitoring O. ochengi-infected calves over a 26-month period, provided a comprehensive kinetic of the humoral response to infection that was strictly correlated with parasite load and occurrence of microfilariae.

Immunohistological studies on neoplasms of female and male Onchocerca volvulus: filarial origin and absence of Wolbachia from tumor cells.

Up to 5% of untreated female Onchocerca volvulus filariae develop potentially fatal pleomorphic neoplasms, whose incidence is increased following ivermectin treatment. We studied the occurrence of 8 filarial proteins and of Wolbachia endobacteria in the tumor cells. Onchocercomas from patients, untreated and treated with antibiotics and anthelminthics, were examined by immunohistology. Neoplasms were diagnosed in 112 of 3587 female and in 2 of 1570 male O. volvulus. The following proteins and other compounds of O. volvulus were expressed in the cells of the neoplasms: glutathione S-transferase 1, lysosomal aspartic protease, cAMP-dependent protein kinase, alpha-enolase, aspartate aminotransferase, ankyrin E1, tropomyosin, heat shock protein 60, transforming growth factor-beta, and prostaglandin E(2). These findings prove the filarial origin of the neoplasms and confirm the pleomorphism of the tumor cells. Signs indicating malignancy of the neoplasms are described. Wolbachia were observed in the hypodermis, oocytes, and embryos of tumor-harbouring filariae using antibodies against Wolbachia surface protein, Wolbachia HtrA-type serine protease, and Wolbachia aspartate aminotransferase. In contrast, Wolbachia were not found in the cells of the neoplasms. Further, neoplasm-containing worms were not observed after more than 10 months after the start of sufficient treatment with doxycycline or doxycycline plus ivermectin.

Comparative characterization of two galectins excreted-secreted from intestine-dwelling parasitic versus free-living females of the soil-transmitted nematode Strongyloides.

Helminths are complex pathogens that ensure their long-term survival by influencing the immune responses of their host. Excretory/secretory products (ESP) can exert immunoregulatory effects which foster parasite survival. Galectins represent a widespread group of ß-galactoside-binding proteins which are involved in a multitude of biological processes operative in parasite-host interaction. We had earlier identified seven galectins in Strongyloides ratti, four of them detected in the ESP of distinct developmental stages of the parasite. In the present report, we focused on the characterization of two of them, Sr-galectin-1 (Sr-Gal-1) and Sr-galectin-3 (Sr-Gal-3). While Sr-Gal-3 expression was strongest in parasitic females, Sr-Gal-1 was predominantly expressed in free-living females. Both proteins were cloned and recombinantly expressed in an E. coli expression system. Their glycan-binding activity was verified by haemagglutination and glycan array analysis. Furthermore, primary immunological activities of the Sr-galectins were initially investigated by the application of an in vitro mucosal 3D-culture model, comprising of mucosa-associated epithelial and dendritic cells. The Sr-galectins stimulated preferentially the release of the type 2 cytokines thymic stromal lymphopoietin and IL-22, a first indication for immunoregulatory activity. In addition, the Sr-galectins dose-dependently fostered cell migration. Our results confirm the importance of these carbohydrate-binding proteins in host-parasite-interaction by indicating possible interaction with the host mucosa-associated cells.

Pathogenesis and host responses in human onchocerciasis: impact of Onchocerca filariae and Wolbachia endobacteria.

Onchocerca volvulus is a tissue-invasive parasitic nematode causing skin and eye pathology in human onchocerciasis. The filariae habour abundant intracellular Wolbachia bacteria, now recognised as obligatory symbionts, and therefore emerging as a novel target for chemotherapy. Recent research demonstrates that both the filariae and endobacteria contribute to the pathogenesis of onchocerciasis, and molecules have been identified that promote inflammatory or counter-inflammatory immune mechanisms, divert the host's immune response or procure evasion of the parasite.

Neutrophil accumulation around Onchocerca worms and chemotaxis of neutrophils are dependent on Wolbachia endobacteria.

