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1.
PLoS Negl Trop Dis ; 14(4): e0008224, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32302296

RESUMO

Giardiasis and other protozoan infections are major worldwide causes of morbidity and mortality, yet development of new antimicrobial agents with improved efficacy and ability to override increasingly common drug resistance remains a major challenge. Antimicrobial drug development typically proceeds by broad functional screens of large chemical libraries or hypothesis-driven exploration of single microbial targets, but both strategies have challenges that have limited the introduction of new antimicrobials. Here, we describe an alternative drug development strategy that identifies a sufficient but manageable number of promising targets, while reducing the risk of pursuing targets of unproven value. The strategy is based on defining and exploiting the incompletely understood adduction targets of 5-nitroimidazoles, which are proven antimicrobials against a wide range of anaerobic protozoan and bacterial pathogens. Comprehensive adductome analysis by modified click chemistry and multi-dimensional proteomics were applied to the model pathogen Giardia lamblia to identify dozens of adducted protein targets common to both 5'-nitroimidazole-sensitive and -resistant cells. The list was highly enriched for known targets in G. lamblia, including arginine deiminase, α-tubulin, carbamate kinase, and heat shock protein 90, demonstrating the utility of the approach. Importantly, over twenty potential novel drug targets were identified. Inhibitors of two representative new targets, NADP-specific glutamate dehydrogenase and peroxiredoxin, were found to have significant antigiardial activity. Furthermore, all the identified targets remained available in resistant cells, since giardicidal activity of the respective inhibitors was not impacted by resistance to 5'-nitroimidazoles. These results demonstrate that the combined use of click chemistry and proteomics has the potential to reveal alternative drug targets for overcoming antimicrobial drug resistance in protozoan parasites.

2.
Nature ; 575(7783): 505-511, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31723265

RESUMO

Chronic liver disease due to alcohol-use disorder contributes markedly to the global burden of disease and mortality1-3. Alcoholic hepatitis is a severe and life-threatening form of alcohol-associated liver disease. The gut microbiota promotes ethanol-induced liver disease in mice4, but little is known about the microbial factors that are responsible for this process. Here we identify cytolysin-a two-subunit exotoxin that is secreted by Enterococcus faecalis5,6-as a cause of hepatocyte death and liver injury. Compared with non-alcoholic individuals or patients with alcohol-use disorder, patients with alcoholic hepatitis have increased faecal numbers of E. faecalis. The presence of cytolysin-positive (cytolytic) E. faecalis correlated with the severity of liver disease and with mortality in patients with alcoholic hepatitis. Using humanized mice that were colonized with bacteria from the faeces of patients with alcoholic hepatitis, we investigated the therapeutic effects of bacteriophages that target cytolytic E. faecalis. We found that these bacteriophages decrease cytolysin in the liver and abolish ethanol-induced liver disease in humanized mice. Our findings link cytolytic E. faecalis with more severe clinical outcomes and increased mortality in patients with alcoholic hepatitis. We show that bacteriophages can specifically target cytolytic E. faecalis, which provides a method for precisely editing the intestinal microbiota. A clinical trial with a larger cohort is required to validate the relevance of our findings in humans, and to test whether this therapeutic approach is effective for patients with alcoholic hepatitis.


Assuntos
Bacteriófagos/fisiologia , Enterococcus faecalis/patogenicidade , Enterococcus faecalis/virologia , Microbioma Gastrointestinal , Hepatite Alcoólica/microbiologia , Hepatite Alcoólica/terapia , Terapia por Fagos , Alcoolismo/complicações , Alcoolismo/microbiologia , Animais , Enterococcus faecalis/isolamento & purificação , Etanol/efeitos adversos , Fígado Gorduroso/complicações , Fígado Gorduroso/microbiologia , Fezes/microbiologia , Feminino , Vida Livre de Germes , Hepatite Alcoólica/complicações , Hepatite Alcoólica/mortalidade , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Perforina/metabolismo
3.
Artigo em Inglês | MEDLINE | ID: mdl-31451503

