Onchocerca volvulus-specific antibody and cellular responses in onchocerciasis patients treated annually with ivermectin for 30 years and exposed to parasite transmission in central Togo.

BACKGROUND: Annual mass drug administrations (MDA) of ivermectin will strongly reduce Onchocerca volvulus microfilariae (mf) in the skin and in the onchocerciasis patients' eyes. Ivermectin treatment will also affect the expression of immunity in patients, such that activated immune defenses may help control and contribute to clearance of mf of O. volvulus. Longitudinal surveys are a prerequisite to determining the impact of ivermectin on the status of anti-parasite immunity, notably in risk zones where parasite transmission and active O. volvulus infections persist. METHODOLOGY/PRINCIPAL FINDINGS: Onchocerciasis patients were treated annually with ivermectin and their Onchocerca volvulus antigen (OvAg) specific IgG and cellular responses were investigated before and at 30 years post initial ivermectin treatment (30yPT). Repeated annual ivermectin treatments eliminated persisting O. volvulus microfilariae (mf) from the skin of patients and abrogated patent infections. The OvAg-specific IgG1 and IgG4 responses were diminished at 30yPT to the levels observed in endemic controls. Prior to starting ivermectin treatment, OvAg-induced cellular productions of IL-10, IFN-γ, CCL13, CCL17 and CCL18 were low in patients, and at 30yPT, cellular cytokine and chemokine responses increased to the levels observed in endemic controls. In contrast, mitogen(PHA)- induced IL-10, IFN-γ, CCL17 and CCL18 cellular production was diminished. This divergent response profile thus revealed increased parasite antigen-specific but reduced polyclonal cellular responsiveness in patients. The transmission of O. volvulus continued at the patients' location in the Mô river basin in central Togo 2018 and 2019 when 0.58% and 0.45%, respectively, of Simulium damnosum s.l. vector blackflies carried O. volvulus infections. CONCLUSIONS/SIGNIFICANCE: Repeated annual ivermectin treatment of onchocerciasis patients durably inhibited their patent O. volvulus infections despite ongoing low-level parasite transmission in the study area. Repeated MDA with ivermectin affects the expression of immunity in patients. O. volvulus parasite-specific antibody levels diminished to levels seen in infection-free endemic controls. With low antibody levels, antibody-dependent cellular cytotoxic responses against tissue-dwelling O. volvulus larvae will weaken. O. volvulus antigen inducible cytokine and chemokine production increased in treated mf-negative patients, while their innate responsiveness to mitogen declined. Such lower innate responsiveness in elderly patients could contribute to reduced adaptive immune responses to parasite infections and vaccines. On the other hand, increased specific cellular chemokine responses in mf-negative onchocerciasis patients could reflect effector cell activation against tissue invasive larval stages of O. volvulus. The annual Simulium damnosum s.l. biting rate observed in the Mô river basin was similar to levels prior to initiation of MDA with ivermectin, and the positive rtPCR results reported here confirm ongoing O. volvulus transmission.

Echinococcus multilocularis specific antibody, systemic cytokine, and chemokine levels, as well as antigen-specific cellular responses in patients with progressive, stable, and cured alveolar echinococcosis: A 10-year follow-up.

BACKGROUND: The infestation with Echinococcus multilocularis larvae may persist in humans for up to decades without evident clinical symptoms. Longitudinal investigations are needed to understand the dynamic immunological processes in alveolar echinococcosis (AE) patients associated with an active and progressive, a stable or a regressive course of disease. METHODOLOGY/PRINCIPAL FINDINGS: This study evaluated the E. multilocularis specific antibody responses, systemic cytokine, and chemokine serum levels over a 10-year follow-up period, as well as cellular responsiveness in AE patients. Our results demonstrate a rapid decrease in antibodies against E. multilocularis specific antigen Em2+. Especially in cured patients, these antibodies remained negative, making them a significant predictor for cured AE. E. multilocularis specific IgG4, and indirect hemagglutination IHA decreased later in time, after around 5 years. While total IgE did not show significant dynamics over the course of disease, E. multilocularis specific IgE decreased after one to two years, and increasing levels were a significant predictor of progressive disease. There was no significant change in systemic IL-8, IL-9, CCL18 or CCL20 serum levels over time. Univariate analysis across groups indicated lower IL-8 levels in cured patients; however, this result could not be confirmed by multivariate analysis. Levels of CCL17 decreased during treatment, especially in cured patients, and thus might serve as a predictive or risk factor for progressive disease. Levels of IL-10 and CCL13 decreased during disease, especially after five and ten years of intervention. The E. multilocularis antigen (EmAg) inducible cellular productions of MCP1(CCL13), TARC(CCL17) and PARC(CCL18) were lowest in patients with cured AE and infection-free controls, while the EmAg inducible cellular production of IFN-γ increased after cure. Significant positive cytokine and chemokine correlations were observed in AE patients for IL-9, IL-10, CCL13(MCP-4), CCL17(TARC) and CCL20(LARC)(for all p<0.001). E. multilocularis specific IgG4 response correlated positively with TARC (p<0.001). Both markers enhanced over time in progressive disease and decreased after cure. The levels of IL-8, IL-10, MCP4 and LARC enhanced with AE regression. CONCLUSIONS/SIGNIFICANCE: Repeated biomarker surveys are advisable to evaluate progression or regression of disease during longitudinal follow-up and such analyses can support imaging techniques and improve staging of AE patients.

