RESUMO
Lymphatic filariasis and onchocerciasis are two major neglected tropical diseases that are responsible for causing severe disability in 50 million people worldwide, whilst veterinary filariasis (heartworm) is a potentially lethal parasitic infection of companion animals. There is an urgent need for safe, short-course curative (macrofilaricidal) drugs to eliminate these debilitating parasite infections. We investigated combination treatments of the novel anti-Wolbachia azaquinazoline small molecule, AWZ1066S, with benzimidazole drugs (albendazole or oxfendazole) in up to four different rodent filariasis infection models: Brugia malayi-CB.17 SCID mice, B. malayi-Mongolian gerbils, B. pahangi-Mongolian gerbils, and Litomosoides sigmodontis-Mongolian gerbils. Combination treatments synergised to elicit threshold (>90%) Wolbachia depletion from female worms in 5 days of treatment, using 2-fold lower dose-exposures of AWZ1066S than monotherapy. Short-course lowered dose AWZ1066S-albendazole combination treatments also delivered partial adulticidal activities and/or long-lasting inhibition of embryogenesis, resulting in complete transmission blockade in B. pahangi and L. sigmodontis gerbil models. We determined that short-course AWZ1066S-albendazole co-treatment significantly augmented the depletion of Wolbachia populations within both germline and hypodermal tissues of B. malayi female worms and in hypodermal tissues in male worms, indicating that anti-Wolbachia synergy is not limited to targeting female embryonic tissues. Our data provides pre-clinical proof-of-concept that sub-seven-day combinations of rapid-acting novel anti-Wolbachia agents with benzimidazole anthelmintics are a promising curative and transmission-blocking drug treatment strategy for filarial diseases of medical and veterinary importance.
RESUMO
Lymphatic filariasis and onchocerciasis are major neglected tropical diseases affecting over 90 million people worldwide with painful and profoundly disfiguring pathologies (such as lymphoedema or blindness). Type 2 inflammation is a hallmark of filarial nematode tissue infection and is implicated both in eosinophil dependent immunity and lymphatic or ocular immunopathologies. Type-2 innate lymphoid cells (ILC2) are known to play an important role in the initiation of type 2 inflammation in helminth infection. We therefore tracked comparative IL-12Rß2+ ILC1, ST2+ ILC2 and NKp46+ natural killer (NK) innate lymphoid cell population expansions during Brugia malayi experimental peritoneal filarial infections using either immunocompetent or immunodeficient mice. In immunocompetent BALB/c animals, NKp46+ NK cells rapidly expanded representing over 90% of the ILC population in the first week of infection, whereas, surprisingly, ST2+ ILC2 failed to expand. NKp46+ NK cell expansions were confirmed in RAG2 deficient mice lacking adaptive immunity. Ablation of the NKp46+ NK cell compartment in RAG2 common gamma chain (gc) mice led to increased susceptibility to chronic adult B. malayi infection. This data was recapitulated using an Onchocerca ochengi male worm peritoneal implant model. When NKp46+ NK cells were depleted in RAG2 deficient mice using anti-NKp46 or asialo GM1 antibody injections over the first five weeks of B. malayi infection, susceptibility to adult B. malayi infection was significantly increased by 2-3 fold with concomitant impairment in eosinophil or neutrophil recruitments. Finally, we demonstrate that in RAG2 deficient mice, drug clearance of a primary adult B. malayi infection followed by challenge infection leads to resistance against early larval B. malayi establishment. This innate resistance is associated with bolstered NK and eosinophils whereby NKp46+ NK cells express markers of memory-like/enhanced activation (increased expression of interferon gamma and Ly6C). Our data promotes a novel functional role for NKp46+ NK cells in immunoprotection against experimental primary and secondary filarial infection which can proceed in the absence of adaptive immune regulation.