RESUMO

Trichomoniasis is a sexually transmitted disease with hundreds of millions of annual cases worldwide. Approved treatment options are limited to two related nitro-heterocyclic compounds, yet resistance to these drugs is an increasing concern. New antimicrobials against the causative agent, Trichomonas vaginalis, are urgently needed. We show here that clinically approved anticancer drugs that inhibit the proteasome, a large protease complex with a critical role in degrading intracellular proteins in eukaryotes, have submicromolar activity against the parasite in vitro and on-target activity against the enriched T. vaginalis proteasome in cell-free assays. Proteomic analysis confirmed that the parasite has all seven α and seven ß subunits of the eukaryotic proteasome although they have only modest sequence identities, ranging from 28 to 52%, relative to the respective human proteasome subunits. A screen of proteasome inhibitors derived from a marine natural product, carmaphycin, revealed one derivative, carmaphycin-17, with greater activity against T. vaginalis than the reference drug metronidazole, the ability to overcome metronidazole resistance, and reduced human cytotoxicity compared to that of the anticancer proteasome inhibitors. The increased selectivity of carmaphycin-17 for T. vaginalis was related to its >5-fold greater potency against the ß1 and ß5 catalytic subunits of the T. vaginalis proteasome than against the human proteasome subunits. In a murine model of vaginal trichomonad infection, proteasome inhibitors eliminated or significantly reduced parasite burden upon topical treatment without any apparent adverse effects. Together, these findings validate the proteasome of T. vaginalis as a therapeutic target for development of a novel class of trichomonacidal agents.

4.
J Am Assoc Lab Anim Sci ; 58(5): 558-568, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31319899

RESUMO

Gnotobiotic animal research has expanded markedly over the past decade. Although germ-free animals were first described more than 100 y ago, little evidence-based guidance is available on best operational procedures. A key aspect of gnotobiotic technology is the sterilization of animal enclosures, most commonly flexible vinyl film isolators. The objective of this study was to determine the most effective methods for chemical sterilization of gnotobiotic isolators and associated equipment. As test microbes, we used bacteria from 4 different accidental isolator contaminations that occurred in a gnotobiotic core facility. Identification by 16S ribotyping revealed facultative anaerobic firmicutes, including several Paenibacillus and Bacillus species, and obligate aerobic actinobacteria, namely Micrococcus luteus, among the contaminants. We selected 6 products commonly used for disinfecting hospital rooms, kitchens, and veterinary facilities to represent chlorine-oxide- and peroxide-based disinfectants and tested the hypothesis that these 2 classes are equally effective. However, evaluation of bactericidal and sporicidal activity in liquid cultures revealed that chlorine oxide-based disinfectants were more effective than peroxide-based disinfectants. In both groups, various products effectively sterilized gnotobiotic isolators by fogging in field tests, although bactericidal concentrations were markedly higher than those in suspension cultures, and effectiveness was contact-time-dependent. In addition, in both groups, some disinfectants were excessively corrosive to ferrous metals and acrylic. These results demonstrate that no single disinfectant has all desirable properties and that the different characteristics of disinfectants must be balanced during their selection. However, chlorine oxide-based disinfectants were generally more effective and less corrosive than peroxide-based products.

5.
Infect Immun ; 87(6)2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30962402

RESUMO

Giardia lamblia, one of the most common protozoal infections of the human intestine, is an important worldwide cause of diarrheal disease, malabsorption, malnutrition, delayed cognitive development in children, and protracted postinfectious syndromes. Despite its medical importance, no human vaccine is available against giardiasis. A crude veterinary vaccine has been developed, and experimental vaccines based on expression of multiple variant-specific surface proteins have been reported, but poorly defined vaccine components and excessive antigen variability are problematic for pharmaceutical vaccine production. To expand the repertoire of antigen candidates for vaccines, we reasoned that surface proteins may provide an enriched source of such antigens since key host effectors, such as secretory IgA, can directly bind to such antigens in the intestinal lumen and interfere with epithelial attachment. Here, we have applied a proteomics approach to identify 23 novel surface antigens of G. lamblia that show >90% amino acid sequence identity between the two human-pathogenic genetic assemblages (A and B) of the parasite. Surface localization of a representative subset of these proteins was confirmed by immunostaining. Four selected proteins, uridine phosphorylase-like protein-1, protein 21.1 (GL50803_27925), α1-giardin, and α11-giardin, were subsequently produced in recombinant form and shown to be immunogenic in mice and G. lamblia-infected humans and confer protection against G. lamblia infection upon intranasal immunization in rodent models of giardiasis. These results demonstrate that identification of conserved surface antigens provides a powerful approach for overcoming a key rate-limiting step in the design and construction of an effective vaccine against giardiasis.


