RESUMO
Recent advances in the in vitro cultivation of Cryptosporidium parvum using hollow fiber bioreactor technology (HFB) have permitted continuous growth of parasites that complete all life cycle stages. The method provides access to all stages of the parasite and provides a method for non-animal production of oocysts for use in clinical trials. Here we examined the effect of long-term (>20 months) in vitro culture on virulence-factors, genome conservation, and in vivo pathogenicity of the host by in vitro cultured parasites. We find low-level sequence variation that is consistent with that observed in calf-passaged parasites. Further using a calf model infection, oocysts obtained from the HFB caused diarrhea of the same volume, duration and oocyst shedding intensity as in vivo passaged parasites.
Assuntos
Criptosporidiose , Cryptosporidium parvum , Cryptosporidium , Animais , Cryptosporidium parvum/genética , Virulência , Criptosporidiose/parasitologia , Oocistos , Genômica , FezesRESUMO
Recent advances on the development of bumped kinase inhibitors for treatment of cryptosporidiosis have focused on the 5-aminopyrazole-4-carboxamide scaffold, due to analogs that have less hERG inhibition, superior efficacy, and strong in vitro safety profiles. Three compounds, BKI-1770, -1841, and -1708, showed strong efficacy in C. parvum infected mice. Both BKI-1770 and BKI-1841 had efficacy in the C. parvum newborn calf model, reducing diarrhea and oocyst excretion. However, both compounds caused hyperflexion of the limbs seen as dropped pasterns. Toxicity experiments in rats and calves dosed with BKI-1770 showed enlargement of the epiphyseal growth plate at doses only slightly higher than the efficacious dose. Mice were used as a screen to check for bone toxicity, by changes to the tibia epiphyseal growth plate, or neurological causes, by use of a locomotor activity box. These results showed neurological effects from both BKI-1770 and BKI-1841 and bone toxicity in mice from BKI-1770, indicating one or both effects may be contributing to toxicity. However, BKI-1708 remains a viable treatment candidate for further evaluation as it showed no signs of bone toxicity or neurological effects in mice.
Assuntos
Antineoplásicos , Antiprotozoários , Criptosporidiose , Cryptosporidium parvum , Animais , Bovinos , Camundongos , Ratos , Criptosporidiose/tratamento farmacológico , Antiprotozoários/farmacologia , Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , OocistosRESUMO
Infection with Cryptosporidium spp. can cause severe diarrhea, leading to long-term adverse impacts and even death in malnourished children and immunocompromised patients. The only FDA-approved drug for treating cryptosporidiosis, nitazoxanide, has limited efficacy in the populations impacted the most by the diarrheal disease, and safe, effective treatment options are urgently needed. Initially identified by a large-scale phenotypic screening campaign, the antimycobacterial therapeutic clofazimine demonstrated great promise in both in vitro and in vivo preclinical models of Cryptosporidium infection. Unfortunately, a phase 2a clinical trial in HIV-infected adults with cryptosporidiosis did not identify any clofazimine treatment effect on Cryptosporidium infection burden or clinical outcomes. To explore whether clofazimine's lack of efficacy in the phase 2a trial may have been due to subtherapeutic clofazimine concentrations, a pharmacokinetic/pharmacodynamic modeling approach was undertaken to determine the relationship between clofazimine in vivo concentrations and treatment effects in multiple preclinical infection models. Exposure-response relationships were characterized using Emax and logistic models, which allowed predictions of efficacious clofazimine concentrations for the control and reduction of disease burden. After establishing exposure-response relationships for clofazimine treatment of Cryptosporidium infection in our preclinical model studies, it was unmistakable that the clofazimine levels observed in the phase 2a study participants were well below concentrations associated with anti-Cryptosporidium efficacy. Thus, despite a dosing regimen above the highest doses recommended for mycobacterial therapy, it is very likely the lack of treatment effect in the phase 2a trial was at least partially due to clofazimine concentrations below those required for efficacy against cryptosporidiosis. It is unlikely that clofazimine will provide a remedy for the large number of cryptosporidiosis patients currently without a viable treatment option unless alternative, safe clofazimine formulations with improved oral absorption are developed. (This study has been registered in ClinicalTrials.gov under identifier NCT03341767.).