Unlike in many other helminth infections, neutrophilic granulocytes are major cellular components in the hosts immune response against filarial worms. The pathways that drive the immune response involving neutrophils are unclear. This study shows that Wolbachia endobacteria (detectable by polyclonal antibodies against endobacterial heat shock protein 60 and catalase and by polymerase chain reaction being sensitive to doxycycline treatment) are direct and indirect sources of signals accounting for neutrophil accumulation around adult Onchocerca volvulus filariae. Worm nodules from untreated onchocerciasis patients displayed a strong neutrophil infiltrate adjacent to the live adult worms. In contrast, in patients treated with doxycycline to eliminate the endobacteria from O. volvulus and to render the worms sterile, the neutrophil accumulation around live adult filariae was drastically reduced. Neutrophils were absent in worm nodules from the deer filaria Onchocerca flexuosa, a species which does not contain endobacteria. Extracts of O. volvulus extirpated from untreated patients showed neutrophil chemotactic activity and in addition, induced strong TNF-alpha and IL-8 production in human monocytes, in contrast to filarial extracts obtained after doxycycline treatment. Thus, neutrophil chemotaxis and activation are induced directly by endobacterial products and also indirectly via chemokine induction by monocytes. These results show that the neutrophil response is a characteristic of endobacteria-containing filariae.

Lipopolysaccharide-like molecules derived from Wolbachia endobacteria of the filaria Onchocerca volvulus are candidate mediators in the sequence of inflammatory and antiinflammatory responses of human monocytes.

The majority of Onchocerca volvulus-infected persons show signs of cellular anergy, and long-time survival of adult and larval parasites in subcutaneous tissue is observed. The mechanisms leading to immunological hyporesponsiveness are poorly understood. Monocytes/macrophages represent a link between the innate and acquired immune system and are candidate cells to promote inflammatory and antiinflammatory processes. In the present study we have shown that products of microfilarial (O. volvulus) and adult (O. volvulus and O. ochengi) parasites affect monocytes in vitro. An early production of TNF-alpha by exposed monocytes was followed by the production of IL-10 and a reduced expression of HLA-DR and the costimulatory molecules B7-1 and B7-2, while other adhesion receptors remained unaffected. Downregulation of the functional membrane receptors failed to occur after treatment of the cells with anti-IL-10 antibodies. The engagement of CD14, a dominant membrane receptor on monocytes and major binding protein for lipopolysaccharides, was indicated by partial blocking of monocyte modulation by neutralizing antibodies to CD14 and by the antagonistic lipid A analog compound 406. Lipopolysaccharide-like molecules were detected in sterile products of O. volvulus stages which could originate from Wolbachia bacteria related to Gram-negative Rickettsiales, known to be abundant in the hypodermis and the female reproductive organs of O. volvulus. The present results indicate that the monocyte/macrophage may be a major target cell for immunomodulatory parasite-derived and intraparasitic, bacteria-derived molecules, thereby contributing to the host's cellular hyporesponsiveness.

The secretory Onchocerca volvulus protein OvS1/Ov20 exhibits the capacity to compete with serum albumin for the host's long-chain fatty acids.

The mechanism by which filarial parasites derive fatty acids bound to the host's carrier protein is poorly understood. The capacity of a secretory protein of Onchocerca volvulus (OvS1/Ov20) to compete with serum albumin for arachidonic and other fatty acids was investigated in this study. Binding affinities of the two proteins for the long-chain fatty acids were determined using displacement assays. The fluorescent probes used included 11-((5-dimethylaminonaphthalene-1-sulfonyl)amino) undecanoic acid (DAUDA) and cis-parinaric acid. OvS1 protein bound arachidonic acid with an affinity five-fold greater than the affinity exhibited by serum albumin. Oleic acid was bound by the parasite protein with an affinity two-fold greater than the affinity shown by serum albumin. Furthermore, the affinities exhibited by OvS1 protein in binding arachidonic and linoleic acid were about two times higher than the affinity for oleic acid. The results suggest that the OvS1 protein has the capacity to compete with the main host's fatty acid carrier protein for the long-chain fatty acids, in particular arachidonic acid, the precursor for eicosanoids.

Hyperreactive onchocerciasis exhibits reduced arachidonate and linoleate levels in serum triglycerides.

The mechanism by which the minority of patients with onchocerciasis exhibiting the hyperreactive (sowda) form of the disease may be able to kill the microfilariae of Onchocerca volvulus is still poorly understood. In this study, the relative amounts of arachidonate and linoleate in serum phospholipids and triglycerides were investigated by gas chromatography both in patients infected with O. volvulus who exhibited either a hyperreactive or a generalized form of onchocerciasis and in persons with no filarial infections. Remarkable differences were observed in the serum triglycerides but not in the phospholipids. In comparison to persons without any filarial infection, significantly lower relative amounts of arachidonate--indicated by elevated triene-tetraene ratios--and of linoleate--indicated by lower diene + tetraene - triene values--were detected in patients with hyperreactive onchocerciasis, and less pronounced differences were found in persons with generalized onchocerciasis. The relationship between reduced amounts of arachidonate and linoleate in serum triglycerides and possible implications on the eicosanoid production in the host-parasite relationship leading to parasite elimination are discussed.