Assuntos
Imunidade Inata , Interferon gama , Animais , Antígenos Ly , Inflamação , Proteína 1 Semelhante a Receptor de Interleucina-1 , Células Matadoras Naturais , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Receptor 1 Desencadeador da Citotoxicidade Natural , FenótipoRESUMO
The development of new drugs targeting adult-stage lymphatic filarial nematodes is hindered by the lack of a robust long-term in vitro culture model. Testing potential direct-acting and anti-Wolbachia therapeutic candidates against adult lymphatic filariae in vitro requires their propagation via chronic infection of gerbils. We evaluated Brugia malayi parasite burden data from male Mongolian gerbils compared with two immune-deficient mouse strains highly susceptible to B. malayi: CB.17 Severe-Combined Immmuno-Deficient (SCID) and interleukin-4 receptor alpha, interleukin-5 double knockout (IL-4Rα-/-IL-5-/-) mice. Adult worms generated in IL-4Rα-/-IL-5-/- mice were tested with different feeder cells (human embryonic kidney cells, human adult dermal lymphatic endothelial cells and human THP-1 monocyte differentiated macrophages) and comparative cell-free conditions to optimise and validate a long-term in vitro culture system. Cultured parasites were compared against those isolated from mice using motility scoring, metabolic viability assay (MTT), ex vivo microfilariae release assay and Wolbachia content by qPCR. A selected culture system was validated as a drug screen using reference anti-Wolbachia (doxycycline, ABBV-4083 / flubentylosin) or direct-acting compounds (flubendazole, suramin). BALB/c IL-4Rα-/-IL-5-/- or CB.17 SCID mice were superior to Mongolian gerbils in generating adult worms and supporting in vivo persistence for periods of up to 52 weeks. Adult females retrieved from BALB/c IL-4Rα-/-IL-5-/- mice could be cultured for up to 21 days in the presence of a lymphatic endothelial cell co-culture system with comparable motility, metabolic activity and Wolbachia titres to those maintained in vivo. Drug studies confirmed significant Wolbachia depletions or direct macrofilaricidal activities could be discerned when female B. malayi were cultured for 14 days. We therefore demonstrate a novel methodology to generate adult B. malayi in vivo and accurately evaluate drug efficacy ex vivo which may be adopted for drug screening with the dual benefit of reducing overall animal use and improving anti-filarial drug development.
Assuntos
Brugia Malayi , Wolbachia , Animais , Técnicas de Cocultura , Modelos Animais de Doenças , Células Endoteliais , Endotélio Linfático , Feminino , Interleucina-5 , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
BACKGROUND: Current treatment options for onchocerciasis are sub-optimal, prompting research and development of a safe cure (macrofilaricide). Onchocerca ochengi, a parasite of cattle, is used as a close surrogate for the human parasite O. volvulus in a murine model for pre-clinical screening of macrofilaricides. Skin from naturally infected cattle have been used in previous studies as a reliable source of parasite material. However, there is limited knowledge on how source-related factors such as the microfilaridermia status of the cattle, the nodule load and nodular worm viability may affect survival of male O. ochengi worms implanted in the rodent hosts. Such relationships were investigated in this study. METHODS: Dermal tissue and nodules were obtained from Gudali cattle, dissected and cultured to obtain migrating microfilariae (mf) and male worms. Emerged male worms were implanted into SCID mice and Gerbils (Meriones unguiculatus) and recovery rates were determined upon 42 days post implantation. Finally, nodules were processed for histology and embryogram analyses to assess the nodular worm viability and fertility, respectively. RESULTS: Of the 69 cattle sampled, 24 (34.8%) were mf+ and 45 (65.2%) were mf-. The mean nodule loads were 180.5 ± 117.7 (mf+) and 110.6 ± 102.7 (mf-) (p = 0.0186). The mean male worm harvest from nodules were 76.8 ± 120.3 and 47.2 ± 33.4 (p = 0.2488) for mf+ and mf- cattle, respectively. The number of male worms per 100 nodules were 57/100 and 46/100 nodules for mf+ and mf- cows, respectively. Female worms from nodules of mf- cows had higher counts of both normal and abnormal embryos with higher proportions of dead nodular worms evinced by histology compared to those from mf+ cows. A total of 651 worms were implanted into mice and gerbils, out of which 129 (19.81%) were recovered. Logistic regression analysis indicated that the microfilaridermia status of the cattle (presence of mf) (OR = 4.3319; P = 0.001) is the single most important predictor of the success of male worm recovery after implantation into rodents. CONCLUSION: Microfilaridermic cattle provide a promising source of adult O. ochengi. Male worms from this group of cattle have a better success rate of survival in a murine implant model. Nevertheless, in the programmatic point of view, amicrofilaridermic Gudali cattle would still constitute an important source of O. ochengi male worms with relatively good viability after implantation into rodents.