Mansonella perstans, Onchocerca volvulus and Strongyloides stercoralis infections in rural populations in central and southern Togo.

BACKGROUND: Mansonella perstans, Onchocerca volvulus and Strongyloides stercoralis are widespread helminth parasites in the tropics. Their distribution remains difficult to determine as it may change during national disease control programs and with regional mass drug administration (MDA). Epidemiological surveys are of importance to evaluate the geographical distribution of these helminth parasites and the diseases they may cause, however, up to date epidemiological evaluations on M. perstans and S. stercoralis in Togo are rare, and surveys on O. volvulus are important especially under the aspect of MDA of ivermectin which is performed since decades. METHODS: Dry blood samples (n = 924) were collected from rural populations in the Régions Central and Plateaux in Togo, and analyzed by parasite-specific real-time PCR and ELISA techniques. RESULTS: Dry blood samples from 733 persons where investigated by real-time PCR tested for DNA of blood-circulating M. perstans microfilaria, and a prevalence of 14.9% was detected. Distinct differences were observed between genders, positivity was higher in men increasing with age, and prevalence was highest in the Région Plateaux in Togo. IgG4 responses to O. volvulus antigen (OvAg) were studied in 924 persons and 59% were found positive. The distribution of parasite infestation between age and gender groups was higher in men increasing with age, and regional differences were detected being highest in the Région Plateaux. The diagnostic approach disclosed 64,5% positive IgG4 responses to S. stercoralis infective third-stage larvae-specific antigen (SsL3Ag) in the surveyed regions. Antigen cross reactivity of SsL3Ag with parasite co-infections may limit the calculated prevalence. Singly IgG4 positive for SsL3Ag were 13.9%, doubly positive for OvAg and SsL3Ag were 35.5% and triply positive for M. perstans, O. volvulus and S. stercoralis were 9.9%. CONCLUSIONS: Mansonelliasis, onchocerciasis and strongyloidiasis remain prevalent in the surveyed regions, yet with local differences. Our observations suggest that transmission of M. perstans, O. volvulus and S. stercoralis may be ongoing. The degree of positive test results in the examined rural communities advocate for the continuation of MDA with ivermectin and albendazole, and further investigations should address the intensity of transmission of these parasites.

Onchocerca volvulus infection and serological prevalence, ocular onchocerciasis and parasite transmission in northern and central Togo after decades of Simulium damnosum s.l. vector control and mass drug administration of ivermectin.

BACKGROUND: Mass drug administration (MDA) of ivermectin has become the main intervention to control onchocerciasis or "river blindness". In Togo, after many years of MDA, Onchocerca volvulus infection has declined dramatically, and elimination appears achievable, but in certain river basins the current situation remains unknown. We have conducted parasitological, serological, ophthalmological, and entomological assessments in northern and central Togo within the river basins of Ôti, Kéran and Mô. METHODOLOGY/PRINCIPAL FINDINGS: Examinations were completed in 1,455 participants from 11 onchocerciasis sentinel villages, and O. volvulus transmission by Simulium damnosum sensu lato (s.l.) was evaluated. In children (aged 1-10 years), the prevalence of microfilariae (Mf) was 2.3% and in adults it ranged from 5.1 to 13.3%. Positive IgG4 responses to O. volvulus adult (crude) worm antigen (OvAg) and the recombinant Ov16 antigen were in all-ages 48.7% and 34.4%, and 29.1% and 14.9% in children, respectively. In the river basin villages of Kéran, Mô and Ôti, the IgG4 seroprevalences to OvAg in children were 51.7%, 23.5% and 12.7%, respectively, and to the Ov16 antigen 33.3% (Kéran) and 5.2% (Ôti). Onchocerciasis ocular lesions (punctate keratitis, evolving iridocyclitis and chorioretinitis) were observed in children and young adults. O. volvulus-specific DNA (Ov150) was detected by poolscreen in vector samples collected from Tchitchira/Kéran(22.8%), Bouzalo/Mô(11.3%), Baghan/Mô(2.9%) and Pancerys/Ôti(4.9%); prevalences of O. volvulus infection in S. damnosum s.l. were, respectively, 1%, 0.5%, 0.1% and 0.2%. CONCLUSIONS/SIGNIFICANCE: In the northern and central river basins in Togo, interruption of O. volvulus transmission has not yet been attained. Patent O. volvulus infections, positive antibody responses, progressive ocular onchocerciasis were diagnosed, and parasite transmission by S. damnosum s.l. occurred close to the survey locations. Future interventions may require approaches selectively targeted to non-complying endemic populations, to the seasonality of parasite transmission and national onchocerciasis control programs should harmonize cross-border MDA as a coordinated intervention.

