scholarly journals Cryptosporidium parvum Pyruvate Kinase Inhibitors With in vivo Anti-cryptosporidial Efficacy

2022 ◽  
Vol 12 ◽  
Author(s):  
Shahbaz M. Khan ◽  
Xuejin Zhang ◽  
William H. Witola
Keyword(s):  
Pyruvate Kinase ◽  
Cryptosporidium Parvum ◽  
Diarrheal Disease ◽  
Severe Disease ◽  
The United States ◽  
Metabolic Energy ◽  
Host Cells ◽  
Infection Model

Cryptosporidium parvum is a highly prevalent protozoan parasite that causes a diarrheal disease in humans and animals worldwide. Thus far, the moderately effective nitazoxanide is the only drug approved by the United States Food and Drug Administration for treating cryptosporidiosis in immunocompetent humans. However, no effective drug exists for the severe disease seen in young children, immunocompromised individuals and neonatal livestock. C. parvum lacks the Krebs cycle and the oxidative phosphorylation steps, making it dependent solely on glycolysis for metabolic energy production. Within its glycolytic pathway, C. parvum possesses two unique enzymes, the bacterial-type lactate dehydrogenase (CpLDH) and the plant-like pyruvate kinase (CpPyK), that catalyze two sequential steps for generation of essential metabolic energy. We have previously reported that inhibitors of CpLDH are effective against C. parvum, both in vitro and in vivo. Herein, we developed an in vitro assay for the enzymatic activity of recombinant CpPyK protein and used it to screen a chemical compound library for inhibitors of CpPyK’s activity. The identified inhibitors were tested (at non-toxic concentrations) for efficacy against C. parvum using in vitro assays, and an in vivo mouse infection model. We identified six CpPyK inhibitors that blocked in vitro growth and proliferation of C. parvum at low micromolar concentrations (EC50 values ranging from 10.29 to 86.01 μM) that were non-toxic to host cells. Among those six compounds, two (NSC252172 and NSC234945) were found to be highly efficacious against cryptosporidiosis in immunocompromised mice at a dose of 10 mg/kg body weight, with very significant reduction in parasite load and amelioration of intestinal pathologies. Together, these findings have unveiled inhibitors for an essential molecular target in C. parvum and demonstrated their efficacy against the parasite in vitro and in vivo. These inhibitors are, therefore, potential lead-compounds for developing efficacious treatments for cryptosporidiosis.

scholarly journals Role of CpSUB1, a Subtilisin-Like Protease, in Cryptosporidium parvum Infection In Vitro

2009 ◽  
Vol 8 (4) ◽  
pp. 470-477 ◽  
Author(s):  
Jane W. Wanyiri ◽  
Patsharaporn Techasintana ◽  
Roberta M. O'Connor ◽  
Michael J. Blackman ◽  
Kami Kim ◽  
...  
Keyword(s):  
Serine Protease ◽  
Cryptosporidium Parvum ◽  
Protease Activity ◽  
Diarrheal Disease ◽  
Genomic Sequence ◽  
Host Cells ◽  
Pcr Analysis ◽  
Apical Region ◽  
Gene Encoding

ABSTRACTThe apicomplexan parasiteCryptosporidiumis a significant cause of diarrheal disease worldwide. Previously, we reported that aCryptosporidium parvumsubtilisin-like serine protease activity with furin-type specificity cleaves gp40/15, a glycoprotein that is proteolytically processed into gp40 and gp15, which are implicated in mediating infection of host cells. Neither the enzyme(s) responsible for the protease activity inC. parvumlysates nor those that process gp40/15 are known. There are no furin or other proprotein convertase genes in theC. parvumgenome. However, a gene encoding CpSUB1, a subtilisin-like serine protease, is present. In this study, we cloned the CpSUB1 genomic sequence and expressed and purified the recombinant prodomain. Reverse transcriptase PCR analysis of RNA fromC. parvum-infected HCT-8 cells revealed that CpSUB1 is expressed throughout infection in vitro. In immunoblots, antiserum to the recombinant CpSUB1 prodomain revealed two major bands, of ∼64 kDa and ∼48 kDa, forC. parvumlysates and proteins “shed” during excystation. In immunofluorescence assays, the antiserum reacted with the apical region of sporozoites and merozoites. The recombinant prodomain inhibited protease activity and processing of recombinant gp40/15 byC. parvumlysates but not by furin. Since prodomains are often selective inhibitors of their cognate enzymes, these results suggest that CpSUB1 may be a likely candidate for the protease activity inC. parvumand for processing of gp40/15. Importantly, the recombinant prodomain inhibitedC. parvuminfection of HCT-8 cells. These studies indicate that CpSUB1 plays a significant role in infection of host cells by the parasite and suggest that this enzyme may serve as a target for intervention.