Assuntos
Doenças dos Bovinos/parasitologia , Onchocerca/fisiologia , Oncocercose/veterinária , Animais , Bovinos , Modelos Animais de Doenças , Feminino , Fertilidade , Gerbillinae , Masculino , Camundongos , Camundongos SCID , Microfilárias/crescimento & desenvolvimento , Microfilárias/fisiologia , Análise Multivariada , Onchocerca/crescimento & desenvolvimento , Oncocercose/parasitologia , Análise de RegressãoRESUMO
Dendritic cells (DCs) encompass several cell subsets that collaborate to initiate and regulate immune responses. Proper DC localization determines their function and requires the tightly controlled action of chemokine receptors. All DC subsets express CXCR4, but the genuine contribution of this receptor to their biology has been overlooked. We addressed this question using natural CXCR4 mutants resistant to CXCL12-induced desensitization and harboring a gain of function that cause the warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome (WS), a rare immunodeficiency associated with high susceptibility to the pathogenesis of human papillomavirus (HPV). We report a reduction in the number of circulating plasmacytoid DCs (pDCs) in WHIM patients, whereas that of conventional DCs is preserved. This pattern was reproduced in an original mouse model of WS, enabling us to show that the circulating pDC defect can be corrected upon CXCR4 blockade and that pDC differentiation and function are preserved, despite CXCR4 dysfunction. We further identified proper CXCR4 signaling as a critical checkpoint for Langerhans cell and DC migration from the skin to lymph nodes, with corollary alterations of their activation state and tissue inflammation in a model of HPV-induced dysplasia. Beyond providing new hypotheses to explain the susceptibility of WHIM patients to HPV pathogenesis, this study shows that proper CXCR4 signaling establishes a migration threshold that controls DC egress from CXCL12-containing environments and highlights the critical and subset-specific contribution of CXCR4 signal termination to DC biology.
Assuntos
Células Dendríticas/fisiologia , Inflamação/patologia , Doenças da Imunodeficiência Primária/fisiopatologia , Receptores CXCR4/fisiologia , Verrugas/fisiopatologia , Alphapapillomavirus/genética , Animais , Benzilaminas/farmacologia , Contagem de Células , Diferenciação Celular , Quimiocina CXCL12/fisiologia , Quimiotaxia , Ciclamos/farmacologia , Células Dendríticas/classificação , Epiderme/patologia , Feminino , Técnicas de Introdução de Genes , Genes Virais , Humanos , Inflamação/metabolismo , Células de Langerhans/fisiologia , Tecido Linfoide/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Transgênicos , Especificidade de Órgãos , Parabiose , Doenças da Imunodeficiência Primária/sangue , Doenças da Imunodeficiência Primária/genética , Doenças da Imunodeficiência Primária/patologia , Proteínas Recombinantes/metabolismo , Verrugas/sangue , Verrugas/genética , Verrugas/patologiaRESUMO
Lymphatic filariasis is the major global cause of nonhereditary lymphedema. We demonstrate that the filarial nematode Brugia malayi induced lymphatic remodeling and impaired lymphatic drainage following parasitism of limb lymphatics in a mouse model. Lymphatic insufficiency was associated with elevated circulating lymphangiogenic mediators, including vascular endothelial growth factor C. Lymphatic insufficiency was dependent on type 2 adaptive immunity, the interleukin-4 receptor, and recruitment of C-C chemokine receptor-2-positive monocytes and alternatively activated macrophages with a prolymphangiogenic phenotype. Oral treatments with second-generation tetracyclines improved lymphatic function, while other classes of antibiotic had no significant effect. Second-generation tetracyclines directly targeted lymphatic endothelial cell proliferation and modified type 2 prolymphangiogenic macrophage development. Doxycycline treatment impeded monocyte recruitment, inhibited polarization of alternatively activated macrophages, and suppressed T cell adaptive immune responses following infection. Our results determine a mechanism of action for the antimorbidity effects of doxycycline in filariasis and support clinical evaluation of second-generation tetracyclines as affordable, safe therapeutics for lymphedemas of chronic inflammatory origin.
Assuntos
Brugia Malayi/imunologia , Filariose Linfática/tratamento farmacológico , Linfangiogênese/imunologia , Receptores de Interleucina-4/imunologia , Tetraciclinas/farmacologia , Imunidade Adaptativa , Animais , Movimento Celular/genética , Movimento Celular/imunologia , Filariose Linfática/genética , Filariose Linfática/imunologia , Filariose Linfática/patologia , Linfangiogênese/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Monócitos/imunologia , Monócitos/patologia , Receptores de Interleucina-4/genética , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
Basic and translational research on loiasis, a filarial nematode infection of medical importance, is impeded by a lack of suitable Loa loa infection models and techniques of obtaining and culturing life cycle stages. We describe the development of a new method for routine production of infective third-stage larvae (L3) of L. loa from the natural intermediate arthropod vector host, Chrysops silacea, following experimental infection with purified microfilariae. At 14-days post-infection of C. silacea, the fly survival rate was 43%. Survival was significantly higher in flies injected with 50 mf (55.2%) than those that received 100 mf (31.0%). However, yield per surviving fly and total yield of L3 was markedly higher in the group of flies inoculated with 100 mf (3474 vs 2462 L3 produced). The abdominal segment hosted the highest percentage recovery of L3 (47.7%) followed by head (34.5%) and thorax (17.9%). L. loa larval survival was higher than 90% after 30 days of in vitro culture. The in vitro moulting success rate to the L4 larval stage was 59.1%. After experimental infection of RAG2-/-IL-2γc-/-mice, the average L. loa juvenile adult worm recovery rate was 10.5% at 62 dpi. More than 87% of the worms were recovered from the muscles and subcutaneous tissues. Worms recovered measured an average 24.3 mm and 11.4 mm in length for females (n = 5) and males (n = 5), respectively. In conclusion, L. loa mf injected into C. silacea intrathoracically develop into infective larvae that remain viable and infective comparable to L3 obtained through natural feeding on the human host. This technique further advances the development of a full laboratory life cycle of L. loa where mf derived from experimentally-infected animals may be utilized to passage life cycle generations via intrathoracic injections of wild-caught vector hosts.