Assuntos
Antiprotozoários , Criptosporidiose , Cryptosporidium , Adulto , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Criança , Clofazimina/farmacologia , Clofazimina/uso terapêutico , Criptosporidiose/tratamento farmacológico , Diarreia/tratamento farmacológico , HumanosRESUMO
This is a review of the development of bumped-kinase inhibitors (BKIs) for the therapy of One Health parasitic apicomplexan diseases. Many apicomplexan infections are shared between humans and livestock, such as cryptosporidiosis and toxoplasmosis, as well as livestock only diseases such as neosporosis. We have demonstrated proof-of-concept for BKI therapy in livestock models of cryptosporidiosis (newborn calves infected with Cryptosporidium parvum), toxoplasmosis (pregnant sheep infected with Toxoplasma gondii), and neosporosis (pregnant sheep infected with Neospora caninum). We discuss the potential uses of BKIs for the treatment of diseases caused by apicomplexan parasites in animals and humans, and the improvements that need to be made to further develop BKIs.
Assuntos
Antiparasitários/farmacologia , Criptosporidiose/tratamento farmacológico , Saúde Única , Piperidinas/farmacologia , Pirimidinas/farmacologia , Quinolinas/farmacologia , Animais , Apicomplexa , HumanosRESUMO
The protozoan parasite Cryptosporidium sp. is a leading cause of diarrheal disease in those with compromised or underdeveloped immune systems, particularly infants and toddlers in resource-poor localities. As an enteric pathogen, Cryptosporidium sp. invades the apical surface of intestinal epithelial cells, where it resides in close proximity to metabolites in the intestinal lumen. However, the effect of gut metabolites on susceptibility to Cryptosporidium infection remains largely unstudied. Here, we first identified which gut metabolites are prevalent in neonatal mice when they are most susceptible to Cryptosporidium parvum infection and then tested the isolated effects of these metabolites on C. parvum invasion and growth in intestinal epithelial cells. Our findings demonstrate that medium or long-chain saturated fatty acids inhibit C. parvum growth, perhaps by negatively affecting the streamlined metabolism in C. parvum, which is unable to synthesize fatty acids. Conversely, long-chain unsaturated fatty acids enhanced C. parvum invasion, possibly by modulating membrane fluidity. Hence, gut metabolites, either from diet or produced by the microbiota, influence C. parvum growth in vitro and may also contribute to the early susceptibility to cryptosporidiosis seen in young animals.IMPORTANCECryptosporidium sp. occupies a unique intracellular niche that exposes the parasite to both host cell contents and the intestinal lumen, including metabolites from the diet and produced by the microbiota. Both dietary and microbial products change over the course of early development and could contribute to the changes seen in susceptibility to cryptosporidiosis in humans and mice. Consistent with this model, we show that the immature gut metabolome influenced the growth of Cryptosporidium parvumin vitro Interestingly, metabolites that significantly altered parasite growth were fatty acids, a class of molecules that Cryptosporidium sp. is unable to synthesize de novo The enhancing effects of polyunsaturated fatty acids and the inhibitory effects of saturated fatty acids presented in this study may provide a framework for future studies into this enteric parasite's interactions with exogenous fatty acids during the initial stages of infection.
Assuntos
Bactérias/metabolismo , Criptosporidiose/parasitologia , Cryptosporidium parvum/fisiologia , Microbioma Gastrointestinal , Mucosa Intestinal/microbiologia , Mucosa Intestinal/parasitologia , Animais , Animais Recém-Nascidos/metabolismo , Animais Recém-Nascidos/microbiologia , Animais Recém-Nascidos/parasitologia , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Criptosporidiose/metabolismo , Criptosporidiose/microbiologia , Cryptosporidium parvum/genética , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/parasitologia , Ácidos Graxos/metabolismo , Feminino , Humanos , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICRRESUMO
Apicomplexan parasites cause severe disease in both humans and their domesticated animals. Since these parasites readily develop drug resistance, development of new, effective drugs to treat infection caused by these parasites is an ongoing challenge for the medical and veterinary communities. We hypothesized that invertebrate-bacterial symbioses might be a rich source of anti-apicomplexan compounds because invertebrates are susceptible to infections with gregarines, parasites that are ancestral to all apicomplexans. We chose to explore the therapeutic potential of shipworm symbiotic bacteria as they are bona fide symbionts, are easily grown in axenic culture and have genomes rich in secondary metabolite loci [1,2]. Two strains of the shipworm symbiotic bacterium, Teredinibacter turnerae, were screened for activity against Toxoplasma gondii and one strain, T7901, exhibited activity against intracellular stages of the parasite. Bioassay-guided fractionation identified tartrolon E (trtE) as the source of the activity. TrtE has an EC50 of 3 nM against T. gondii, acts directly on the parasite itself and kills the parasites after two hours of treatment. TrtE exhibits nanomolar to picomolar level activity against Cryptosporidium, Plasmodium, Babesia, Theileria, and Sarcocystis; parasites representing all branches of the apicomplexan phylogenetic tree. The compound also proved effective against Cryptosporidium parvum infection in neonatal mice, indicating that trtE may be a potential lead compound for preclinical development. Identification of a promising new compound after such limited screening strongly encourages further mining of invertebrate symbionts for new anti-parasitic therapeutics.
