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

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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Anti-Inflammatory and Antibacterial Activity Constituents from the Stem of Cinnamomum validinerve

Molecules ◽

10.3390/molecules25153382 ◽

2020 ◽

Vol 25 (15) ◽

pp. 3382 ◽

Cited By ~ 1

Author(s):

Chi-Lung Yang ◽

Ho-Cheng Wu ◽

Tsong-Long Hwang ◽

Chu-Hung Lin ◽

Yin-Hua Cheng ◽

...

Keyword(s):

Bacterial Infections ◽

Immune Cell ◽

Intraperitoneal Administration ◽

In Vitro Study ◽

Human Neutrophils ◽

Infection Model ◽

Ic50 Values ◽

Anti Inflammatory

One new dibenzocycloheptene, validinol (1), and one butanolide firstly isolated from the natural source, validinolide (2), together with 17 known compounds were isolated from the stem of Cinnamomum validinerve. Among the isolates, lincomolide A (3), secosubamolide (7), and cinnamtannin B1 (19) exhibited potent inhibition on both superoxide anion generation (IC50 values of 2.98 ± 0.3 µM, 4.37 ± 0.38 µM, and 2.20 ± 0.3 µM, respectively) and elastase release (IC50 values of 3.96 ± 0.31 µM, 3.04 ± 0.23 µM, and 4.64 ± 0.71 µM, respectively) by human neutrophils. In addition, isophilippinolide A (6), secosubamolide (7), and cinnamtannin B1 (19) showed bacteriostatic effects against Propionibacterium acnes in in vitro study, with minimal inhibitory concentration (MIC) values at 16 μg/mL, 16 μg/mL, and 500 μg/mL, respectively. Further investigations using the in vivo ear P. acnes infection model showed that the intraperitoneal administration of the major component cinnamtannin B1 (19) reduced immune cell infiltration and pro-inflammatory cytokines TNF-α and IL-6 at the infection sites. The results demonstrated the potential of cinnamtannin B1 (19) for acne therapy. In summary, these results demonstrated the anti-inflammatory potentials of Formosan C. validinerve during bacterial infections.

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In vivo analysis of influenza A mRNA secondary structures identifies critical regulatory motifs

Nucleic Acids Research ◽

10.1093/nar/gkz318 ◽

2019 ◽

Vol 47 (13) ◽

pp. 7003-7017 ◽

Cited By ~ 15

Author(s):

Lisa Marie Simon ◽

Edoardo Morandi ◽

Anna Luganini ◽

Giorgio Gribaudo ◽

Luis Martinez-Sobrido ◽

...

Keyword(s):

Secondary Structure ◽

Influenza A ◽

Host Cells ◽

Messenger Rnas ◽

Infected Cells ◽

In Vivo Analysis ◽

First Time ◽

Negative Sense

AbstractThe influenza A virus (IAV) is a continuous health threat to humans as well as animals due to its recurring epidemics and pandemics. The IAV genome is segmented and the eight negative-sense viral RNAs (vRNAs) are transcribed into positive sense complementary RNAs (cRNAs) and viral messenger RNAs (mRNAs) inside infected host cells. A role for the secondary structure of IAV mRNAs has been hypothesized and debated for many years, but knowledge on the structure mRNAs adopt in vivo is currently missing. Here we solve, for the first time, the in vivo secondary structure of IAV mRNAs in living infected cells. We demonstrate that, compared to the in vitro refolded structure, in vivo IAV mRNAs are less structured but exhibit specific locally stable elements. Moreover, we show that the targeted disruption of these high-confidence structured domains results in an extraordinary attenuation of IAV replicative capacity. Collectively, our data provide the first comprehensive map of the in vivo structural landscape of IAV mRNAs, hence providing the means for the development of new RNA-targeted antivirals.

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Effect of bovine lactoferrin onChlamydia trachomatisinfection and inflammation

Biochemistry and Cell Biology ◽

10.1139/bcb-2016-0049 ◽

2017 ◽

Vol 95 (1) ◽

pp. 34-40 ◽

Cited By ~ 22

Author(s):

Rosa Sessa ◽

Marisa Di Pietro ◽

Simone Filardo ◽

Alessia Bressan ◽

Luigi Rosa ◽

...

Keyword(s):

Acute Infection ◽

Host Cells ◽

Developmental Cycle ◽

Bovine Lactoferrin ◽

Bacterial Disease ◽

Chronic Infections ◽

Sexually Transmitted ◽

Obligate Intracellular Pathogen

Chlamydia trachomatis is an obligate, intracellular pathogen responsible for the most common sexually transmitted bacterial disease worldwide, causing acute and chronic infections. The acute infection is susceptible to antibiotics, whereas the chronic one needs prolonged therapies, thus increasing the risk of developing antibiotic resistance. Novel alternative therapies are needed. The intracellular development of C. trachomatis requires essential nutrients, including iron. Iron-chelating drugs inhibit C. trachomatis developmental cycle. Lactoferrin (Lf), a pleiotropic iron binding glycoprotein, could be a promising candidate against C. trachomatis infection. Similarly to the efficacy against other intracellular pathogens, bovine Lf (bLf) could both interfere with C. trachomatis entry into epithelial cells and exert an anti-inflammatory activity. In vitro and in vivo effects of bLf against C. trachomatis infectious and inflammatory process has been investigated. BLf inhibits C. trachomatis entry into host cells when incubated with cell monolayers before or at the moment of the infection and down-regulates IL-6/IL-8 synthesized by infected cells. Six out of 7 pregnant women asymptomatically infected by C. trachomatis, after 30 days of bLf intravaginal administration, were negative for C. trachomatis and showed a decrease of cervical IL-6 levels. This is the first time that the bLf protective effect against C. trachomatis infection has been demonstrated.