Assuntos
Dípteros/parasitologia , Vetores de Doenças , Larva/parasitologia , Loa/isolamento & purificação , Loíase/parasitologia , Animais , Modelos Animais de Doenças , Feminino , Humanos , Larva/crescimento & desenvolvimento , Estágios do Ciclo de Vida , Loa/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microfilárias , Taxa de SobrevidaRESUMO
Helminth infections are accompanied by eosinophilia in parasitized tissues. Eosinophils are effectors of immunity to tissue helminths. We previously reported that in the context of experimental filarial nematode infection, optimum tissue eosinophil recruitment was coordinated by local macrophage populations following IL-4R-dependent in situ proliferation and alternative activation. However, in the current study, we identify that control of chronic adult filarial worm infection is evident in IL-4Rα-deficient (IL-4Rα-/-) mice, whereby the majority of infections do not achieve patency. An associated residual eosinophilia was apparent in infected IL-4Rα-/- mice. By treating IL-4Rα-/- mice serially with anti-CCR3 Ab or introducing a compound deficiency in CCR3 within IL-4Rα-/- mice, residual eosinophilia was ablated, and susceptibility to chronic adult Brugia malayi infection was established, promoting a functional role for CCR3-dependent eosinophil influx in immune control in the absence of IL-4/IL-13-dependent immune mechanisms. We investigated additional cytokine signals involved in residual eosinophilia in the absence IL-4Rα signaling and defined that IL-4Rα-/-/IL-5-/- double-knockout mice displayed significant eosinophil deficiency compared with IL-4Rα-/- mice and were susceptible to chronic fecund adult filarial infections. Contrastingly, there was no evidence that either IL-4R-dependent or IL-4R-independent/CCR3/IL-5-dependent immunity influenced B. malayi microfilarial loads in the blood. Our data demonstrate multiplicity of Th2-cytokine control of eosinophil tissue recruitment during chronic filarial infection and that IL-4R-independent/IL-5- and CCR3-dependent pathways are sufficient to control filarial adult infection via an eosinophil-dependent effector response prior to patency.
Assuntos
Brugia Malayi/imunologia , Eosinófilos/imunologia , Filariose/imunologia , Receptores de Superfície Celular/imunologia , Células Th2/imunologia , Animais , Eosinófilos/patologia , Filariose/genética , Filariose/patologia , Gerbillinae , Interleucina-13/genética , Interleucina-13/imunologia , Interleucina-4/genética , Interleucina-4/imunologia , Interleucina-5/genética , Interleucina-5/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptores CCR3/genética , Receptores CCR3/imunologia , Receptores de Superfície Celular/genética , Células Th2/patologiaRESUMO
Skin snip evaluation for onchocerciasis has insufficient sensitivity when skin microfilarial (mf) densities are low, such as following ivermectin treatment. Mf density is suitable for assessing microfilaricidal efficacy but only serves as an indirect indicator of macrofilaricidal activity. We assessed circulating nucleic acids from Onchocerca volvulus as an alternative to skin snips. We screened a plasma sample set of infected individuals followed up at four, 12 and 21 months after microfilaricidal (ivermectin, n = four), macrofilaricidal (doxycycline, n = nine), or combination treatment (n = five). Two parasite-derived miRNAs, cel-miR-71-5p and bma-lin-4, and O-150 repeat DNA were assessed. Highly abundant DNA repeat families identified in the O. volvulus genome were also evaluated. miRNAs were detected in two of 72 plasma samples (2.8%) and two of 47 samples (4.3%) with microfilaridermia using RT-qPCR. O-150 DNA was detected in eight (44.4%) baseline samples by qPCR and the number of positives declined post-treatment. One doxycycline-treated individual remained O-150 positive. However, only 11 (23.4%) samples with microfilaridermia were qPCR-positive. Analysis by qPCR showed novel DNA repeat families were comparatively less abundant than the O-150 repeat. Circulating parasite-derived nucleic acids are therefore insufficient as diagnostic tools or as biomarkers of treatment efficacy for O. volvulus.