Assuntos
Antiprotozoários , Apicomplexa/crescimento & desenvolvimento , Bivalves/microbiologia , Gammaproteobacteria/metabolismo , Simbiose , Animais , Antiprotozoários/metabolismo , Antiprotozoários/farmacologia , Camundongos , Infecções por Protozoários/tratamento farmacológicoRESUMO
Bumped Kinase Inhibitors, targeting Calcium-dependent Protein Kinase 1 in apicomplexan parasites with a glycine gatekeeper, are promising new therapeutics for apicomplexan diseases. Here we will review advances, as well as challenges and lessons learned regarding efficacy, safety, and pharmacology that have shaped our selection of pre-clinical candidates.
Assuntos
Apicomplexa/efeitos dos fármacos , Coccidiose/tratamento farmacológico , Inibidores de Proteínas Quinases , Animais , Apicomplexa/metabolismo , Criptosporidiose/tratamento farmacológico , Cryptosporidium/efeitos dos fármacos , Cryptosporidium/metabolismo , Humanos , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/efeitos dos fármacos , Proteínas Quinases/metabolismo , Toxoplasma/efeitos dos fármacos , Toxoplasma/metabolismo , Toxoplasmose/tratamento farmacológicoRESUMO
Cryptosporidiosis, caused by the apicomplexan parasite Cryptosporidium parvum, is a moderate-to-severe diarrheal disease now recognized as one of the leading causes of morbidity and mortality in livestock globally, and in humans living in resource-limited parts of the world, particularly those with AIDS or malnourished individuals. This recognition has fueled efforts for the discovery of effective therapeutics. While recent progress in drug discovery has been encouraging, there are presently no acceptably effective parasite-specific drugs for the disease. The urgent need for new drug discovery or drug repurposing has also increased the need for refined animal models of clinical disease for therapeutic efficacy evaluation. Here, we describe an acute model of cryptosporidiosis using newborn calves to evaluate well-defined clinical and parasitological parameter outcomes, including the effect on diarrhea severity and duration, oocyst numbers produced, and multiple measures of clinical health. The model is highly reproducible and provides unequivocal direct measures of treatment efficacy on diarrhea severity and parasite replication.
Assuntos
Doenças dos Bovinos/tratamento farmacológico , Criptosporidiose/tratamento farmacológico , Cryptosporidium parvum/efeitos dos fármacos , Diarreia/veterinária , Modelos Animais de Doenças , Oocistos/efeitos dos fármacos , Animais , Bovinos , Doenças dos Bovinos/parasitologia , Doenças dos Bovinos/patologia , Doenças dos Bovinos/urina , Criptosporidiose/parasitologia , Criptosporidiose/patologia , Criptosporidiose/urina , Cryptosporidium parvum/crescimento & desenvolvimento , Cryptosporidium parvum/parasitologia , Diarreia/tratamento farmacológico , Diarreia/parasitologia , Diarreia/patologia , Fezes/parasitologia , Humanos , Recém-Nascido , Oocistos/crescimento & desenvolvimento , Oocistos/isolamento & purificação , Oocistos/metabolismo , Fluxo de TrabalhoRESUMO
Stem-cell-derived organoids recapitulate in vivo physiology of their original tissues, representing valuable systems to model medical disorders such as infectious diseases. Cryptosporidium, a protozoan parasite, is a leading cause of diarrhoea and a major cause of child mortality worldwide. Drug development requires detailed knowledge of the pathophysiology of Cryptosporidium, but experimental approaches have been hindered by the lack of an optimal in vitro culture system. Here, we show that Cryptosporidium can infect epithelial organoids derived from human small intestine and lung. The parasite propagates within the organoids and completes its complex life cycle. Temporal analysis of the Cryptosporidium transcriptome during organoid infection reveals dynamic regulation of transcripts related to its life cycle. Our study presents organoids as a physiologically relevant in vitro model system to study Cryptosporidium infection.