Assuntos
Antígenos de Protozoários/imunologia , Giardia lamblia/imunologia , Giardíase/parasitologia , Proteoma/imunologia , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/imunologia , Adulto , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Reações Cruzadas , Feminino , Giardia lamblia/química , Giardia lamblia/genética , Giardíase/imunologia , Giardíase/prevenção & controle , Humanos , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Proteoma/química , Proteoma/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Vacinas Protozoárias/química , Vacinas Protozoárias/genética , Adulto Jovem
6.
Diabetes Ther ; 10(2): 673-681, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30788807

RESUMO

INTRODUCTION: Despite the widespread use of glucagon-like peptide-1 (GLP-1) receptor agonists (GLP-1ras) to improve glycemic regulation, with a low risk of hypoglycemia and weight reduction, their effectiveness varies among individuals. This study aimed to identify predictors of the efficacy of GLP-1ra on Hemoglobin A1c (HbA1c) in patients with insulin-independent diabetes. METHODS: In total, 58 patients with insulin-independent diabetes were included. Patients were included if their ß-cell function was evaluated via a glucagon stimulation test (GST) before the introduction of GLP-1ra therapy. ß-Cell function-related indices, such as the C-peptide index (CPI), increments in C-peptide immunoreactivity (CPR) after glucagon stimulation (ΔCPR), and the area under the CPR curve (CPR-AUC) during the GST, were evaluated. HbA1c and body weight (BW) were measured at 6 and 12 months after the initiation of GLP-1ra. RESULTS: A univariate regression analysis revealed a significant correlation between CPR-AUC and changes in HbA1c at 6 months and with changes in BW at 6 and 12 months. A multivariate regression analysis revealed that CPR-AUC was significantly correlated with changes in HbA1c at 6 months. A receiver-operating characteristic analysis revealed that 21.9 ng/ml·min CPR-AUC was the optimal cut-off value to predict an HbA1c level < 7%, i.e., 53 mmol/mol. CONCLUSION: Residual ß-cell function, as assessed via CPR-AUC in the GST, is an effective predictor of the efficacy of GLP-1ras.

7.
Int J Parasitol Drugs Drug Resist ; 8(3): 394-402, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30103207

RESUMO

Babesiosis is a tick-transmitted zoonosis caused by apicomplexan parasites of the genus Babesia. Treatment of this emerging malaria-related disease has relied on antimalarial drugs and antibiotics. The proteasome of Plasmodium, the causative agent of malaria, has recently been validated as a target for anti-malarial drug development and therefore, in this study, we investigated the effect of epoxyketone (carfilzomib, ONX-0914 and epoxomicin) and boronic acid (bortezomib and ixazomib) proteasome inhibitors on the growth and survival of Babesia. Testing the compounds against Babesia divergens ex vivo revealed suppressive effects on parasite growth with activity that was higher than the cytotoxic effects on a non-transformed mouse macrophage cell line. Furthermore, we showed that the most-effective compound, carfilzomib, significantly reduces parasite multiplication in a Babesia microti infected mouse model without noticeable adverse effects. In addition, treatment with carfilzomib lead to an ex vivo and in vivo decrease in proteasome activity and accumulation of polyubiquitinated proteins compared to untreated control. Overall, our results demonstrate that the Babesia proteasome is a valid target for drug development and warrants the design of potent and selective B. divergens proteasome inhibitors for the treatment of babesiosis.


Assuntos
Babesia microti/efeitos dos fármacos , Babesia/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , Proteoma/efeitos dos fármacos , Animais , Babesia/genética , Babesia/crescimento & desenvolvimento , Babesia microti/genética , Babesia microti/crescimento & desenvolvimento , Babesiose/tratamento farmacológico , Ácidos Borônicos/farmacologia , Linhagem Celular , Modelos Animais de Doenças , Feminino , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Camundongos , Oligopeptídeos/farmacologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Inibidores de Proteassoma/administração & dosagem , Inibidores de Proteassoma/efeitos adversos , Proteoma/genética
8.
J Clin Lipidol ; 12(2): 331-337, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29397342