The effects of taxanes, vorinostat and doxorubicin on growth and proliferation of Echinococcus multilocularis metacestodes assessed with magnetic resonance imaging and simultaneous positron emission tomography.

Cytostatic drugs used in cancer therapy were evaluated for their capacity to inhibit Echinococcus multilocularis metacestode growth and proliferation. Metacestode tissues were exposed in vitro to docetaxel, doxorubicin, navelbine, paclitaxel, and vorinostat for 1 week, then incubated in drug-free culture, and thereafter metacestodes were injected into the peritoneum of Meriones unguiculatus. Magnetic resonance imaging (MRI) and simultaneous positron emission tomography (PET) were applied to monitor in vivo growth of drug-exposed E. multilocularis in Meriones. At 3 month p.i., docetaxel (at 10 µM, 5 µM and 2 µM) inhibited in vivo growth and proliferation of E. multilocularis, and at 5 months p.i., only in the 2 µM docetaxel exposure group 0.3 cm 3 of parasite tissue was found. With paclitaxel and navelbine the in vivo growth of metacestodes was suppressed until 3 months p.i., thereafter, parasite tissues enlarged up to 3 cm 3 in both groups. E. multilocularis tissues of more than 10 g developed in Meriones injected with metacestodes which were previously exposed in vitro to doxorubicin, navelbine, paclitaxel or vorinostat. In Meriones infected with metacestodes previously exposed to docetaxel, the in vivo grown parasite tissues weighted 0.2 g. In vitro cultured E. multilocularis metacestodes exposed to docetaxel did not produce vesicles until 7 weeks post drug exposure, while metacestodes exposed to doxorubicin, navelbine and vorinostat proliferated continuously. In summary, docetaxel, and less efficaciously paclitaxel, inhibited in vivo and in vitro parasite growth and proliferation, and these observations suggest further experimental studies with selected drug combinations which may translate into new treatment options against alveolar echinococcosis.

Analysis of age-dependent trends in Ov16 IgG4 seroprevalence to onchocerciasis.

BACKGROUND: Diagnostics provide a means to measure progress toward disease elimination. Many countries in Africa are approaching elimination of onchocerciasis after successful implementation of mass drug administration programs as well as vector control. An understanding of how markers for infection such as skin snip microfilaria and Onchocerca volvulus-specific seroconversion perform in near-elimination settings informs how to best use these markers. METHODS: All-age participants from 35 villages in Togo were surveyed in 2013 and 2014 for skin snip Onchocerca volvulus microfilaria and IgG4 antibody response by enzyme-linked immunosorbent assay (ELISA) to the Onchocerca volvulus-specific antigen Ov16. A Gaussian mixture model applying the expectation-maximization (EM) algorithm was used to determine seropositivity from Ov16 ELISA data. For a subset of participants (n = 434), polymerase chain reaction (PCR) was performed on the skin snips taken during surveillance. RESULTS: Within the 2,005 participants for which there was Ov16 ELISA data, O. volvulus microfilaremia prevalence and Ov16 seroprevalence were, 2.5 and 19.7 %, respectively, in the total population, and 1.6 and 3.6 % in children under 11. In the subset of 434 specimens for which ELISA, PCR, and microscopy data were generated, it was found that in children under 11 years of age, the anti-Ov16 IgG4 antibody response demonstrate a sensitivity and specificity of 80 and 97 %, respectively, against active infections as determined by combined PCR and microscopy on skin snips. Further analysis was performed in 34 of the 35 villages surveyed. These villages were stratified by all-age seroprevalence into three clusters: < 15 %; 15-20 %; and > 20 %. Age-dependence of seroprevalence for each cluster was best reflected by a two-phase force-of-infection (FOI) catalytic model. In all clusters, the lower of the two phases of FOI was associated with a younger age group, as reflected by the seroconversion rates for each phase. The age at which transition from lower to higher seroconversion, between the two phases of FOI, was found to be highest (older) for the cluster of villages with < 15 % seroprevalence and lowest (younger) for the cluster with the highest all-age seroprevalence. CONCLUSIONS: The anti-Ov16 IgG4 antibody response is an accurate marker for active infection in children under 11 years of age in this population. Applying Ov16 surveillance to a broader age range provides additional valuable information for understanding progression toward elimination and can inform where targeted augmented interventions may be needed. Clustering of villages by all-age sero-surveillance allowed application of a biphasic FOI model to differentiate seroconversion rates for different age groups within the village cluster categories.

Cellular gene expression induced by parasite antigens and allergens in neonates from parasite-infected mothers.