Assuntos
Biomarcadores/sangue , MicroRNA Circulante/sangue , DNA/sangue , Onchocerca volvulus/fisiologia , Oncocercose/tratamento farmacológico , Oncocercose/genética , Adulto , Animais , Humanos , Masculino , Oncocercose/sangue , Parasitos/genética , Resultado do TratamentoRESUMO
Parasitic filarial nematodes cause debilitating infections in people in resource-limited countries. A clinically validated approach to eliminating worms uses a 4- to 6-week course of doxycycline that targets Wolbachia, a bacterial endosymbiont required for worm viability and reproduction. However, the prolonged length of therapy and contraindication in children and pregnant women have slowed adoption of this treatment. Here, we describe discovery and optimization of quinazolines CBR417 and CBR490 that, with a single dose, achieve >99% elimination of Wolbachia in the in vivo Litomosoides sigmodontis filarial infection model. The efficacious quinazoline series was identified by pairing a primary cell-based high-content imaging screen with an orthogonal ex vivo validation assay to rapidly quantify Wolbachia elimination in Brugia pahangi filarial ovaries. We screened 300,368 small molecules in the primary assay and identified 288 potent and selective hits. Of 134 primary hits tested, only 23.9% were active in the worm-based validation assay, 8 of which contained a quinazoline heterocycle core. Medicinal chemistry optimization generated quinazolines with excellent pharmacokinetic profiles in mice. Potent antiwolbachial activity was confirmed in L. sigmodontis, Brugia malayi, and Onchocerca ochengi in vivo preclinical models of filarial disease and in vitro selectivity against Loa loa (a safety concern in endemic areas). The favorable efficacy and in vitro safety profiles of CBR490 and CBR417 further support these as clinical candidates for treatment of filarial infections.
Assuntos
Antibacterianos/uso terapêutico , Descoberta de Drogas , Filariose/tratamento farmacológico , Filariose/parasitologia , Filarioidea/fisiologia , Quinazolinas/uso terapêutico , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Modelos Animais de Doenças , Feminino , Filarioidea/efeitos dos fármacos , Filarioidea/microbiologia , Ensaios de Triagem em Larga Escala , Camundongos , Fenótipo , Quinazolinas/química , Quinazolinas/farmacologia , Bibliotecas de Moléculas Pequenas , Wolbachia/efeitos dos fármacosRESUMO
There is an urgent global need for a safe macrofilaricide drug to accelerate elimination of the neglected tropical diseases onchocerciasis and lymphatic filariasis. From an anti-infective compound library, the macrolide veterinary antibiotic, tylosin A, was identified as a hit against Wolbachia This bacterial endosymbiont is required for filarial worm viability and fertility and is a validated target for macrofilaricidal drugs. Medicinal chemistry was undertaken to develop tylosin A analogs with improved oral bioavailability. Two analogs, A-1535469 and A-1574083, were selected. Their efficacy was tested against the gold-standard second-generation tetracycline antibiotics, doxycycline and minocycline, in mouse and gerbil infection models of lymphatic filariasis (Brugia malayi and Litomosoides sigmodontis) and onchocerciasis (Onchocerca ochengi). A 1- or 2-week course of oral A-1535469 or A-1574083 provided >90% Wolbachia depletion from nematodes in infected animals, resulting in a block in embryogenesis and depletion of microfilarial worm loads. The two analogs delivered comparative or superior efficacy compared to a 3- to 4-week course of doxycycline or minocycline. A-1574083 (now called ABBV-4083) was selected for further preclinical testing. Cardiovascular studies in dogs and toxicology studies in rats and dogs revealed no adverse effects at doses (50 mg/kg) that achieved plasma concentrations >10-fold above the efficacious concentration. A-1574083 (ABBV-4083) shows potential as an anti-Wolbachia macrolide with an efficacy, pharmacology, and safety profile that is compatible with a short-term oral drug course for treating lymphatic filariasis and onchocerciasis.
Assuntos
Filariose Linfática/tratamento farmacológico , Filariose Linfática/microbiologia , Macrolídeos/administração & dosagem , Macrolídeos/uso terapêutico , Oncocercose/tratamento farmacológico , Oncocercose/microbiologia , Wolbachia/fisiologia , Administração Oral , Animais , Modelos Animais de Doenças , Filariose Linfática/sangue , Feminino , Macrolídeos/efeitos adversos , Masculino , Camundongos Endogâmicos BALB C , Camundongos SCID , Oncocercose/sangue , Resultado do Tratamento , Tilosina/sangue , Tilosina/síntese química , Tilosina/química , Tilosina/uso terapêuticoRESUMO
Elimination of the helminth disease, river blindness, remains challenging due to ivermectin treatment-associated adverse reactions in loiasis co-infected patients. Here, we address a deficit in preclinical research tools for filarial translational research by developing Loa loa mouse infection models. We demonstrate that adult Loa loa worms in subcutaneous tissues, circulating microfilariae (mf) and presence of filarial biomarkers in sera occur following experimental infections of lymphopenic mice deficient in interleukin (IL)-2/7 gamma-chain signaling. A microfilaraemic infection model is also achievable, utilizing immune-competent or -deficient mice infused with purified Loa mf. Ivermectin but not benzimidazole treatments induce rapid decline (>90%) in parasitaemias in microfilaraemic mice. We identify up-regulation of inflammatory markers associated with allergic type-2 immune responses and eosinophilia post-ivermectin treatment. Thus, we provide validation of murine research models to identify loiasis biomarkers, to counter-screen candidate river blindness cures and to interrogate the inflammatory etiology of loiasis ivermectin-associated adverse reactions.