scholarly journals Postantibiotic and Sub-MIC Effects of Exebacase (Lysin CF-301) Enhance Antimicrobial Activity againstStaphylococcus aureus

2019 ◽  
Vol 63 (6) ◽  
Author(s):  
Jun Taek Oh ◽  
Cara Cassino ◽  
Raymond Schuch
Keyword(s):  
Antimicrobial Activity ◽  
Cell Wall ◽  
The United States ◽  
Infection Model ◽  
Postantibiotic Effect ◽  
Content Type ◽  
Wall Ultrastructure ◽  
Bacterial Fitness

ABSTRACTCF-301 (exebacase) is a recombinantly produced bacteriophage-derived lysin (cell wall hydrolase) and is the first agent of this class to enter clinical development in the United States for treating bacteremia including endocarditis due toStaphylococcus aureus. Whereas rapid bactericidal activity is the hallmarkin vitroandin vivoresponse to CF-301 at exposures higher than the MIC, prolonged antimicrobial activity, mediated by cell wall damage, is predicted at concentrations less than the MIC. In the current study, a series ofin vitropharmacodynamic parameters, including the postantibiotic effect (PAE), postantibiotic sub-MIC effect (PA-SME), and sub-MIC effect (SME), were studied to determine how short-duration and sub-MIC CF-301 exposures affect the growth of surviving staphylococci and extend its antimicrobial activity. Mean PAE, PA-SME, and SME values up to 4.8, 9.3, and 9.8 h, respectively, were observed against 14 staphylococcal strains tested in human serum; growth delays were extended by 6 h in the presence of daptomycin. Exposures to CF-301 at sub-MIC levels as low as 0.001× to 0.01× MIC (∼1 to 10 ng/ml) resulted in aberrant cell wall ultrastructure, increased membrane permeability, dissipation of membrane potential, and inhibition of virulence phenotypes, including agglutination and biofilm formation. A mouse thigh infection model designed to study the PAE was used to confirm our findings and demonstratein vivogrowth delays of ≥19.3 h. Our findings suggest that at CF-301 concentrations less than the MIC during therapeutic use, sustained reductions in bacterial fitness and virulence may substantially enhance efficacy.

scholarly journals Generating Robust and Informative NonclinicalIn VitroandIn VivoBacterial Infection Model Efficacy Data To Support Translation to Humans

2019 ◽  
Vol 63 (5) ◽  
Author(s):  
Jürgen B. Bulitta ◽  
William W. Hope ◽  
Ann E. Eakin ◽  
Tina Guina ◽  
Vincent H. Tam ◽  
...  
Keyword(s):  
Critical Role ◽  
The United States ◽  
Future Research ◽  
Infection Model ◽  
Published Data ◽  
Optimal Dosage ◽  
Open Communication ◽  
Dosage Regimens

ABSTRACTIn June 2017, the National Institute of Allergy and Infectious Diseases, part of the National Institutes of Health, organized a workshop entitled “Pharmacokinetics-Pharmacodynamics (PK/PD) for Development of Therapeutics against Bacterial Pathogens.” The aims were to discuss details of various PK/PD models and identify sound practices for deriving and utilizing PK/PD relationships to design optimal dosage regimens for patients. Workshop participants encompassed individuals from academia, industry, and government, including the United States Food and Drug Administration. This and the accompanying review on clinical PK/PD summarize the workshop discussions and recommendations. Nonclinical PK/PD models play a critical role in designing human dosage regimens and are essential tools for drug development. These includein vitroandin vivoefficacy models that provide valuable and complementary information for dose selection and translation from the laboratory to human. It is crucial that studies be designed, conducted, and interpreted appropriately. For antibacterial PK/PD, extensive published data and expertise are available. These have been leveraged to develop recommendations, identify common pitfalls, and describe the applications, strengths, and limitations of various nonclinical infection models and translational approaches. Despite these robust tools and published guidance, characterizing nonclinical PK/PD relationships may not be straightforward, especially for a new drug or new class. Antimicrobial PK/PD is an evolving discipline that needs to adapt to future research and development needs. Open communication between academia, pharmaceutical industry, government, and regulatory bodies is essential to share perspectives and collectively solve future challenges.

scholarly journals In VivoPharmacodynamics of Cefquinome in a Neutropenic Mouse Thigh Model of Streptococcus suis Serotype 2 at Varied Initial Inoculum Sizes