Analysis of renal function in onchocerciasis patients before and after therapy.

The occurrence of renal abnormalities was investigated in patients with onchocerciasis in comparison to individuals without onchocerciasis in Guinea. Serum creatinine levels, excretion of urinary marker proteins, and kidney size by ultrasound were determined. A high prevalence of glomerular as well as tubular dysfunctions was observed; however, no association with onchocerciasis could be detected. We also hypothesized that patients with hyperreactive onchocerciasis might be prone to develop immune-mediated glomerular disorders; however, this could not be verified. Following treatment with ivermectin, a slight but significant increase in the excretion of urinary albumin and alpha1-microglobulin was seen five days after treatment in all treated patients, whereas levels of proteinuria were significantly higher five days after treatment only in patients with high microfilarial densities. Our results indicate that ivermectin can cause glomerular and tubular disturbances in patients with onchocerciasis; however, these are minor and do not seem to be clinically relevant.

A simple and sensitive fluorometric immunoassay for the measurement of immunoglobulins in culture medium of mitogen-stimulated lymphocytes.

A sensitive fluorescent immunoassay (FIA) for the quantification of immunoglobulins (IgG, IgM) in culture supernatants of human lymphocytes has been developed and compared with an ELISA method. FIA, like the ELISA assay, detects nanogram amounts of immunoglobulin (Ig), and has a high degree of specificity and reproducibility. The intra-assay coefficient of variation was about 5% in both test systems; the inter-assay coefficient of variation was approximately 9% in FIA and 13% in ELISA. Quantification of supernatants of lymphocyte cultures by both systems showed a high degree of concordance (correlation coefficient r = 0.93). The advantages of the FIA procedure are the short assay time and the more direct acquisition of signal.

Demonstration of immunoglobulin G antibodies against Onchocerca volvulus excretory-secretory antigens in different forms and stages of onchocerciasis.

The excretory-secretory (E-S) products of helminths are considered to comprise immunogenic molecules of high diagnostic value. In the present study, the serodiagnostic potential of the E-S products released in vitro by cultured female Onchocerca volvulus was investigated by enzyme-linked immunosorbent assay (ELISA) and Western blotting using 190 serum samples from persons infected with O. volvulus and unexposed persons. The sensitivity of detection of anti-O. volvulus E-S antibodies was 94% for sera from patients with the generalized form of onchocerciasis and 100% for sera from patients with the chronic hyperreactive form (sowda). 95% of the sera from amicrofilaridermic persons, who subsequently became microfilaridermic within 2 years, reacted with O. volvulus E-S antigens and the donors were therefore regarded as having had a prepatent infection when first examined. These sera gave higher (P < 0.05) ELISA optical densities than sera from the same persons obtained when they had become patent, indicating a loss of antibody reactivity after emergence of microfilariae. The specificity of the E-S ELISA was 100% when sera of subjects infected with Wuchereria bancrofti were used, and at least 88% for Mansonella perstans sera. In Western blot analysis, the sera of persons with generalized onchocerciasis recognized 7 protein bands. Many E-S proteins were stained less intensely by the sera of subjects with generalized onchocerciasis than by the sera of sowda patients. Similar antigen bands were demonstrated using sera from the persons with prepatent infections.

Serum-dependent interaction of granulocytes with Onchocerca volvulus microfilariae in generalized and chronic hyper-reactive onchocerciasis and its modulation by diethylcarbamazine.

The adherence and cytotoxicity of granulocytes to microfilariae of Onchocerca volvulus were examined in vitro. Reactivity and modulation by diethylcarbamazine of isolated eosinophilic and neutrophilic granulocytes from patients with generalized and chronic hyper-reactive onchocerciasis (sowda or localized form) from endemic foci in Liberia were evaluated under varying serum conditions. In the presence of pooled sera from patients with generalized onchocerciasis granulocytes from both polar groups of patients exhibited similar adherence rates, whereas immobilization rates were higher for eosinophils than for neutrophils. In localized onchocerciasis, the use of autologous serum resulted in a significant decrease in adherence and immobilization rates for both eosinophils and neutrophils. After preincubation of eosinophils, but not of microfilariae, with diethylcarbamazine autologous serum-mediated adherence and cytotoxicity were enhanced to rates similar to those found with pooled serum from individuals with generalized onchocerciasis. These results suggest that granulocytes from both forms of onchocerciasis did not differ with respect to their anti-parasitic reactivity and that antibodies as well as additional serum factors appear to contribute to the functional activity of these effector cells. The findings indicate that predominantly eosinophils, compared to neutrophils, damage the larvae of O. volvulus and support earlier observations which suggest that diethylcarbamazine influences the effector cells rather than the parasite itself.