RESUMO

BACKGROUND: Angiopoietin-like protein 8 (ANGPTL8) is considered to be metabolically multifunctional. One notable function still to be elucidated definitively is a betatrophic role in protecting and preserving pancreatic beta-cell function. There is, however, a paucity of data regarding the role of ANGPTL8 in the etiology of type 2 diabetes (T2D), but some findings of human research have suggested the potential for significant involvement. OBJECTIVE: To examine the frequency of T2D and impaired glucose tolerance (IGT) in Japanese subjects with the ANGPTL8 R59W variant. METHODS: ANGPTL8 R59W (Rs2278426, c.194C > T) was determined by polymerase chain reaction-restriction fragment length polymorphism using the restriction enzyme FokI in 797 consecutive Japanese individuals. Subjects with triglyceride levels greater than or equal to 150 mg/dL were considered to be hypertriglyceridemic. RESULTS: Genotype frequencies of ANGPTL8 R59W were as follows: wild-type RR (C/C) 53.5%, RW (C/T) 36.6%, and WW (T/T) 9.9%. T2D and IGT were significantly prevalent in WW and RW subjects relative to RR among all 797 subjects (P = .0138) and also in hypertriglyceridemic subjects (P = .0015). In multiple logistic regression models for the existence of T2D and IGT in hypertriglyceridemic subjects, the odds ratio for heterozygote RW and homozygote WW genotypes to wild-type RR was 2.406 (P = .0017) after controlling the risk factors of age, gender, and body mass index as covariates. CONCLUSIONS: The frequency of ANGPTL8 R59W is significantly higher in Japanese subjects than in other ethnic groups. The rates of T2D and IGT were greater in subjects with the R59W variant. These findings indicate that ANGPTL8 is a participant in diabetes and a potential therapeutic target for T2D prevention, especially in East Asians.


Assuntos
Proteínas Semelhantes a Angiopoietina/genética , Diabetes Mellitus Tipo 2/epidemiologia , Intolerância à Glucose/epidemiologia , Mutação de Sentido Incorreto , Hormônios Peptídicos/genética , Adulto , Idoso , Grupo com Ancestrais do Continente Asiático/genética , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/etnologia , Feminino , Genótipo , Intolerância à Glucose/etnologia , Humanos , Hipertrigliceridemia/epidemiologia , Hipertrigliceridemia/etnologia , Hipertrigliceridemia/genética , Japão , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único
9.
PLoS Negl Trop Dis ; 12(2): e0006266, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29470490

RESUMO

Diarrheal diseases are a major cause of morbidity and mortality worldwide. In many cases, antibiotic therapy is either ineffective or not recommended due to concerns about emergence of resistance. The pathogenesis of several of the most prevalent infections, including cholera and enteroxigenic Escherichia coli, is dominated by enterotoxins produced by lumen-dwelling pathogens before clearance by intestinal defenses. Toxins gain access to the host through critical host receptors, making these receptors attractive targets for alternative antimicrobial strategies that do not rely on conventional antibiotics. Here, we developed a new nanotechnology strategy as a countermeasure against cholera, one of the most important and prevalent toxin-mediated enteric infections. The key host receptor for cholera toxin, monosialotetrahexosylganglioside (GM1), was coated onto the surface of polymeric nanoparticles. The resulting GM1-polymer hybrid nanoparticles were shown to function as toxin decoys by selectively and stably binding cholera toxin, and neutralizing its actions on epithelial cells in vitro and in vivo. Furthermore, the GM1-coated nanoparticle decoys attenuated epithelial 3',5'-cyclic adenosine monophosphate production and fluid responses to infection with live Vibrio cholera in cell culture and a murine infection model. Together, these studies illustrate that the new nanotechnology-based platform can be employed as a non-traditional antimicrobial strategy for the management of enteric infections with enterotoxin-producing pathogens.


Assuntos
Toxina da Cólera/metabolismo , Cólera/tratamento farmacológico , Gangliosídeo G(M1)/metabolismo , Nanopartículas , Vibrio cholerae/patogenicidade , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Cólera/microbiologia , Cólera/fisiopatologia , Toxina da Cólera/química , AMP Cíclico/metabolismo , Feminino , Gangliosídeo G(M1)/química , Mucosa Intestinal/metabolismo , Intestino Delgado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/química , Nanotecnologia/métodos
10.
Dig Endosc ; 30(3): 380-387, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29181859

RESUMO

BACKGROUND AND AIM: Cholecystitis is a major complication after self-expandable metallic stent (SEMS) placement for malignant biliary obstruction. Ischemia is one of the risk factors for cholecystitis, but little is known about the influence of tumor invasion to the feeding artery of the gallbladder on the onset of cholecystitis after SEMS placement. The aim of the present study was to identify risk factors for cholecystitis after SEMS placement. METHODS: Incidence and nine predictive factors of cholecystitis were retrospectively evaluated in 107 patients who underwent SEMS placement for unresectable distal malignant biliary obstruction at Kyoto University Hospital and Otsu Red Cross Hospital between January 2012 and June 2016. RESULTS: Cholecystitis occurred in 13 of 107 patients (12.1%) after SEMS placement during the median follow-up period of 262 days. Univariate analyses showed that tumor invasion to the feeding artery of the gallbladder and tumor involvement to the orifice of the cystic duct were significant predictors of cholecystitis (P = 0.001 and P < 0.001). Multivariate analysis confirmed that these two factors were significant and independent risks for cholecystitis with odds ratios of 22.13 (95% CI, 3.57-137.18; P = 0.001) and 25.26 (95% CI, 4.12-154.98; P < 0.001), respectively. CONCLUSIONS: This study showed for the first time that tumor invasion to the feeding artery of the gallbladder as well as tumor involvement to the orifice of the cystic duct are independent risk factors for cholecystitis after SEMS placement.