2015 ◽  
Vol 60 (2) ◽  
pp. 1114-1120 ◽  
Author(s):  
Chunna Guo ◽  
Xiaoping Liao ◽  
Mingru Wang ◽  
Feng Wang ◽  
Chaoqun Yan ◽  
...  
Keyword(s):  
Severe Disease ◽  
Streptococcus Suis ◽  
Dose Fractionation ◽  
Fourth Generation ◽  
Maximum Effect ◽  
Infection Model ◽  
Human Beings ◽  
Content Type

ABSTRACTStreptococcus suisserotype 2 is an emerging zoonotic pathogen and causes severe disease in both pigs and human beings. Cefquinome (CEQ), a fourth-generation cephalosporin, exhibits broad-spectrum activity against Gram-positive bacteria such asS. suis. This study evaluated thein vitroandin vivoantimicrobial activities of CEQ against four strains ofS. suisserotype 2 in a murine neutropenic thigh infection model. We investigated the effect of varied inoculum sizes (106to 108CFU/thigh) on the pharmacokinetic (PK)/pharmacodynamic (PD) indices and magnitudes of a particular PK/PD index or dose required for efficacy. Dose fractionation studies included total CEQ doses ranging from 0.625 to 640 mg/kg/24 h. Data were analyzed via a maximum effect (Emax) model using nonlinear regression. The PK/PD studies demonstrated that the percentage of time that serum drug levels were above the MIC of free drug (%ƒT>MIC) in a 24-h dosing interval was the primary index driving the efficacy of both inoculum sizes (R2= 91% andR2= 63%). CEQ doses of 2.5 and 40 mg/kg body weight produced prolonged postantibiotic effects (PAEs) of 2.45 to 8.55 h. Inoculum sizes had a significant influence on CEQ efficacy. Compared to the CEQ exposure and dosages in tests using standard inocula, a 4-fold dose (P= 0.006) and a 2-fold exposure time (P= 0.01) were required for a 1-log kill using large inocula of 108CFU/thigh.

scholarly journals Pharmacodynamic Evaluation of the Activities of Six Parenteral Vancomycin Products Available in the United States

2014 ◽  
Vol 59 (1) ◽  
pp. 622-632 ◽  
Author(s):  
Arnold Louie ◽  
Michael T. Boyne ◽  
Vikram Patel ◽  
Clayton Huntley ◽  
Weiguo Liu ◽  
...  
Keyword(s):  
United States ◽  
Drug Exposure ◽  
The United States ◽  
Mouse Serum ◽  
Infection Model ◽  
Population Pharmacokinetic ◽  
Bacterial Killing ◽  
The Impact

ABSTRACTA recent report found that generic parenteral vancomycin products may not havein vivoefficacies equivalent to those of the innovator in a neutropenic murine thigh infection model despite having similarin vitromicrobiological activities and murine serum pharmacokinetics. We compared thein vitroandin vivoactivities of six of the parenteral vancomycin products available in the United States. Thein vitroassessments for the potencies of the vancomycin products included MIC/minimal bactericidal concentration (MBC) determinations, quantifying the impact of human and murine serum on the MIC values, and time-kill studies. Also, the potencies of the vancomycin products were quantified with a biological assay, and the human and mouse serum protein binding rates for the vancomycin products were measured. Thein vivostudies included dose-ranging experiments with the 6 vancomycin products for three isolates ofStaphylococcus aureusin a neutropenic mouse thigh infection model. The pharmacokinetics of the vancomycin products were assessed in infected mice by population pharmacokinetic modeling. No differences were seen across the vancomycin products with regard to anyin vitroevaluation. Inhibitory sigmoid maximal bacterial kill (Emax) modeling of the relationship between vancomycin dosage and the killing of the bacteria in micein vivoyielded similarEmaxand EC50(drug exposure driving one-halfEmax) values for bacterial killing. Further, there were no differences in the pharmacokinetic clearances of the 6 vancomycin products from infected mice. There were no important pharmacodynamic differences in thein vitroorin vivoactivities among the six vancomycin products evaluated.

scholarly journals The dual function monoclonal antibodies VIR-7831 and VIR-7832 demonstrate potent in vitro and in vivo activity against SARS-CoV-2

2021 ◽  
Author(s):  
Andrea L. Cathcart ◽  
Colin Havenar-Daughton ◽  
Florian A. Lempp ◽  
Daphne Ma ◽  
Michael Schmid ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Severe Acute Respiratory Syndrome ◽  
Host Cells ◽  
Infection Model ◽  
Dual Function ◽  
Resistance Mutations ◽  
Viral Respiratory Infection ◽  
Dual Action