Prenatal exposure to parasite antigens or allergens will influence the profile and strength of postnatal immune responses, such contact may tolerize and increase susceptibility to future infections or sensitize to environmental allergens. Exposure in utero to parasite antigens will distinctly alter cellular gene expression in newborns. Gene microarrays were applied to study gene expression in umbilical cord blood cell (UCBC) from parasite-exposed (Para-POS) and non-exposed (Para-NEG) neonates. UCBC were activated with antigens of helminth (Onchocerca volvulus), amoeba (Entamoeba histolytica) or allergens of mite (Dermatophagoides farinae). When UCBC from Para-POS and Para-NEG newborns were exposed to helminth antigens or allergens consistent differences occurred in the expression of genes encoding for MHC class I and II alleles, signal transducers of activation and transcription (STATs), cytokines, chemokines, immunoglobulin heavy and light chains, and molecules associated with immune regulation (SOCS, TLR, TGF), inflammation (TNF, CCR) and apoptosis (CASP). Expression of genes associated with innate immune responses were enhanced in Para-NEG, while in Para-POS, the expression of MHC class II and STAT genes was reduced. Within functional gene networks for cellular growth, proliferation and immune responses, Para-NEG neonates presented with significantly higher expression values than Para-POS. In Para-NEG newborns, the gene cluster and pathway analyses suggested that gene expression profiles may predispose for the development of immunological, hematological and dermatological disorders upon postnatal helminth parasite infection or allergen exposure. Thus, prenatal parasite contact will sensitize without generating aberrant inflammatory immune responses, and increased pro-inflammatory but decreased regulatory gene expression profiles will be present in those neonates lacking prenatal parasite antigen encounter.

A Recombinant Positive Control for Serology Diagnostic Tests Supporting Elimination of Onchocerca volvulus.

BACKGROUND: Serological assays for human IgG4 to the Onchocerca volvulus antigen Ov16 have been used to confirm elimination of onchocerciasis in much of the Americas and parts of Africa. A standardized source of positive control antibody (human anti-Ov16 IgG4) will ensure the quality of surveillance data using these tests. METHODOLOGY/PRINCIPAL FINDINGS: A recombinant human IgG4 antibody to Ov16 was identified by screening against a synthetic human Fab phage display library and converted into human IgG4. This antibody was developed into different positive control formulations for enzyme-linked immunosorbent assay (ELISA) and rapid diagnostic test (RDT) platforms. Variation in ELISA results and utility as a positive control of the antibody were assessed from multiple laboratories. Temperature and humidity conditions were collected across seven surveillance activities from 2011-2014 to inform stability requirements for RDTs and positive controls. The feasibility of the dried positive control for RDT was evaluated during onchocerciasis surveillance activity in Togo, in 2014. When the anti-Ov16 IgG4 antibody was used as a standard dilution in horseradish peroxidase (HRP) and alkaline phosphatase (AP) ELISAs, the detection limits were approximately 1ng/mL by HRP ELISA and 10ng/mL by AP ELISA. Positive control dilutions and spiked dried blood spots (DBS) produced similar ELISA results. Used as a simple plate normalization control, the positive control antibody may improve ELISA data comparison in the context of inter-laboratory variation. The aggregate temperature and humidity monitor data informed temperature parameters under which the dried positive control was tested and are applicable inputs for testing of diagnostics tools intended for sub-Saharan Africa. As a packaged positive control for Ov16 RDTs, stability of the antibody was demonstrated for over six months at relevant temperatures in the laboratory and for over 15 weeks under field conditions. CONCLUSIONS: The recombinant human anti-Ov16 IgG4 antibody-based positive control will benefit inter-laboratory validation of ELISA assays and serve as quality control (QC) reagents for Ov16 RDTs at different points of the supply chain from manufacturer to field use.

Cellular cytokine and chemokine responses to parasite antigens and fungus and mite allergens in children co-infected with helminthes and protozoa parasites.

BACKGROUND: In sub-Saharan Africa poly-parasite infections are frequently observed in children, and with poly-parasitism modulating immune mechanisms, mediated by cytokines and chemokines, are required to prevent overwhelming inflammation and host tissue damage. We analyzed in children co-infected with helminthes and protozoan parasites their cellular production of regulatory and pro-inflammatory cytokines and chemokines in response to parasite antigens and allergens. METHODS: Intestinal and intravascular parasite infections were detected in stool and urines samples. The in vitro cellular cytokine and chemokine responses of peripheral blood mononuclear cells (PBMC) to parasite antigens and allergens were analysed in children (n = 87) with single and poly-parasite infection, and skin prick test reactivity to fungus and mite allergens was determined in singly and poly-parasitized children (n = 509). RESULTS: In children Entamoeba histolytica/dispar (62%), Necator americanus (31%), Schistosoma haematobium (28%), S. mansoni (21%), Hymenolepis nana (2%) and Strongyloides stercoralis (1%) were diagnosed. Singly infected were 37%, 47% were positive for 2 or more parasite species and 16% were infection-free. When PBMC were stimulated in vitro with parasite antigens and allergens, regulatory-type cytokine IL-27 and alarmin-type IL-33 enhanced with poly-parasite infections whilst IL-10 and pro-inflammatory MIP3-α/CCL20 and MIG/CXCL9 were produced in similar amounts in singly or poly-parasitized children. The co-stimulation in vitro of PBMC with mite allergens and Ascaris lumbricoides antigens depressed the allergen-induced pro-inflammatory IL-27, IL-33 and MIP3-α/CCL20 responses while regulatory IL-10 remained unaffected. Post albendazole and/or praziquantel treatment, the cellular release of IL-10, IL-33, MIP3-α/CCL20 and MIG/CXCL9 lessened significantly in all children infection groups. Skin prick test (SPT) reactivity to fungus Aspergillus fumigatus and mite Dermatophagoides pteronyssinus allergens was investigated in 509 children, and positive SPT responses were found in 23% of the infection-free, and in 47%, 53% and 56% of the singly, doubly and poly-parasite infected, respectively. CONCLUSIONS: In children co-infected with helminthes and protozoan parasites a mixed cellular response profile of both inflammatory and regulatory chemokines and cytokines was stimulated by individual antigens and allergens, pro-inflammatory cytokines and chemokines enhanced with an increasing number of parasite infections, and in poly-parasitized children skin prick test reactivity to allergens extracts was highest.