Delayed-type hypersensitivity reactions to Onchocerca volvulus antigens in exposed and non-exposed African individuals.

Although considered of critical importance, the mode of helper T-lymphocyte function in Onchocerca volvulus infection is still unclear including the role of the Th1/Th2 dichotomy. We studied the delayed-type hypersensitivity (DTH) reaction, which is the classical Th1 response, to O. volvulus antigens in Africans exposed and not exposed to the infection. DTH reactions were found in a small percentage of patients with generalized onchocerciasis, but in a high percentage of patients with localized onchocerciasis, in putatively immune subjects, and also in non-exposed individuals, which may be due to cross-reactivity with other nematodes. These findings support the notions of (i) prenatal influence of maternal O. volvulus infection preventing development of Th1 responses and/or (ii) suppression of Th1 responses by the infection itself.

Ultrastructural study of the interaction between eosinophilic granulocytes and third and fourth stage larvae of Onchocerca volvulus.

The interaction in vitro between eosinophil effector cells and third and fourth stage larvae of Onchocerca volvulus was studied by electron microscopy. The morphological observations demonstrated different mechanisms of attack of eosinophil cells that are dependent upon the time of incubation. Rapid adherence to the cuticle of the target, flattening, secretion of granule contents, vacuole formation and, finally, complete degranulation of the eosinophils were seen after incubation with third stage larvae and moulting stages. Alterations of the epicuticular and cuticular structures could be found near the attachment site of the cells. The eosinophils, however, showed no interactions with fourth stage larvae of this filarial parasite.

Modulatory effects of antifilarial drugs ivermectin, CGP 6140 and CGP 20376 on the oxidative burst of eosinophilic granulocytes.

Three recently introduced anthelmintic agents, the macrocyclic lactone, ivermectin, the amoscanate derivative CGP 6140 and the benzothiazole compound CGP 20376, were investigated for their in vitro modulatory effects on eosinophilic effector cells. The investigation comprised studies on the generation of the toxic oxygen intermediates superoxide anion and hydrogen peroxide which are major effector products of granulocytes. Eosinophils were obtained from 19 patients infected with the filarial parasite Onchocerca volvulus. Inhibitory effects on the generation of toxic oxygen intermediates were demonstrated for ivermectin and CGP 20376 at concentrations higher than 200 ng/ml (0.5 microM) and for CGP 6140 at concentrations higher than 1000 ng/ml (2.7 microM). An increased production of the reactive oxygen metabolites was demonstrated at low doses of ivermectin (20-40 ng/ml; corresponding to 0.02-0.04 microM) and CGP 6140 (40-100 ng/ml; 0.1-0.3 microM), respectively. The results reveal a dual, dose-dependent modulatory in vitro effect of the investigated anthelmintic drugs on the respiratory burst of eosinophilic effector cells indicating that these compounds may modulate host defense in vivo.

Serum levels of eosinophil cationic protein, eosinophil-derived neurotoxin and myeloperoxidase in infections with filariae and schistosomes.

The serum levels of three major granulocyte proteins were measured in patients with onchocerciasis, bancroftian filariasis and intestinal schistosomiasis and compared to controls from patients with malaria, Africans living in areas not endemic for these infections and healthy Germans. The investigation comprised the determination of the eosinophil granule proteins eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN/EPX), and the neutrophil/monocyte granule protein myeloperoxidase (MPO). ECP and EDN/EPX levels were found elevated only in the three helminth infections that are associated with eosinophilia, while MPO was found elevated in all tested disease groups. The levels of eosinophil granule proteins observed in the helminth diseases by far exceeded those described for bronchial asthma and atopic dermatitis. ECP, EDN/EPX and MPO serum levels reflect the ongoing disease and are related to functional activity of the respective leukopoetic system. ECP and EDN/EPX appear to be markers of the eosinophil effector system and MPO a marker of the neutrophil and/or monocyte/macrophage effector system. Significantly higher ECP levels in chronic hyperreactive onchodermatitis (sowda) versus generalized onchocerciasis seem to reflect an augmented degree of antigenic stimulation, eosinophil activation and eosinophil turnover rates, indicating a more active mechanism of parasite clearance in sowda patients.