Assuntos
Colecistite/epidemiologia , Colestase/cirurgia , Vesícula Biliar/irrigação sanguínea , Neoplasias Pancreáticas/patologia , Complicações Pós-Operatórias/epidemiologia , Stents Metálicos Autoexpansíveis/efeitos adversos , Adulto , Idoso , Idoso de 80 Anos ou mais , Colestase/etiologia , Feminino , Vesícula Biliar/patologia , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Neoplasias Pancreáticas/cirurgia , Estudos Retrospectivos , Fatores de Risco
11.
Artigo em Inglês | MEDLINE | ID: mdl-28396548

RESUMO

Giardia lamblia is an important and ubiquitous cause of diarrheal disease. The primary agents in the treatment of giardiasis are nitroheterocyclic drugs, particularly the imidazoles metronidazole and tinidazole and the thiazole nitazoxanide. Although these drugs are generally effective, treatment failures occur in up to 20% of cases, and resistance has been demonstrated in vivo and in vitro Prior work had suggested that side chain modifications of the imidazole core can lead to new effective 5-nitroimidazole drugs that can combat nitro drug resistance, but the full potential of nitroheterocycles other than imidazole to yield effective new antigiardial agents has not been explored. Here, we generated derivatives of two clinically utilized nitroheterocycles, nitrothiazole and nitrofuran, as well as a third heterocycle, nitropyrrole, which is related to nitroimidazole but has not been systematically investigated as an antimicrobial drug scaffold. Click chemistry was employed to synthesize 442 novel nitroheterocyclic compounds with extensive side chain modifications. Screening of this library against representative G. lamblia strains showed a wide spectrum of in vitro activities, with many of the compounds exhibiting superior activity relative to reference drugs and several showing >100-fold increase in potency and the ability to overcome existing forms of metronidazole resistance. The majority of new compounds displayed no cytotoxicity against human cells, and several compounds were orally active against murine giardiasis in vivo These findings provide additional impetus for the systematic development of nitroheterocyclic compounds with nonimidazole cores as alternative and improved agents for the treatment of giardiasis and potentially other infectious agents.


Assuntos
Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Antiprotozoários/química , Antiprotozoários/farmacologia , Química Click/métodos , Giardia lamblia/efeitos dos fármacos , Nitrofuranos/química , Nitrofuranos/farmacologia , Pirróis/farmacologia , Tiazóis/farmacologia , Testes de Sensibilidade Parasitária , Pirróis/química , Relação Estrutura-Atividade , Tiazóis/química
12.
J Allergy Clin Immunol ; 139(4): 1205-1216.e6, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27746235

RESUMO

BACKGROUND: Mast cell (MC) progenitors leave the bone marrow, enter the circulation, and settle in the skin and other tissues. Their maturation in tissues is influenced by the surrounding microenvironment. OBJECTIVE: We tested the hypothesis that environmental factors play a role in MC maturation in the skin. METHODS: MCs were numerically, phenotypically, and functionally compared between germ-free (GF), specific pathogen-free, and GF mice reconstituted with microbiota. The maturity of MCs was then correlated with skin levels of stem cell factor (SCF), a critical MC differentiation factor, and lipoteichoic acid (LTA), a Toll-like receptor 2 ligand. MCs were also evaluated in mice with keratinocyte-specific deletion of Scf. RESULTS: We found that GF mice express abnormally low amounts of SCF, a critical MC differentiation factor, and contain MCs that are largely undifferentiated. Reconstituting the GF microbiota reverted this MC phenotype to normal, indicating that the phenotype is related to ongoing interactions of the microbiota and skin. Consistent with the immaturity of GF MCs, degranulation-provoking compound 48/80 induced less edema in the skin of GF mice than in conventional mice. Our results show that the skin microbiome drives SCF production in keratinocytes, which triggers the differentiation of dermal MCs. Because the skin microbiome is a rich source of LTA, a Toll-like receptor 2 ligand, we mimicked the GF microbiome's effect on MCs by applying LTA to the skin of GF mice. We also demonstrated that MC migration within the skin depends exclusively on keratinocyte-produced SCF. CONCLUSION: This study has revealed a novel mechanism by which the skin microbiota signals the recruitment and maturation of MCs within the dermis through SCF production by LTA-stimulated keratinocytes.