Evaluation of the Metabolic Activity of Echinococcus multilocularis in Rodents Using Positron Emission Tomography Tracers.

PURPOSE: 2-Deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) has been used as a standard clinical positron emission tomography (PET) tracer for the follow-up of the rare but life-threatening parasitic disease alveolar echinococcosis (AE). Given that the disease is endemic in many countries in the northern hemisphere and the diagnosis is still challenging, the aim of our study was to evaluate further clinically relevant PET tracers as possible diagnostic tools for AE in vitro and in vivo. PROCEDURES: Various clinically used PET tracers were evaluated in vitro and assessed in an in vivo AE animal model based on PET/magnetic resonance (MR) measurements. RESULTS: In vitro binding assays displayed high uptake of [(18)F]FDG in a cell suspension of E. multilocularis tissue, whereas 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT) and [(11)C]choline were found to be taken up strongly by E. multilocularis vesicles. [(18)F]FDG and [(18)F]FLT displayed an elevated uptake in vivo, which appeared as several foci throughout the parasite tissue as opposed to [(18)F]fluoro-azomycinarabinofuranoside ([(18)F]FAZA) and [(11)C]choline. CONCLUSIONS: Our data clearly demonstrate that the clinically applied PET tracer [(18)F]FDG is useful for the diagnosis and disease staging of AE but also has drawbacks in the assessment of currently inactive or metabolically weak parasitic lesions. The different tested PET tracers do not show the potential for the replacement or supplementation of current diagnostic strategies. Hence, there is still the need for novel diagnostic tools.

Sustainable control of onchocerciasis: ocular pathology in onchocerciasis patients treated annually with ivermectin for 23 years: a cohort study.

UNLABELLED: The evolution and persistence of ocular pathology was assessed in a cohort of Onchocerca volvulus infected patients treated annually with ivermectin for 23 years. Patients were resident in rural Central and Kara Region of Togo and ocular examinations included testing of visual acuity, slit lamp examination of the anterior eye segment and the eye fundus by ophthalmoscopy. Before ivermectin treatment, vivid O.volvulus microfilariae (MF) were observed in the right and left anterior eye chamber in 52% and 42% of patients (n = 82), and dead MF were seen in the right and left cornea in 24% and 15% of cases, respectively. At 23 years post initial treatment (PIT), none of the patients (n = 82) presented with MF in the anterior chamber and cornea. A complete resolution of punctate keratitis (PK) lesions without observable corneal scars was present at 23 years PIT (p<0.0001), and sclerosing keratitits (SK) lessened by half, but mainly in patients with lesions at early stage of evolution. Early-stage iridocyclitis diminished from 42%(rE) and 40%(lE) to 13% (rE+lE)(p<0.0001), but advanced iridocyclitis augmented (p<0.001) at 23 years PIT compared to before ivermectin. Advanced-stage papillitis and chorioretinitis did not regress, while early-stage papillitis present in 28%(rE) and 27%(lE) of patients at before ivermectin regressed to 17%(rE) and 18%(lE), and early-stage chorioretinitis present in 51%(rE+lE) of cases at before ivermectin was observed in 12%(rE) and 13%(lE) at 23 years PIT (p<0.0001). Thus, regular annual ivermectin treatment eliminated and prevented the migration of O. volvulus microfilariae into the anterior eye chamber and cornea; keratitis punctata lesions resolved completely and early-stage sclerosing keratitits and iridocyclitis regressed, whilst advanced lesions of the anterior and posterior eye segment remained progressive. In conclusion, annual ivermectin treatments may prevent the emergence of ocular pathology in those populations still exposed to O.volvulus infection. TRIAL REGISTRATION: www.pactr.org PACTR201303000464219).

Distinctive cytokine, chemokine, and antibody responses in Echinococcus multilocularis-infected patients with cured, stable, or progressive disease.