Differences in eosinophil and neutrophil chemotactic responses in sowda and generalized form of onchocerciasis.

Extravasation of host's leukocytes from blood vessels into inflammatory tissues represents a prerequisite for a subsequent interaction with invaded parasites. The migratory responses of eosinophilic and neutrophilic granulocytes in the polar forms of the filarial infection onchocerciasis were investigated. The hyporeactive, generalized form, the chronic hyperreactive (sowda) form and persons without signs of onchocerciasis from a hypoendemic area for onchocerciasis were compared. Eosinophils from sowda patients responded more strongly to the inflammatory mediator platelet-activating factor (PAF) than those from generalized patients and persons without onchocerciasis. The most significant differences were found between the sowda group and a subgroup of the generalized form with 16-80 microfilariae/mg skin (P<0.05) while patients with low microfilarial density exhibited chemotactic responses similar to the sowda group. In contrast to the strong eosinophil response, neutrophils from sowda patients appeared unreactive to PAF and the tripeptide activator formyl-methionyl-leucyl-phenylalanine (fMLP), thereby differing from both other groups. Laboratory data confirmed a state of hyperreactivity in sowda patients similarly found in generalized patients with low microfilarial density and contrasting with those with a microfilarial density of 16-80 mf/mg. The results revealed an inverse chemotactic responsiveness of eosinophil and neutrophil granulocytes in sowda patients and strengthened the observation of a spectrum of host's responsiveness in onchocerciasis.

Detection of a chemotactic factor for neutrophils in extracts of female Onchocerca volvulus.

Neutrophilic granulocytes and macrophages are the dominant inflammatory cell types observed in the vicinity of and attached to adult Onchocerca volvulus in the subcutaneous nodules. Crude extract from female O. volvulus was examined for chemotactic activity for peripheral neutrophils from healthy individuals by use of an endogenous component chemotactic assay in Boyden chambers. Significant chemotactic responses of neutrophils were detected using O. volvulus extracts at > or = 15 microg/ml in a dose-dependent manner. Checkerboard analysis demonstrated low chemokinetic in addition to chemotactic activity. Neutrophil migration was also elicited by excretory-secretory products of vital females. Fractionation of the female worm extract by FPLC revealed two components with chemotactic activity, one with a molecular mass less than 12 kDa and another with a molecular mass of > 200 kDa. Immunohistological examination of onchocercomas containing only one adult alive filarial worm demonstrated that neutrophils were accumulated near and attached to the cuticle of immature females, females producing microfilariae and males.

Eosinophils, eosinophil cationic protein and eosinophil-derived neurotoxin in serum and urine of patients with onchocerciasis coinfected with intestinal nematodes and in urinary schistosomiasis.

Eosinophils, eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN/EPX), myeloperoxidase (MPO) and IgE were measured in blood, serum and/or urine in Schistosoma haematobium- and Onchocerca volvulus-infected Guineans and O. volvulus- and S. haematobium-negative Guineans coinfected or infected with intestinal nematodes. The number of eosinophils and levels of eosinophil granule proteins but not of MPO were found to be strongly elevated in all Africans as compared to European controls. The highest serum ECP and serum and urinary EDN/EPX levels were observed in the hyperreactive form of onchocerciasis (sowda). Onchocerciasis patients and O. volvulus-negative Africans coinfected or infected with intestinal nematodes (hookworm and/or Ascaris lumbricoides) revealed higher serum granule protein concentrations and/or absolute eosinophil counts and urinary ECP than those without nematode infections. Statistical differences between both sections were found for the absolute eosinophil counts and for serum EDN/EPX and IgE in generalized onchocerciasis, and for urinary ECP in sowda, indicating stimulation of the eosinophil potential of O. volvulus-positive patients by coexistent hookworm infection. This worm species, in contrast to A. lumbricoides, causes especially high eosinophil counts and EDN/EPX and IgE levels. From these results it is concluded that in nematode diseases, ECP and EDN/EPX levels reflect the degree of antigenic stimulation, eosinophil activation and eosinophil turnover rates. Serum ECP and serum and urinary EDN/EPX may, therefore, serve as parameters to monitor helminth infection. Urinary ECP may be a marker of eosinophiluria secondary to urogenital manifestation of S. haematobium. It is elevated in hyperreactive onchocerciasis activated by intestinal nematodes.