Assuntos
Criptosporidiose/genética , Cryptosporidium/patogenicidade , Perfilação da Expressão Gênica/métodos , Organoides/parasitologia , Criptosporidiose/parasitologia , Cryptosporidium/crescimento & desenvolvimento , Regulação da Expressão Gênica , Humanos , Intestino Delgado/parasitologia , Pulmão/parasitologia , Modelos Biológicos , Técnicas de Cultura de Órgãos , Análise de Sequência de RNA , Análise Espaço-TemporalRESUMO
Improvements have been made to the safety and efficacy of bumped kinase inhibitors, and they are advancing toward human and animal use for treatment of cryptosporidiosis. As the understanding of bumped kinase inhibitor pharmacodynamics for cryptosporidiosis therapy has increased, it has become clear that better compounds for efficacy do not necessarily require substantial systemic exposure. We now have a bumped kinase inhibitor with reduced systemic exposure, acceptable safety parameters, and efficacy in both the mouse and newborn calf models of cryptosporidiosis. Potential cardiotoxicity is the limiting safety parameter to monitor for this bumped kinase inhibitor. This compound is a promising pre-clinical lead for cryptosporidiosis therapy in animals and humans.
Assuntos
Criptosporidiose/tratamento farmacológico , Cryptosporidium parvum/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Administração Oral , Animais , Animais Recém-Nascidos , Bovinos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Coração/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Interferon gama/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Testes de Mutagenicidade , Gravidez , Ligação Proteica , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/sangue , Inibidores de Proteínas Quinases/toxicidade , SegurançaRESUMO
There is a substantial need for novel therapeutics to combat the widespread impact caused by Crytosporidium infection. However, there is a lack of knowledge as to which drug pharmacokinetic (PK) characteristics are key to generate an in vivo response, specifically whether systemic drug exposure is crucial for in vivo efficacy. To identify which PK properties are correlated with in vivo efficacy, we generated physiologically based PK models to simulate systemic and gastrointestinal drug concentrations for a series of bumped kinase inhibitors (BKIs) that have nearly identical in vitro potency against Cryptosporidium but display divergent PK properties. When BKI concentrations were used to predict in vivo efficacy with a neonatal model of Cryptosporidium infection, these concentrations in the large intestine were the sole predictors of the observed in vivo efficacy. The significance of large intestinal BKI exposure for predicting in vivo efficacy was further supported with an adult mouse model of Cryptosporidium infection. This study suggests that drug exposure in the large intestine is essential for generating a superior in vivo response, and that physiologically based PK models can assist in the prioritization of leading preclinical drug candidates for in vivo testing.
Assuntos
Criptosporidiose/tratamento farmacológico , Trato Gastrointestinal/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacocinética , Animais , Cryptosporidium parvum/efeitos dos fármacos , Cryptosporidium parvum/isolamento & purificação , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Trato Gastrointestinal/metabolismo , Concentração Inibidora 50 , Camundongos , Camundongos Knockout , Modelos Teóricos , Naftalenos/farmacocinética , Piperidinas/farmacocinética , Inibidores de Proteínas Quinases/sangue , Pirazóis/farmacocinéticaRESUMO
Cryptosporidium parvum calcium-dependent protein kinase 1 (CpCDPK1) is a promising target for drug development against cryptosporidiosis. We report a series of low-nanomolar CpCDPK1 5-aminopyrazole-4-carboxamide (AC) scaffold inhibitors that also potently inhibit C. parvum growth in vitro Correlation between anti-CpCDPK1 and C. parvum growth inhibition, as previously reported for pyrazolopyrimidines, was not apparent. Nonetheless, lead AC compounds exhibited a substantial reduction of parasite burden in the neonatal mouse cryptosporidiosis model when dosed at 25 mg/kg.