Metacestode larvae of the tapeworm Echinococcus multilocularis can cause alveolar echinococcosis (AE), a severe parasitic disease in man, which, if it remains untreated, may cause organ failure and death. Spontaneous and parasite antigen-induced cellular responses were studied in patients with cured, stable, and progressive AE to differentiate the response profiles between the distinct states of infection. Antibody reactivity was evaluated in AE patients with cured, stable, and progressive disease. The spontaneous cellular release of pro-inflammatory IL-31 and IL-33 was clearly depressed in all AE patients, while regulatory IL-27, anti-inflammatory SDF-1/CXCL12, and eosinophil granulocyte attracting Eotaxin-1, Eotaxin-2, and Eotaxin-3 (CCL11, CCL24, CCL26) were enhanced with disease progression. Such distinctive response profiles could be applied for monitoring of AE disease progression or regression. E. multilocularis metacestode (Em) antigens (entire metacestode EmAg as well as EmVesicles) stimulated in vitro IL-31, IL-33, Eotaxin-1, Eotaxin-3, and CXCL12 cytokine and chemokine responses, which were similarly present in all AE patient groups, while regulatory IL-27 was suppressed and pro-inflammatory Eotaxin-2 was enhanced. E. multilocularis metacestode-specific IgG1, IgG3, and IgE responses progressively diminished with regression from active to stable and cured AE. IgG2 and IgG4 reactivity remained similarly high in stable and progressive cases, and lessened only with cured AE. Antibody reactivity against E. multilocularis vesicle antigen distinctively separated between cured, stable, or progressive AE, with the exception of IgG4. In sum, the combined and longitudinal study of several cytokines and chemokines, together with the evaluation of E. multilocularis vesicle-specific antibody responses, should provide a better understanding of the immune response during progression and regression of AE, and may help to improve the staging of AE patients.

Cytokine and chemokine responses to helminth and protozoan parasites and to fungus and mite allergens in neonates, children, adults, and the elderly.

BACKGROUND: In rural sub-Saharan Africa, endemic populations are often infected concurrently with several intestinal and intravascular helminth and protozoan parasites. A specific, balanced and, to an extent, protective immunity will develop over time in response to repeated parasite encounters, with immune responses initially being poorly adapted and non-protective. The cellular production of pro-inflammatory and regulatory cytokines and chemokines in response to helminth, protozoan antigens and ubiquitous allergens were studied in neonates, children, adults and the elderly. RESULTS: In children schistosomiasis prevailed (33%) while hookworm and Entamoeba histolytica/E. dispar was found in up to half of adults and the elderly. Mansonella perstans filariasis was only present in adults (24%) and the elderly (25%). Two or more parasite infections were diagnosed in 41% of children, while such polyparasitism was present in 34% and 38% of adults and the elderly. Cytokine and chemokine production was distinctively inducible by parasite antigens; pro-inflammatory Th2-type cytokine IL-19 was activated by Entamoeba and Ascaris antigens, being low in neonates and children while IL-19 production enhanced "stepwise" in adults and elderly. In contrast, highest production of MIP-1delta/CCL15 was present in neonates and children and inducible by Entamoeba-specific antigens only. Adults and the elderly had enhanced regulatory IL-27 cytokine responses, with Th2-type chemokines (MCP-4/CCL13, Eotaxin-2/CCL24) and cytokines (IL-33) being notably inducible by helminth- and Entamoeba-specific antigens and fungus-derived allergens. The lower cellular responsiveness in neonates and children highlighted the development of a parasite-specific cellular response profile in response to repeated episodes of exposure and re-infection. CONCLUSIONS: Following repeated exposure to parasites, and as a consequence of host inability to prevent or eliminate intestinal helminth or protozoa infections, a repertoire of immune responses will evolve with lessened pro-inflammatory and pronounced regulatory cytokines and chemokines; this is required for partial parasite control as well as for preventing inadequate and excessive host tissue and organ damage.

In-vitro characterization of novel and functional regulatory SNPs in the promoter region of IL2 and IL2R alpha in a Gabonese population.

BACKGROUND: The selection pressure imposed by the parasite has a functional consequence on the immune genes, leading to altered immune function in which regulatory T cells (Tregs) induced by parasites during infectious challenges modulate or thwart T effector cell mechanism. METHODS: We identified and investigated regulatory polymorphisms in the immune gene IL2 and its receptor IL2R alpha (also known as CD25) in Gabonese individuals exposed to plentiful parasitic infections. RESULTS: We identified two reported variants each for IL2 and its receptor IL2R alpha gene loci. Also identified were two novel variants, -83 /-84 CT deletions (ss410961576) for IL2 and -409C/T (ss410961577) for IL2R alpha. We further validated all identified promoter variants for their allelic gene expression using transient transfection assays. Three promoter variants of the IL2 locus revealed no significant expression of the reporter gene. The identified novel variant (ss410961577C/T) of the IL2R alpha revealed a significant higher expression of the reporter gene in comparison to the major allele (P<0.05). In addition, the rs12722616C/T variant of the IL2R alpha locus altered the transcription factor binding site TBP (TATA box binding protein) and C/EBP beta (CCAAT/enhancer binding protein beta) that are believed to regulate the Treg function. CONCLUSIONS: The identification and validation of such regulatory polymorphisms in the immune genes may provide a basis for future studies on parasite susceptibility in a population where T cell functions are compromised.

Parasite-specific IL-17-type cytokine responses and soluble IL-17 receptor levels in Alveolar Echinococcosis patients.