ABSTRACTVIR-7831 and VIR-7832 are dual action monoclonal antibodies (mAbs) targeting the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). VIR-7831 and VIR-7832 were derived from a parent antibody (S309) isolated from memory B cells of a 2003 severe acute respiratory syndrome coronavirus (SARS-CoV) survivor. Both mAbs contain an “LS” mutation in the Fc region to prolong serum half-life and potentially enhance distribution to the respiratory mucosa. In addition, VIR-7832 encodes an Fc GAALIE mutation that has been shown previously to evoke CD8+ T-cells in the context of an in vivo viral respiratory infection. VIR-7831 and VIR-7832 potently neutralize live wild-type SARS-CoV-2 in vitro as well as pseudotyped viruses encoding spike protein from the B.1.1.7, B.1.351 and P.1 variants. In addition, they retain activity against monoclonal antibody resistance mutations that confer reduced susceptibility to currently authorized mAbs. The VIR-7831/VIR-7832 epitope does not overlap with mutational sites in the current variants of concern and continues to be highly conserved among circulating sequences consistent with the high barrier to resistance observed in vitro. Furthermore, both mAbs can recruit effector mechanisms in vitro that may contribute to clinical efficacy via elimination of infected host cells. In vitro studies with these mAbs demonstrated no enhancement of infection. In a Syrian Golden hamster proof-of concept wildtype SARS-CoV-2 infection model, animals treated with VIR-7831 had less weight loss, and significantly decreased total viral load and infectious virus levels in the lung compared to a control mAb. Taken together, these data indicate that VIR-7831 and VIR-7832 are promising new agents in the fight against COVID-19.

scholarly journals Novel Bioengineered Three-Dimensional Human Intestinal Model for Long-Term Infection of Cryptosporidium parvum

2017 ◽  
Vol 85 (3) ◽  
Author(s):  
Maria A DeCicco RePass ◽  
Ying Chen ◽  
Yinan Lin ◽  
Wenda Zhou ◽  
David L. Kaplan ◽  
...  
Keyword(s):  
Cryptosporidium Parvum ◽  
Drug Targets ◽  
Diarrheal Disease ◽  
Three Dimensional ◽  
Content Type ◽  
Tissue Model ◽  
Culture Models

ABSTRACT Cryptosporidium spp. are apicomplexan parasites of global importance that cause human diarrheal disease. In vitro culture models that may be used to study this parasite and that have physiological relevance to in vivo infection remain suboptimal. Thus, the pathogenesis of cryptosporidiosis remains poorly characterized, and interventions for the disease are limited. In this study, we evaluated the potential of a novel bioengineered three-dimensional (3D) human intestinal tissue model (which we developed previously) to support long-term infection by Cryptosporidium parvum. Infection was assessed by immunofluorescence assays and confocal and scanning electron microscopy and quantified by quantitative reverse transcription-PCR. We found that C. parvum infected and developed in this tissue model for at least 17 days, the extent of the study time used in the present study. Contents from infected scaffolds could be transferred to fresh scaffolds to establish new infections for at least three rounds. Asexual and sexual stages and the formation of new oocysts were observed during the course of infection. Additionally, we observed ablation, blunting, or distortion of microvilli in infected epithelial cells. Ultimately, a 3D model system capable of supporting continuous Cryptosporidium infection will be a useful tool for the study of host-parasite interactions, identification of putative drug targets, screening of potential interventions, and propagation of genetically modified parasites.

scholarly journals Antiviral activity of a novel mixture of natural antimicrobials, in vitro, and in a chicken infection model in vivo

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Igori Balta ◽  
Lavinia Stef ◽  
Ioan Pet ◽  
Patrick Ward ◽  
Todd Callaway ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Clinical Signs ◽  
Production Performance ◽  
Host Cells ◽  
Infection Model ◽  
Natural Antimicrobials ◽  
Infectious Bronchitis ◽  
Experimental Group