Assuntos
Antiprotozoários/farmacologia , Criptosporidiose/tratamento farmacológico , Cryptosporidium parvum/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Animais , Antiprotozoários/química , Criptosporidiose/parasitologia , Cryptosporidium parvum/crescimento & desenvolvimento , Camundongos , Proteínas de Protozoários/metabolismo , Pirazóis/química , Pirazóis/farmacologiaRESUMO
Bumped kinase inhibitors (BKIs) of Cryptosporidium parvum calcium-dependent protein kinase 1 (CpCDPK1) are leading candidates for treatment of cryptosporidiosis-associated diarrhea. Potential cardiotoxicity related to anti-human ether-à-go-go potassium channel (hERG) activity of the first-generation anti-Cryptosporidium BKIs triggered further testing for efficacy. A luminescence assay adapted for high-throughput screening was used to measure inhibitory activities of BKIs against C. parvum in vitro. Furthermore, neonatal and interferon γ knockout mouse models of C. parvum infection identified BKIs with in vivo activity. Additional iterative experiments for optimum dosing and selecting BKIs with minimum levels of hERG activity and frequencies of other safety liabilities included those that investigated mammalian cell cytotoxicity, C. parvum proliferation inhibition in vitro, anti-human Src inhibition, hERG activity, in vivo pharmacokinetic data, and efficacy in other mouse models. Findings of this study suggest that fecal concentrations greater than parasite inhibitory concentrations correlate best with effective therapy in the mouse model of cryptosporidiosis, but a more refined model for efficacy is needed.
Assuntos
Antiprotozoários/administração & dosagem , Criptosporidiose/tratamento farmacológico , Cryptosporidium parvum/efeitos dos fármacos , Inibidores de Proteínas Quinases/administração & dosagem , Administração Oral , Animais , Diarreia/tratamento farmacológico , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Knockout , Camundongos SCIDRESUMO
Many life-cycle processes in parasites are regulated by protein phosphorylation. Hence, disruption of essential protein kinase function has been explored for therapy of parasitic diseases. However, the difficulty of inhibiting parasite protein kinases to the exclusion of host orthologues poses a practical challenge. A possible path around this difficulty is the use of bumped kinase inhibitors for targeting calcium-dependent protein kinases that contain atypically small gatekeeper residues and are crucial for pathogenic apicomplexan parasites' survival and proliferation. In this article, we review efficacy against the kinase target, parasite growth in vitro, and in animal infection models, as well as the relevant pharmacokinetic and safety parameters of bumped kinase inhibitors.
Assuntos
Antiprotozoários/farmacologia , Apicomplexa/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Infecções por Protozoários/tratamento farmacológico , Animais , Antiprotozoários/uso terapêutico , Apicomplexa/enzimologia , Benzimidazóis/química , Humanos , Imidazóis/química , Inibidores de Proteínas Quinases/uso terapêutico , Infecções por Protozoários/prevenção & controle , Piridinas/químicaRESUMO
Cryptosporidiosis, caused by the apicomplexan parasite Cryptosporidium parvum, is a diarrheal disease that has produced a large global burden in mortality and morbidity in humans and livestock. There are currently no consistently effective parasite-specific pharmaceuticals available for this disease. Bumped kinase inhibitors (BKIs) specific for parasite calcium-dependent protein kinases (CDPKs) have been shown to reduce infection in several parasites having medical and veterinary importance, including Toxoplasma gondii, Plasmodium falciparum, and C. parvum In the present study, BKIs were screened for efficacy against C. parvum infection in the neonatal mouse model. Three BKIs were then selected for safety and clinical efficacy evaluation in the calf model for cryptosporidiosis. Significant BKI treatment effects were observed for virtually all clinical and parasitological scoring parameters, including diarrhea severity, oocyst shedding, and overall health. These results provide proof of concept for BKIs as therapeutic drug leads in an animal model for human cryptosporidiosis.