Alveolar Echinococcosis (AE) caused by the cestode Echinococcus multilocularis, is a severe helminth infection of man, where unrestricted parasite growth will ultimately result in organ failure and fatality. The tissue-infiltrative growth of the larval metacestode and the limited efficacy of available drugs complicate successful intervention in AE; patients often need life-long medication, and if possible, surgical resection of affected tissues and organs. Resistance to AE has been reported, but the determinants which confer protection are not known. ln this study, we analyzed in patients at distinct stages of Alveolar Echirococcosis, that is cured, stable and progressive AE, as well as in infection-free controls, the cellular production and plasma levels of pro-inflammatory cytokines lL-17A, lL-17B, lL-17F and their soluble receptors lL-17RA (slL-17RA) and IL-17RB (sIL-17RB). Significantly elevated levels of IL-17B and slL-17RB were observed, whilst lL-17F and slL-17RA were reduced in patients with AE. Similarly, the cellular production of lL-17F and slL-L7RA in response to E. multilocularis antigens was low in AE patients, while levels of slL-17RB were highly enhanced. These observations suggest immune-modulating properties of E. multitocularis on lL-17 cytokine-mediated pro-inflammatory immune responses; this may facilitate the tissue infiltrative growth of the parasite and its persistence in the human host.

Chemokines and cytokines in patients with an occult Onchocerca volvulus infection.

Repeated ivermectin treatment will clear microfilaria (Mf) of Onchocerca volvulus from skin and eyes of onchocerciasis patients while adult filaria remains alive and reproductive, and such occult O. volvulus infection may persist for years. To investigate the effect of residual adult filaria on the immune response profile, chemokines and cytokines were quantified 1) in onchocerciasis patients who developed an occult O. volvulus infection (Mf-negative) due to repeated ivermectin treatments, 2) patients who became Mf-negative without ivermectin treatments due to missing re-infection, and 3) endemic and non-endemic O. volvulus Mf-negative controls. With occult O. volvulus infection, serum levels of pro-inflammatory chemokines MCP-1/CCL2, MIP-1α/CCL3, MIP-1ß/CCL4, MPIF-1/CCL23 and CXCL8/IL-8 enhanced and approached higher concentrations as determined in infection-free controls, whilst regulatory and Th2-type cytokines and chemokines MCP-4/CCL13, MIP-1δ/CCL15, TARC/CCL17 and IL-13 lessened. Levels of Eotaxin-2/CCL24, MCP-3/CCL7 and BCA-1/CXCL13 remained unchanged. At 3 days post-initial ivermectin treatment, MCP-1/CCL2, MCP-4/CCL13, MPIF-1/CCL23 and Eotaxin-2/CCL24 were strongly enhanced, suggesting that monocytes and eosinophil granulocytes have mediated Mf clearance. In summary, with occult and expiring O. volvulus infections the serum levels of inflammatory chemokines enhanced over time while regulatory and Th2-type-promoting cytokines and chemokines lessened; these changes may reflect a decreasing effector cell activation against Mf of O. volvulus, and in parallel, an enhancing inflammatory immune responsiveness.

Effects of in vitro exposure of Echinococcus multilocularis metacestodes to cytostatic drugs on in vivo growth and proliferation of the parasite.

The cytostatic drugs Vincristine (VCR), Navelbine (NAV), and Methotrexate (MTX) were evaluated for their growth inhibitory potential against metacestodes of Echinococcus multilocularis (Em) by in vitro and in vivo assays. In vitro cultures of E. multilocularis were exposed to IC 90, IC 80, and IC 5 concentrations of VCR, NAV, or MTX for 1 week, then parasite tissue cultures were kept for 1 week without drug exposure in vitro, and thereafter, metacestode tissues were injected intra-peritoneally into Meriones unguiculatus. Metacestode growth was monitored for several months post-infection (p.i.) by body weight control, magnetic resonance imaging (MRI), and autopsy at 5 months p.i. Weight monitoring of infected M. unguiculatus did not provide conclusive evidence for Em-metacestode growth, while MRI could detect growing Em-metacestode in the MTX-treated group at 8 weeks (p.i.), whereas metacestodes exposed to VCR and NAV were at 17 weeks (p.i.) detectable. MRI disclosed progressive and massive growth of Em-metacestode in the VCR- and MTX-exposed groups, while the NAV-pretreated Em-metacestodes' volume did not exceed 4 cm(3). At autopsy, Em-metacestodes of less than 4 cm(3) were found in infected M. unguiculatus, which was not detected by MRI. In summary, the cytostatic drugs Methotrexate, Navelbine, and Vincristine--as applied in the present work--did not show parasitocidal or clear parasitostatic effects on metacestodes of E. multilocularis. While parasite growth in vivo was inhibited in NAV- and VCR-pretreated Em-metacestodes, MTX pretreatment seemed to enhance parasite proliferation. Magnetic resonance imaging appears suitable to monitor in vivo the effects of drugs on growth progression and regression only of larger Em-metacestode tissues.