Assuntos
Diferenciação Celular/fisiologia , Queratinócitos/metabolismo , Mastócitos/citologia , Pele/microbiologia , Fator de Células-Tronco/biossíntese , Animais , Separação Celular , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Vida Livre de Germes , Humanos , Microdissecção e Captura a Laser , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Animais , Reação em Cadeia da Polimerase em Tempo Real , Pele/citologia , Pele/metabolismo , Ácidos Teicoicos/farmacologia
13.
Int J Antimicrob Agents ; 48(6): 690-694, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27839893

RESUMO

Trichomoniasis, caused by the protozoan parasite Trichomonas vaginalis, is the most common, non-viral, sexually transmitted infection in the world, but only two closely related nitro drugs are approved for its treatment. New antimicrobials against trichomoniasis remain an urgent need. Several organic gold compounds were tested for activity against T. vaginalis thioredoxin reductase (TrxR) in cell-free systems as well as for activity against different trichomonads in vitro and in a murine infection model. The organic gold(I) compounds auranofin and chloro(diethylphenylphosphine)gold(I) inhibited TrxR in a concentration-dependent manner in assays with recombinant purified reductase and in cytoplasmic extracts of T. vaginalis transfected with a haemagglutinin epitope-tagged form of the reductase. Auranofin potently suppressed the growth of three independent clinical T. vaginalis isolates as well as several strains of another trichomonad (Tritrichomonas foetus) in a 24 h-assay, with 50% inhibitory concentrations of 0.7-2.5 µM and minimum lethal concentrations of 2-6 µM. The drug also compromised the ability of the parasite to overcome oxidant stress, supporting the notion that auranofin acts, in part, by inactivating TrxR-dependent antioxidant defences. Chloro(diethylphenylphosphine)gold(I) was 10-fold less effective against T. vaginalis in vitro than auranofin. Oral administration of auranofin for 4 days cleared the parasites in a murine model of vaginal T. foetus infection without displaying any apparent adverse effects. The approved human drug auranofin may be a promising agent as an alternative treatment of trichomoniasis in cases when standard nitro drug therapies have failed.


Assuntos
Antiprotozoários/farmacologia , Auranofina/farmacologia , Inibidores Enzimáticos/farmacologia , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Vaginite por Trichomonas/tratamento farmacológico , Trichomonas vaginalis/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Reposicionamento de Medicamentos , Feminino , Concentração Inibidora 50 , Camundongos Endogâmicos C57BL , Resultado do Tratamento , Vaginite por Trichomonas/parasitologia , Trichomonas vaginalis/enzimologia , Trichomonas vaginalis/crescimento & desenvolvimento , Tritrichomonas foetus/efeitos dos fármacos , Tritrichomonas foetus/crescimento & desenvolvimento
15.
Front Microbiol ; 6: 1208, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26635732

RESUMO

Diarrheal diseases are among the leading causes of morbidity and mortality in the world, particularly among young children. A limited number of infectious agents account for most of these illnesses, raising the hope that advances in the treatment and prevention of these infections can have global health impact. The two most important parasitic causes of diarrheal disease are Cryptosporidium and Giardia. Both parasites infect predominantly the small intestine and colonize the lumen and epithelial surface, but do not invade deeper mucosal layers. This review discusses the therapeutic challenges, current treatment options, and drug development efforts against cryptosporidiosis and giardiasis. The goals of drug development against Cryptosporidium and Giardia are different. For Cryptosporidium, only one moderately effective drug (nitazoxanide) is available, so novel classes of more effective drugs are a high priority. Furthermore, new genetic technology to identify potential drug targets and better assays for functional evaluation of these targets throughout the parasite life cycle are needed for advancing anticryptosporidial drug design. By comparison, for Giardia, several classes of drugs with good efficacy exist, but dosing regimens are suboptimal and emerging resistance begins to threaten clinical utility. Consequently, improvements in potency and dosing, and the ability to overcome existing and prevent new forms of drug resistance are priorities in antigiardial drug development. Current work on new drugs against both infections has revealed promising strategies and new drug leads. However, the primary challenge for further drug development is the underlying economics, as both parasitic infections are considered Neglected Diseases with low funding priority and limited commercial interest. If a new urgency in medical progress against these infections can be raised at national funding agencies or philanthropic organizations, meaningful and timely progress is possible in treating and possibly preventing cryptosporidiosis and giardiasis.