Assuntos
Loíase/patologia , Animais , Doença Crônica , Modelos Animais de Doenças , Eosinofilia/complicações , Eosinofilia/tratamento farmacológico , Eosinofilia/parasitologia , Feminino , Inflamação/complicações , Inflamação/tratamento farmacológico , Inflamação/patologia , Ivermectina/uso terapêutico , Loa/efeitos dos fármacos , Loa/fisiologia , Loíase/complicações , Loíase/tratamento farmacológico , Linfopenia/complicações , Linfopenia/parasitologia , Linfopenia/patologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos SCID , Microfilárias/efeitos dos fármacos , Parasitemia/complicações , Parasitemia/parasitologiaRESUMO
The Onchocerca ochengi adult implant and Brugia malayi microfilariemic Severe-Combined Immunodeficient (SCID) mouse models are validated screens to measure macrofilaricidal and microfilaricidal activities of candidate onchocerciasis drugs. The purpose of this study was to assess whether 5 daily sub-cutaneous (s.c.) injections of standard flubendazole (FBZ) suspension (10mg/kg), a single s.c. injection (10mg/kg) or 5 daily repeated oral doses of FBZ amorphous solid dispersion (ASD) formulation (0.2, 1.5 or 15mg/kg) mediated macrofilaricidal efficacy against O. ochengi male worms implanted into SCID mice. The direct microfilaricidal activity against circulating B. malayi microfilariae of single dose FBZ ASD formulation (2 or 40 mg/kg) was also evaluated and compared against the standard microfilaricide, ivermectin (IVM). Systemic exposures of FBZ/FBZ metabolites achieved following dosing were measured by pharmacokinetic (PK) bioanalysis. At necropsy, five weeks following start of FBZ SC injections, there were significant reductions in burdens of motile O. ochengi worms following multiple injections (93%) or single injection (82%). Further, significant proportions of mice dosed following multiple injections (5/6; 83%) or single injection (6/10; 60%) were infection negative (drug-cured). In comparison, no significant reduction in recovery of motile adult O. ochengi adult worms was obtained in any multiple-oral dosage group. Single oral-dosed FBZ did not mediate any significant microfilaricidal activity against circulating B. malayi mf at 2 or 7 days compared with >80% efficacy of single dose IVM. In conclusion, multiple oral FBZ formulation doses, whilst achieving substantial bioavailability, do not emulate the efficacy delivered by the parenteral route in vivo against adult O. ochengi. PK analysis determined FBZ efficacy was related to sustained systemic drug levels rather than achievable Cmax. PK modelling predicted that oral FBZ would have to be given at low dose for up to 5 weeks in the mouse model to achieve a matching efficacious exposure profile.
Assuntos
Brugia Malayi/efeitos dos fármacos , Filaricidas/farmacologia , Mebendazol/análogos & derivados , Microfilárias/efeitos dos fármacos , Onchocerca/efeitos dos fármacos , Oncocercose/tratamento farmacológico , Administração Oral , Animais , Modelos Animais de Doenças , Filaricidas/administração & dosagem , Ivermectina/administração & dosagem , Ivermectina/farmacologia , Masculino , Mebendazol/administração & dosagem , Mebendazol/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Carga ParasitáriaRESUMO
Controlling condensation conditions by surface topography is of prime interest. The aim of this work is to investigate the behavior of water droplets condensing on oriented sub-micrometric structures representing ripples with wavelengths around 800â¯nm. Droplet behavior was studied on different ripples heights and on untextured surfaces. It was specifically looked at how the presence of ripples creates geometrical confinement, and how that influences the deformation and the orientation of single droplets. Results show that the condensed droplets follow the orientation of textured features, especially with high structures height (150â¯nm). This is shown by the decreasing of droplet roundness with ripples height. The relative number of circular droplets (roundness near to 1) is around 0.6 for 70â¯nm high ripples and decrease to around 0.2 for 150â¯nm high ripples. The corresponding relative number on untextured surface is around 0.5. A mechanism, based on droplets pinning and hysteresis, is proposed to explain the influence of the ripples orientation in a vertical plane, onto the droplet deformation during coalescence step. Finally, the presence of ripples is shown to barely impact breath figure dynamics. Number of droplets and mean droplet radius for the textured and untextured surfaces present a comparable evolution.