Assuntos
Antiprotozoários/administração & dosagem , Doenças dos Bovinos/tratamento farmacológico , Criptosporidiose/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Animais , Animais Recém-Nascidos , Antiprotozoários/efeitos adversos , Bovinos , Cryptosporidium parvum/efeitos dos fármacos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Camundongos Endogâmicos BALB C , Inibidores de Proteínas Quinases/efeitos adversos , Resultado do TratamentoRESUMO
Attaching and effacing (A/E) pathogens adhere intimately to intestinal enterocytes and efface brush border microvilli. A key virulence strategy of A/E pathogens is the type III secretion system (T3SS)-mediated delivery of effector proteins into host cells. The secreted protein EspZ is postulated to promote enterocyte survival by regulating the T3SS and/or by modulating epithelial signaling pathways. To explore the role of EspZ in A/E pathogen virulence, we generated an isogenic espZ deletion strain (ΔespZ) and corresponding cis-complemented derivatives of rabbit enteropathogenic Escherichia coli and compared their abilities to regulate the T3SS and influence host cell survival in vitro. For virulence studies, rabbits infected with these strains were monitored for bacterial colonization, clinical signs, and intestinal tissue alterations. Consistent with data from previous reports, espZ-transfected epithelial cells were refractory to infection-dependent effector translocation. Also, the ΔespZ strain induced greater host cell death than did the parent and complemented strains. In rabbit infections, fecal ΔespZ strain levels were 10-fold lower than those of the parent strain at 1 day postinfection, while the complemented strain was recovered at intermediate levels. In contrast to the parent and complemented mutants, ΔespZ mutant fecal carriage progressively decreased on subsequent days. ΔespZ mutant-infected animals gained weight steadily over the infection period, failed to show characteristic disease symptoms, and displayed minimal infection-induced histological alterations. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining of intestinal sections revealed increased epithelial cell apoptosis on day 1 after infection with the ΔespZ strain compared to animals infected with the parent or complemented strains. Thus, EspZ-dependent host cell cytoprotection likely prevents epithelial cell death and sloughing and thereby promotes bacterial colonization.
Assuntos
Enterócitos/microbiologia , Escherichia coli Enteropatogênica/genética , Escherichia coli Enteropatogênica/patogenicidade , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Microvilosidades/microbiologia , Animais , Apoptose , Carga Bacteriana , Sistemas de Secreção Bacterianos/genética , Enterócitos/patologia , Escherichia coli Enteropatogênica/metabolismo , Infecções por Escherichia coli/patologia , Proteínas de Escherichia coli/metabolismo , Fezes/microbiologia , Deleção de Genes , Expressão Gênica , Teste de Complementação Genética , Interações Hospedeiro-Patógeno , Humanos , Masculino , Microvilosidades/patologia , Coelhos , VirulênciaRESUMO
Host defence peptides (HDPs) are expressed throughout the animal and plant kingdoms. They have multifunctional roles in the defence against infectious agents of mammals, possessing both bactericidal and immune-modulatory activities. We have identified a novel family of molecules secreted by helminth parasites (helminth defence molecules; HDMs) that exhibit similar structural and biochemical characteristics to the HDPs. Here, we have analyzed the functional activities of four HDMs derived from Schistosoma mansoni and Fasciola hepatica and compared them to human, mouse, bovine and sheep HDPs. Unlike the mammalian HDPs the helminth-derived HDMs show no antimicrobial activity and are non-cytotoxic to mammalian cells (macrophages and red blood cells). However, both the mammalian- and helminth-derived peptides suppress the activation of macrophages by microbial stimuli and alter the response of B cells to cytokine stimulation. Therefore, we hypothesise that HDMs represent a novel family of HDPs that evolved to regulate the immune responses of their mammalian hosts by retaining potent immune modulatory properties without causing deleterious cytotoxic effects.
Assuntos
Peptídeos Catiônicos Antimicrobianos/imunologia , Fasciola hepatica/imunologia , Proteínas de Helminto/imunologia , Interações Hospedeiro-Patógeno , Fatores Imunológicos/imunologia , Macrófagos/efeitos dos fármacos , Schistosoma mansoni/imunologia , Animais , Antibacterianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Bactérias/efeitos dos fármacos , Bovinos , Células Cultivadas , Citotoxinas/metabolismo , Eritrócitos/efeitos dos fármacos , Proteínas de Helminto/metabolismo , Humanos , Fatores Imunológicos/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , CatelicidinasRESUMO
Cryptosporidium parvum is one of the main causes of diarrhea in neonatal calves resulting in significant morbidity and economic losses for producers worldwide. We have previously demonstrated efficacy of a new class of antimicrobial antibody fusions in a neonatal mouse model for C. parvum infection. Here, we extend efficacy testing of these products to experimental infection in calves, the principal target species. Neonatal calves were challenged with C. parvum oocysts and concomitantly treated with antibody-biocide fusion 4H9-G1-LL37 over the course of four days. This resulted in reduced severity of the disease when compared to control animals. Overall clinical health parameters showed significant improvement in treated animals. Oocyst shedding was reduced in treated when compared to control animals. Control of oocyst shedding is a prerequisite for breaking the cycle of re-infection on dairy farms. Antibody-biocide fusion products thus have the potential to reduce the impact of the infection in both individual animals and in the herd.