16.
Alcohol Clin Exp Res ; 39(12): 2313-23, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26556636

RESUMO

BACKGROUND: Our aim is to investigate the physiological relevance of the intestinal microbiota in alcohol-induced liver injury. Chronic alcohol abuse is associated with intestinal bacterial overgrowth, increased intestinal permeability, and translocation of microbial products from the intestine to the portal circulation and liver. Translocated microbial products contribute to experimental alcoholic liver disease. METHODS: We subjected germ-free and conventional C57BL/6 mice to a model of acute alcohol exposure that mimics binge drinking. RESULTS: Germ-free mice showed significantly greater liver injury and inflammation after oral gavage of ethanol (EtOH) compared with conventional mice. In parallel, germ-free mice exhibited increased hepatic steatosis and up-regulated expression of genes involved in fatty acid and triglyceride synthesis compared with conventional mice after acute EtOH administration. The absence of microbiota was also associated with increased hepatic expression of EtOH-metabolizing enzymes, which led to faster EtOH elimination from the blood and lower plasma EtOH concentrations. Intestinal levels of EtOH-metabolizing genes showed regional expression differences and were overall higher in germ-free mice relative to conventional mice. CONCLUSIONS: Our findings indicate that absence of the intestinal microbiota increases hepatic EtOH metabolism and the susceptibility to binge-like alcohol drinking.


Assuntos
Etanol/toxicidade , Vida Livre de Germes/fisiologia , Hepatopatias Alcoólicas/microbiologia , Hepatopatias Alcoólicas/prevenção & controle , Microbiota/fisiologia , Animais , Bebedeira/complicações , Bebedeira/microbiologia , Feminino , Vida Livre de Germes/efeitos dos fármacos , Hepatopatias Alcoólicas/etiologia , Camundongos , Camundongos Endogâmicos C57BL , Microbiota/efeitos dos fármacos
17.
J Dermatol ; 42(11): 1083-6, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26074124

RESUMO

We report a case of sarcomatoid carcinoma of the skin in a 63-year-old man who was treated with the carbon dioxide snow freezing method for a huge congenital pigmented nevus that extended from the right upper extremity to the right trunk during childhood. He had an exophytic red tumor on the nevus in the right upper extremity that grew slowly for 4 years and rapidly recently. Histological and immunohistochemical studies revealed both epithelial and mesenchymal malignancy in the same tumor. The epithelial component was composed of basaloid cells forming multiple nests with peripheral palisading, positive for keratins and BerEP4, implying basal cell carcinoma. The mesenchymal component was composed of spindle-shaped cells negative for keratins and positive for vimentin, suggesting sarcoma. This is, to our knowledge, the first report of sarcomatoid carcinoma arising in the primary pigmented nevus that had been treated by the carbon dioxide snow freezing method.


Assuntos
Carcinoma/etiologia , Nevo Pigmentado/congênito , Sarcoma/etiologia , Neoplasias Cutâneas/congênito , Carcinoma/patologia , Carcinoma Basocelular/etiologia , Carcinoma Basocelular/patologia , Gelo-Seco/efeitos adversos , Gelo-Seco/uso terapêutico , Humanos , Masculino , Pessoa de Meia-Idade , Segunda Neoplasia Primária/etiologia , Segunda Neoplasia Primária/patologia , Nevo Pigmentado/terapia , Sarcoma/patologia , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/terapia
18.
Exp Parasitol ; 156: 68-78, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26071205

RESUMO

Giardia lamblia is a leading protozoan cause of diarrheal disease worldwide. It colonizes the lumen and epithelial surface of the small intestine, but does not invade the mucosa. Acute infection causes only minimal mucosal inflammation. Effective immune defenses exist, yet their identity and mechanisms remain incompletely understood. Interleukin (IL)-17A has emerged as an important cytokine involved in inflammation and antimicrobial defense against bacterial pathogens at mucosal surfaces. In this study, we demonstrate that IL-17A has a crucial function in host defense against Giardia infection. Using murine infection models with G. muris and G. lamblia, we observed marked and selective induction of intestinal IL-17A with peak expression after 2 weeks. Th17 cells in the lamina propria and innate immune cells in the epithelial compartment of the small intestine were responsible for the IL-17A response. Experiments in gene-targeted mice revealed that the cytokine, and its cognate receptor IL-17RA, were required for eradication of the parasite. The actions of the cytokine were mediated by hematopoietic cells, and were required for the transport of IgA into the intestinal lumen, since IL-17A deficiency led to marked reduction of fecal IgA levels, as well as for increased intestinal expression of several other potential effectors, including ß-defensin 1 and resistin-like molecule ß. In contrast, intestinal hypermotility, another major antigiardial defense mechanism, was not impacted by IL-17A loss. Taken together, these findings demonstrate that IL-17A and IL-17 receptor signaling are essential for intestinal defense against the important lumen-dwelling intestinal parasite Giardia.