Abstract The aim of this study was to test in vitro the ability of a mixture of citrus extract, maltodextrin, sodium chloride, lactic acid and citric acid (AuraShield L) to inhibit the virulence of infectious bronchitis, Newcastle disease, avian influenza, porcine reproductive and respiratory syndrome (PRRS) and bovine coronavirus viruses. Secondly, in vivo, we have investigated its efficacy against infectious bronchitis using a broiler infection model. In vitro, these antimicrobials had expressed antiviral activity against all five viruses through all phases of the infection process of the host cells. In vivo, the antimicrobial mixture reduced the virus load in the tracheal and lung tissue and significantly reduced the clinical signs of infection and the mortality rate in the experimental group E2 receiving AuraShield L. All these effects were accompanied by a significant reduction in the levels of pro-inflammatory cytokines and an increase in IgA levels and short chain fatty acids (SCFAs) in both trachea and lungs. Our study demonstrated that mixtures of natural antimicrobials, such AuraShield L, can prevent in vitro viral infection of cell cultures. Secondly, in vivo, the efficiency of vaccination was improved by preventing secondary viral infections through a mechanism involving significant increases in SCFA production and increased IgA levels. As a consequence the clinical signs of secondary infections were significantly reduced resulting in recovered production performance and lower mortality rates in the experimental group E2.

scholarly journals In vitro infection of Madin-Darby bovine kidney (MDBK) cells with Eimeria acervulina sporozoites: quantitative analysis of parasite cellular invasion and replication using real-time polymerase chain reaction (PCR)

2021 ◽  
Author(s):  
Shahinaz Taha ◽  
Tran Nguyen-Ho-Bao ◽  
Arwid Daugschies ◽  
Zaida Rentería-Solís
Keyword(s):  
Real Time ◽  
Global Scale ◽  
Host Cells ◽  
Infection Model ◽  
In Vitro Cultivation ◽  
Bovine Kidney ◽  
Mdbk Cells ◽  
Parasite Reproduction

AbstractPoultry coccidiosis causes considerable economical losses to the livestock industry. Eimeria parasites are responsible for this disease. On a global scale, E. acervulina and E. tenella are amongst the most common Eimeria spp. infecting broilers. E. tenella is commonly used as infection model in in vivo and in vitro studies. On the other hand, E. acervulina has barely been studied under in vitro conditions. A well established and widely used in vitro model for E. tenella infection is the Madin-Darby bovine kidney cell line (MDBK); however, little is known regarding suitability of MDBK cells as host cells for E. acervulina. We infected MDBK monolayers with two different doses, 5 × 104 and 2 × 105, of E. acervulina sporozoites and evaluated cultures at 24 and 96 h post infection (hpi). For comparison, we ran an identical infection assay using E. tenella sporozoites. To assess parasite reproduction, the number of DNA copies of E. acervulina SCAR marker and E. tenella ITS-1 gene was quantified using real-time quantitative PCR. We found that the number of E. acervulina copies increased significantly at 24 hpi in comparison to E. tenella (p < 0.05). After 96 hpi, E. acervulina gene copies were considerably reduced while E. tenella continued to multiply (p < 0.05). Our results show that MDBK monolayers could be used for in vitro research aimed to study E. acervulina sporozoite cell invasion. Nevertheless, modifications of in vitro cultivation appear necessary to allow qualitative and quantitative studies over longer periods of parasite reproduction.

scholarly journals In Vitro Activities of Azithromycin and Ofloxacin againstChlamydia pneumoniae in a Continuous-Infection Model

1999 ◽  
Vol 43 (9) ◽  
pp. 2268-2272 ◽  
Author(s):  
Andrei Kutlin ◽  
Patricia M. Roblin ◽  
Margaret R. Hammerschlag
Keyword(s):  
Chlamydia Pneumoniae ◽  
In Vitro Study ◽  
Community Acquired Pneumonia ◽  
Host Cells ◽  
Infection Model ◽  
Chronic Infections ◽  
Adults And Children ◽  
Infected Cells

ABSTRACT Chlamydia pneumoniae is a well-established cause of community-acquired pneumonia and bronchitis in adults and children. Chronic infections with C. pneumoniae have been implicated in the development of atherosclerosis and other diseases in humans. Methods currently used for the culture and propagation ofC. pneumoniae are not analogous to the infection as it occurs in vivo. We have established a model of continuous C. pneumoniae infection in vitro. HEp-2 cells inoculated with CM-1 and TW-183 strains have been persistently infected for periods of over 1.5 and 2 years, respectively. The cultures were maintained without centrifugation or the addition of cycloheximide, fresh host cells, or chlamydia. We observed cycles of host cell lysis, detachment, and regrowth with both strains of C. pneumoniae. Continuous C. pneumoniae infections may more closely resemble the actual events as they occur in vivo and, therefore, may be a better model for the in vitro study of C. pneumoniae infection. When we used continuously infected cells to determine the effects of azithromycin and ofloxacin on C. pneumoniae propagation in vitro, we found that both drugs reduced but did not completely eliminate the organism. This may be an important observation, as the failure of antibiotic therapy against C. pneumoniae infection in humans has been described.

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