RESUMO
Eosinophils are effectors in immunity to tissue helminths but also induce allergic immunopathology. Mechanisms of eosinophilia in non-mucosal tissues during infection remain unresolved. Here we identify a pivotal function of tissue macrophages (MÏ) in eosinophil anti-helminth immunity using a BALB/c mouse intra-peritoneal Brugia malayi filarial infection model. Eosinophilia, via C-C motif chemokine receptor (CCR)3, was necessary for immunity as CCR3 and eosinophil impairments rendered mice susceptible to chronic filarial infection. Post-infection, peritoneal MÏ populations proliferated and became alternatively-activated (AAMÏ). Filarial AAMÏ development required adaptive immunity and interleukin-4 receptor-alpha. Depletion of MÏ prior to infection suppressed eosinophilia and facilitated worm survival. Add back of filarial AAMÏ in MÏ-depleted mice recapitulated a vigorous eosinophilia. Transfer of filarial AAMÏ into Severe-Combined Immune Deficient mice mediated immunological resistance in an eosinophil-dependent manner. Exogenous IL-4 delivery recapitulated tissue AAMÏ expansions, sustained eosinophilia and mediated immunological resistance in MÏ-intact SCID mice. Co-culturing Brugia with filarial AAMÏ and/or filarial-recruited eosinophils confirmed eosinophils as the larvicidal cell type. Our data demonstrates that IL-4/IL-4Rα activated AAMÏ orchestrate eosinophil immunity to filarial tissue helminth infection.
Assuntos
Brugia Malayi/patogenicidade , Eosinofilia/imunologia , Filariose/imunologia , Interleucina-4/farmacologia , Macrófagos/imunologia , Receptores CCR3/metabolismo , Animais , Antineoplásicos/farmacologia , Brugia Malayi/efeitos dos fármacos , Citocinas/genética , Citocinas/metabolismo , Eosinofilia/tratamento farmacológico , Eosinofilia/parasitologia , Feminino , Filariose/tratamento farmacológico , Filariose/parasitologia , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Receptores CCR3/genéticaRESUMO
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RESUMO
Filarial infections are tropical diseases caused by nematodes of the Onchocercidae family such as Mansonella perstans. The infective larvae (L3) are transmitted into the skin of vertebrate hosts by blood-feeding vectors. Many filarial species settle in the serous cavities including M. perstans in humans and L. sigmodontis, a well-established model of filariasis in mice. L. sigmodontis L3 migrate to the pleural cavity where they moult into L4 around day 9 and into male and female adult worms around day 30. Little is known of the early phase of the parasite life cycle, after the L3 is inoculated in the dermis by the vector and enters the afferent lymphatic vessels and before the moulting processes in the pleural cavity. Here we reveal a pulmonary phase associated with lung damage characterized by haemorrhages and granulomas suggesting L3 reach the lung via pulmonary capillaries and damage the endothelium and parenchyma by crossing them to enter the pleural cavity. This study also provides evidence for a transient inflammation in the lung characterized by a very early recruitment of neutrophils associated with high expression levels of S100A8 and S100A9 proteins.
Assuntos
Calgranulina B/análise , Filariose/patologia , Filarioidea/crescimento & desenvolvimento , Filarioidea/imunologia , Pulmão/patologia , Neutrófilos/imunologia , Animais , Modelos Animais de Doenças , Feminino , Filariose/parasitologia , Humanos , Pulmão/parasitologia , Camundongos Endogâmicos BALB CRESUMO
Lymphatic filariasis (LF) and onchocerciasis are priority neglected tropical diseases targeted for elimination. The only safe drug treatment with substantial curative activity against the filarial nematodes responsible for LF (Brugia malayi, Wuchereria bancrofti) or onchocerciasis (Onchocerca volvulus) is doxycycline. The target of doxycycline is the essential endosymbiont, Wolbachia. Four to six weeks doxycycline therapy achieves >90% depletion of Wolbachia in worm tissues leading to blockade of embryogenesis, adult sterility and premature death 18-24 months post-treatment. Long treatment length and contraindications in children and pregnancy are obstacles to implementing doxycycline as a public health strategy. Here we determine, via preclinical infection models of Brugia malayi or Onchocerca ochengi that elevated exposures of orally-administered rifampicin can lead to Wolbachia depletions from filariae more rapidly than those achieved by doxycycline. Dose escalation of rifampicin achieves >90% Wolbachia depletion in time periods of 7 days in B. malayi and 14 days in O. ochengi. Using pharmacokinetic-pharmacodynamic modelling and mouse-human bridging analysis, we conclude that clinically relevant dose elevations of rifampicin, which have recently been determined as safe in humans, could be administered as short courses to filariasis target populations with potential to reduce anti-Wolbachia curative therapy times to between one and two weeks.