Assuntos
Anticorpos Antiprotozoários/biossíntese , Giardia/imunologia , Giardíase/imunologia , Imunoglobulina A/biossíntese , Interleucina-17/metabolismo , Animais , Anticorpos Antiprotozoários/imunologia , Linfócitos T CD4-Positivos/imunologia , Quimera , Giardia lamblia/imunologia , Células-Tronco Hematopoéticas/imunologia , Imunoglobulina A/imunologia , Interleucina-17/genética , Mucosa Intestinal/imunologia , Mucosa Intestinal/parasitologia , Intestino Delgado/imunologia , Intestino Delgado/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Transdução de Sinais/imunologia , Organismos Livres de Patógenos Específicos , Células Th17/imunologia
19.
Eur J Med Chem ; 101: 96-102, 2015 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-26117821

RESUMO

Metronidazole has been used clinically for over 50 years as an antiparasitic and broad-spectrum antibacterial agent effective against anaerobic bacteria. However resistance to metronidazole in parasites and bacteria has been reported, and improved second-generation metronidazole analogues are needed. The copper catalysed Huigsen azide-alkyne 1,3-dipolar cycloaddition offers a way to efficiently assemble new libraries of metronidazole analogues. Several new metronidazole-triazole conjugates (Mtz-triazoles) have been identified with excellent broad spectrum antimicrobial and antiparasitic activity targeting Clostridium difficile, Entamoeba histolytica and Giardia lamblia. Cross resistance to metronidazole was observed against stable metronidazole resistant C. difficile and G. lamblia strains. However for the most potent Mtz-triazoles, the activity remained in a therapeutically relevant window.


Assuntos
Antibacterianos/farmacologia , Antiparasitários/farmacologia , Clostridium difficile/efeitos dos fármacos , Metronidazol/química , Metronidazol/farmacologia , Parasitos/efeitos dos fármacos , Triazóis/química , Triazóis/farmacologia , Animais , Antibacterianos/síntese química , Antibacterianos/química , Antiparasitários/síntese química , Antiparasitários/química , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Entamoeba histolytica/efeitos dos fármacos , Giardia lamblia/efeitos dos fármacos , Células HEK293 , Células Hep G2 , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade
20.
FASEB J ; 29(3): 1043-55, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25466902

RESUMO

Translocation of bacteria and their products across the intestinal barrier is common in patients with liver disease, and there is evidence that experimental liver fibrosis depends on bacterial translocation. The purpose of our study was to investigate liver fibrosis in conventional and germ-free (GF) C57BL/6 mice. Chronic liver injury was induced by administration of thioacetamide (TAA) in the drinking water for 21 wk or by repeated intraperitoneal injections of carbon tetrachloride (CCl4). Increased liver fibrosis was observed in GF mice compared with conventional mice. Hepatocytes showed more toxin-induced oxidative stress and cell death. This was accompanied by increased activation of hepatic stellate cells, but hepatic mediators of inflammation were not significantly different. Similarly, a genetic model using Myd88/Trif-deficient mice, which lack downstream innate immunity signaling, had more severe fibrosis than wild-type mice. Isolated Myd88/Trif-deficient hepatocytes were more susceptible to toxin-induced cell death in culture. In conclusion, the commensal microbiota prevents fibrosis upon chronic liver injury in mice. This is the first study describing a beneficial role of the commensal microbiota in maintaining liver homeostasis and preventing liver fibrosis.


Assuntos
Hepatócitos/efeitos dos fármacos , Inflamação/prevenção & controle , Cirrose Hepática/prevenção & controle , Microbiota , Substâncias Protetoras , Transdução de Sinais/efeitos dos fármacos , Tioacetamida/farmacologia , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cromatografia Líquida , Modelos Animais de Doenças , Hepatócitos/citologia , Hepatócitos/microbiologia , Humanos , Técnicas Imunoenzimáticas , Inflamação/induzido quimicamente , Inflamação/microbiologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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