Applied field research for comprehensive helminth infection control.

The Institute for Tropical Medicine at University of Tübingen has established 30 years ago in Togo a Research Centre and Onchocerciasis Reference Laboratory (ORL). Onchocerca volvulus infection control and of other neglected tropical diseases has been the focus of activities, and those were performed together with the National Institute of Hygiene in Togo, the Medical Faculty at University of Lomé, national disease control programs and district and regional hospitals. The ORL contributed significantly to the assessment of ivermectin as the prime choice for onchocerciasis treatment, and 24 years of repeated annual treatment with ivermectin has progressively reduced disease prevalence and notably the level of ocular and dermal manifestations of onchocerciasis in the endemic population. The ORL has shown that large parts of the rural population in Togo is concurrently infected with intestinal and intravascular protozoan and helminth parasites, notably school children. The application of repeated treatments with albendazole and praziquantel against Schistosoma spp. and instestinal helminthes for several years has reduced infection intensities by more than 80%. Longitudinal investigations of the cellular immune responses in adults and children have found that parasite co-infections will generate prominent pro-inflammatory responses, and a single or few interventions will not suffice to eliminate co-infections and not establish an appropriately balanced immunity.

Cytokine and chemokine responses in adults, newborns and children exposed to Entamoeba histolytica/dispar, Onchocerca volvulus and Plasmodium falciparum.

Cytokine and chemokine response profiles were studied in newborns, 10-yr-old children and post partum mothers. All study groups were repeatedly exposed to Entamoeba histolytica, Onchocerca volvulus and Plasmodium falciparum infections as indicated by their Immunoglobulin (IgG) responses to parasite-specific antigens. As key indicators for regulatory and pro-inflammatory cytokine and chemokine responses, Interferon (IFN)gamma and regulatory IL-10 were investigated, along with the chemokines MIP-1 alpha/CCL3, MIP-1 beta/CCL4, MDC/CCL22 and TARC/CCL17. Entamoeba histolytica antigens (EhAg) strongly activated pro-inflammatory MIP-1 alpha/CCL3 and MIP-1 beta/CCL4 responses of similar magnitude in mothers, children and neonates alike. Plasmodium falciparum antigens (PfAg) enhanced MIP-1 alpha/CCL3, MIP-1 beta/CCL4 and MDC/CCL22 production in neonates, but did not trigger these chemokines in mothers or 10-yr-old children. Onchocerca volvulus antigens (OvAg) activated IFN-gamma and TARC/CCL17 production in mothers but not in neonates and children. Crude IL-10 production [i.e., without subtracting spontaneous cellular release (baseline)] was highest in mothers and somewhat lower in neonates, while the lowest IL-10 amounts of all were released by peripheral blood mononuclear cells from 10-yr-old children. In summary, strong inflammatory chemokine responses to plasmodia and ameba antigens in newborns and 10-yr-old children suggest that adequately balanced immune regulatory mechanisms may not have developed yet in these age groups and that repeated exposure to parasite infections and immune maturation during childhood is required to generate similar cytokine and chemokine profiles as in adults.

Coinfections with Schistosoma haematobium, Necator americanus, and Entamoeba histolytica/Entamoeba dispar in children: chemokine and cytokine responses and changes after antiparasite treatment.

The effect of polyparasite infections on cytokine and chemokine responses as well as the effect of antiparasite treatment was studied in children without parasite infection (the G0 group), in children singly infected with Schistosoma haematobium (the G1 group), and in children multiply infected with S. haematobium/Schistosoma mansoni, Entamoeba histolytica/Entamoeba dispar, and Necator americanus (the G3+ group). Linear regression analysis disclosed a significant risk for coinfection with hookworm and Schistosoma species. Polyparasite infections detected in 23% of children before treatment were present in 5% at 15 months after treatment. Chemokine responses to S. mansoni adult worm antigen (SmAg) diminished after treatment for macrophage inflammatory chemokine (MIP)-1alpha/chemokine (C-C motif) ligand (CCL)-3 (among G3+ children, by a factor of 200 [95% confidence interval {CI}, 33-1111]) and for MIP-1beta/CCL-4 (among G3+ children, by a factor of 26 [95% CI, 6-117]) but were enhanced for thymus- and activation-regulated chemokine/CCL-17 (among G3+ children, by a factor of 10 [95% CI, 3-32]) (P < .001 for all). In response to E. histolytica antigen, interleukin (IL)-13 levels increased after treatment among G1 children by a factor of 138 (95% CI, 12-1569) and among G3+ children by a factor of 21 (95% CI, 7-64) (P < .001 for both). Cellular production of interferon (IFN)-gamma in response to SmAg decreased 4 weeks after treatment among G3+ children, whereas T helper cell type 2 (Th2) IL-13 production was enhanced among G1 and G3+ children. In summary, polyparasite infections with S. haematobium/S. mansoni, E. histolytica/E. dispar, and N. americanus generated prominent proinflammatory cytokine and chemokine responses, and, after antihelminth treatment, the inflammatory chemokine response lessened as the Th2 responsiveness in coinfected children increased.