Assuntos
Antibacterianos/administração & dosagem , Filariose Linfática/tratamento farmacológico , Filarioidea/microbiologia , Oncocercose/tratamento farmacológico , Rifampina/administração & dosagem , Wolbachia/efeitos dos fármacos , Administração Oral , Animais , Antibacterianos/farmacologia , Brugia Malayi/efeitos dos fármacos , Brugia Malayi/microbiologia , Brugia Malayi/fisiologia , DNA Bacteriano/efeitos dos fármacos , Modelos Animais de Doenças , Filariose Linfática/parasitologia , Desenvolvimento Embrionário/efeitos dos fármacos , Filarioidea/efeitos dos fármacos , Filarioidea/fisiologia , Humanos , Camundongos , Onchocerca volvulus/efeitos dos fármacos , Onchocerca volvulus/microbiologia , Onchocerca volvulus/fisiologia , Oncocercose/parasitologia , Rifampina/farmacologia , Resultado do Tratamento , Wolbachia/genética , Wuchereria bancrofti/efeitos dos fármacos , Wuchereria bancrofti/microbiologia , Wuchereria bancrofti/fisiologiaRESUMO
The endosymbiotic bacteria, Wolbachia, induce neutrophilic responses to the human helminth pathogen Onchocerca volvulus. The formation of Neutrophil Extracellular Traps (NETs), has been implicated in anti-microbial defence, but has not been identified in human helminth infection. Here, we demonstrate NETs formation in human onchocerciasis. Extracellular NETs and neutrophils were visualised around O. volvulus in nodules excised from untreated patients but not in nodules from patients treated with the anti-Wolbachia drug, doxycycline. Whole Wolbachia or microspheres coated with a synthetic Wolbachia lipopeptide (WoLP) of the major nematode Wolbachia TLR2/6 ligand, peptidoglycan associated lipoprotein, induced NETosis in human neutrophils in vitro. TLR6 dependency of Wolbachia and WoLP NETosis was demonstrated using purified neutrophils from TLR6 deficient mice. Thus, we demonstrate for the first time that NETosis occurs during natural human helminth infection and demonstrate a mechanism of NETosis induction via Wolbachia endobacteria and direct ligation of Wolbachia lipoprotein by neutrophil TLR2/6.
Assuntos
Armadilhas Extracelulares/metabolismo , Neutrófilos/metabolismo , Oncocercose/microbiologia , Simbiose , Wolbachia/fisiologia , Animais , Micropartículas Derivadas de Células/metabolismo , Doxiciclina/farmacologia , Doxiciclina/uso terapêutico , Armadilhas Extracelulares/efeitos dos fármacos , Feminino , Imunofluorescência , Humanos , Lipopeptídeos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microesferas , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Oncocercose/tratamento farmacológico , Oncocercose/patologia , Simbiose/efeitos dos fármacos , Receptor 6 Toll-Like/metabolismo , Wolbachia/efeitos dos fármacosRESUMO
Our knowledge and control of the pathogenesis induced by the filariae remain limited due to experimental obstacles presented by parasitic nematode biology and the lack of selective prophylactic or curative drugs. Here we thought to investigate the role of neutrophils in the host innate immune response to the infection caused by the Litomosoides sigmodontis murine model of human filariasis using mice harboring a gain-of-function mutation of the chemokine receptor CXCR4 and characterized by a profound blood neutropenia (Cxcr4(+/1013)). We provided manifold evidence emphasizing the major role of neutrophils in the control of the early stages of infection occurring in the skin. Firstly, we uncovered that the filarial parasitic success was dramatically decreased in Cxcr4(+/1013) mice upon subcutaneous delivery of the infective stages of filariae (infective larvae, L3). This protection was linked to a larger number of neutrophils constitutively present in the skin of the mutant mice herein characterized as compared to wild type (wt) mice. Indeed, the parasitic success in Cxcr4(+/1013) mice was normalized either upon depleting neutrophils, including the pool in the skin, or bypassing the skin via the intravenous infection of L3. Second, extending these observations to wt mice we found that subcutaneous delivery of L3 elicited an increase of neutrophils in the skin. Finally, living L3 larvae were able to promote in both wt and mutant mice, an oxidative burst response and the release of neutrophil extracellular traps (NET). This response of neutrophils, which is adapted to the large size of the L3 infective stages, likely directly contributes to the anti-parasitic strategies implemented by the host. Collectively, our results are demonstrating the contribution of neutrophils in early anti-filarial host responses through their capacity to undertake different anti-filarial strategies such as oxidative burst, degranulation and NETosis.