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Deubiquitinating Function of Adenovirus Proteinase

Journal of Virology ◽

10.1128/jvi.76.12.6323-6331.2002 ◽

2002 ◽

Vol 76 (12) ◽

pp. 6323-6331 ◽

Cited By ~ 77

Author(s):

Maxim Y. Balakirev ◽

Michel Jaquinod ◽

Arthur L. Haas ◽

Jadwiga Chroboczek

Keyword(s):

Structural Model ◽

Proteolytic Processing ◽

Host Cells ◽

Cellular Proteins ◽

Viral Gene ◽

Cellular Processes ◽

Infected Cells ◽

Substrate Binding Sites

ABSTRACT The invasion strategy of many viruses involves the synthesis of viral gene products that mimic the functions of the cellular proteins and thus interfere with the key cellular processes. Here we show that adenovirus infection is accompanied by an increased ubiquitin-cleaving (deubiquitinating) activity in the host cells. Affinity chromatography on ubiquitin aldehyde (Ubal), which was designed to identify the deubiquitinating proteases, revealed the presence of adenovirus L3 23K proteinase (Avp) in the eluate from adenovirus-infected cells. This proteinase is known to be necessary for the processing of viral precursor proteins during virion maturation. We show here that in vivo Avp deubiquitinates a number of cellular proteins. Analysis of the substrate specificity of Avp in vitro demonstrated that the protein deubiquitination by this enzyme could be as efficient as proteolytic processing of viral proteins. The structural model of the Ubal-Avp interaction revealed some similarity between S1-S4 substrate binding sites of Avp and ubiquitin hydrolases. These results may reflect the acquisition of an advantageous property by adenovirus and may indicate the importance of ubiquitin pathways in viral infection.

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

Frontiers in Microbiology ◽

10.3389/fmicb.2021.800293 ◽

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.

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

10.1101/2021.03.09.434607 ◽

2021 ◽

Cited By ~ 7

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.

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

Scientific Reports ◽

10.1038/s41598-020-73916-1 ◽

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.

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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)

Parasitology Research ◽

10.1007/s00436-021-07211-x ◽

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.

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Selection and Characterization of ssDNA Aptamers Targeting Largemouth Bass Virus Infected Cells With Antiviral Activities

Frontiers in Microbiology ◽

10.3389/fmicb.2021.785318 ◽

2021 ◽

Vol 12 ◽

Author(s):

Qing Yu ◽

Mengmeng Li ◽

Mingzhu Liu ◽

Shuaishuai Huang ◽

Gaoxue Wang ◽

...

Keyword(s):

Largemouth Bass ◽

Dissociation Constants ◽

High Sensitivity ◽

Molecular Probes ◽

Host Cells ◽

Target Cells ◽

Antiviral Activities ◽

Infected Cells

Largemouth bass virus (LMBV) is one of the most devastating viral pathogens in farmed Largemouth bass. Aptamers are novel molecule probes and have been widely applied in the field of efficient therapeutic and diagnostic agents development. LMBV-infected fathead minnow cells (LMBV-FHM) served as target cells in this study, and three DNA aptamers (LBVA1, LBVA2, and LBVA3) were generated against target cells by SELEX technology. The selected aptamers could specifically bind to LMBV-FHM cells, with rather high calculated dissociation constants (Kd) of 890.09, 517.22, and 249.31 nM for aptamers LBVA1, LBVA2, and LBVA3, respectively. Three aptamers displayed efficient antiviral activities in vitro. It indicates that the selected aptamers have great potentials in developing efficient anti-viruses treatments. The targets of aptamers LBVA1, LBVA2, and LBVA3 could be membrane proteins on host cells. The targets of aptamers (LBVA1, LBVA2, and LBVA3) come out on the cells surface at 8, 10, 8 h post-infection. As novel molecular probes for accurate recognition, aptamer LBVA3 could detect LMBV infection in vitro and in vivo, it indicates that the selected aptamers could be applied in the development of rapid detective technologies, which are characterized by high sensitivity, accuracy, and easy operation.

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Characterization of an operon required for growth on cellobiose in Clostridioides difficile

10.1101/2021.06.26.450058 ◽

2021 ◽

Author(s):

Md Kamrul Hasan ◽

Babita Adhikari Dhungel ◽

Revathi Govind

Keyword(s):

Mutant Strain ◽

Gene Knockout ◽

Recurrent Infection ◽

In Vitro Study ◽

Upstream Region ◽

Infection Model ◽

Cellobiose Metabolism

Cellobiose metabolism is linked to the virulence properties in numerous bacterial pathogens. Here, we characterized a putative cellobiose PTS operon of Clostridiodes difficile to investigate the role of cellobiose metabolism in C. difficile pathogenesis. Our gene knockout experiments demonstrated that the putative cellobiose operon enables uptake of cellobiose into C. difficile and allows growth when cellobiose is provided as the sole carbon source in minimal medium. Additionally, using reporter gene fusion assays and DNA pull-down experiments, we show that its transcription is regulated by CelR, a novel transcriptional repressor protein, which directly binds to the upstream region of the cellobiose operon to control its expression. We have also identified cellobiose metabolism to play a significant role in C. difficile physiology as observed by the reduction of sporulation efficiency when cellobiose uptake was compromised in the mutant strain. In corroboration to in vitro study findings, our in vivo hamster challenge experiment showed a significant reduction of pathogenicity by the cellobiose mutant strain in both the primary and the recurrent infection model- substantiating the role of cellobiose metabolism in C. difficile pathogenesis.

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