Assuntos
Doenças dos Bovinos/parasitologia , Criptosporidiose/veterinária , Cryptosporidium parvum , Proteínas Recombinantes/uso terapêutico , Animais , Anticorpos Antiprotozoários/sangue , Especificidade de Anticorpos , Bovinos , Doenças dos Bovinos/sangue , Criptosporidiose/parasitologia , Indústria de Laticínios , Fezes/parasitologia , Masculino , Camundongos , Oocistos , Contagem de Ovos de Parasitas , Proteínas Recombinantes/administração & dosagemRESUMO
The apicomplexan parasite Cryptosporidium parvum is an important cause of diarrhea in humans and cattle, and it can persistently infect immunocompromised hosts. No consistently effective parasite-specific pharmaceuticals or immunotherapies for control of cryptosporidiosis are presently available. The innate immune system represents the first line of host defense against a range of infectious agents, including parasitic protozoa. Several types of antimicrobial peptides and proteins, collectively referred to herein as biocides, constitute a major effector component of this system. In the present study, we evaluated lactoferrin, lactoferrin hydrolysate, 5 cationic peptides (lactoferricin B, cathelicidin LL37, indolicidin, ß-defensin 1, ß-defensin 2), lysozyme, and 2 phospholipases (phospholipase A2, and phosphatidylinositol-specific phospholipase C) for anti-cryptosporidial activity. The biocides were evaluated either alone or in combination with 3E2, a monoclonal antibody (MAb) against C. parvum that inhibits sporozoite attachment and invasion. Sporozoite viability and infectivity were used as indices of anti-cryptosporidial activity in vitro. All biocides except lactoferrin had a significant effect on sporozoite viability and infectivity. Lactoferrin hydrolysate and each of the 5 cationic peptides were highly parasiticidal and strongly reduced sporozoite infectivity. While each phospholipase also had parasiticidal activity, it was significantly less than that of lactoferrin hydrolysate and each of the cationic peptides. However, each phospholipase reduced sporozoite infectivity comparably to that observed with lactoferrin hydrolysate and the cationic peptides. Moreover, when 3 of the cationic peptides (cathelicidin LL37, ß-defensin 1, and ß-defensin 2) were individually combined with MAb 3E2, a significantly greater reduction of sporozoite infectivity was observed over that by 3E2 alone. In contrast, reduction of sporozoite infectivity by a combination of either phospholipase with MAb 3E2 was no greater than that by 3E2 alone. These collective observations suggest that cationic peptides and phospholipases neutralize C. parvum by mechanisms that are predominantly either parasiticidal or non-parasiticidal, respectively.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Criptosporidiose/tratamento farmacológico , Cryptosporidium parvum/efeitos dos fármacos , Fosfolipases/farmacologia , Animais , Animais Recém-Nascidos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Células CACO-2 , Bovinos , Cryptosporidium parvum/imunologia , Cryptosporidium parvum/fisiologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Quimioterapia Combinada , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Fosfolipases/uso terapêutico , Organismos Livres de Patógenos Específicos , Esporozoítos/efeitos dos fármacos , Esporozoítos/fisiologiaRESUMO
Cryptosporidiosis in calves is an ongoing problem, primarily because of the high prevalence and high morbidity associated with the infection. This article summarizes current knowledge of the host/parasite interactions associated with cryptosporidiosis. The infection process in intestinal mucosa, the pathophysiology of the disease process, and the immune responses initiated in the calf to control the infection are discussed. Methods for diagnosing C. parvum infection, treatments that have been tried, and management controls are also examined.