Enrichment and Recovery of Mammalian Cells from Contaminated Cultures Using Aqueous Two-Phase Systems.

This Article describes a density-based method for removing contaminants, including microorganisms and nonviable cells, from mammalian cell cultures using an aqueous two-phase system (ATPS). The properties of a 7% w/w polyethylene glycol (PEG)-11% w/w Ficoll ATPS can be tuned to prepare a biocompatible system that removes contaminants with little to no adverse effects on the viability or growth of the cultured cells after treatment. This system can be used to enrich cell culture populations for viable cells and to reduce the number of microorganism contaminants in a culture, which increases the chances of subsequent antibiotic treatments being successful. We test the effectiveness of our method in model contaminated cultures of both adherent (HeLa) and suspension (HL-60 II) mammalian cells contaminated with bacteria (E. coli) and yeast (S. cerevisiae). An average of 70.2 ± 4.6% of HeLa cells added to the system are subsequently recovered, and 55.9 ± 2.1% of HL-60 II cells are recovered. After sedimenting to the interface of the ATPS, these cells have an average viability of 98.0 ± 0.2% and 95.3 ± 2.2%, respectively. By removing unwanted cells, desired cell populations can be recovered, and cultures that would otherwise need to be discarded can continue to be used.

Assessing the prevalence of mycoplasma contamination in cell culture via a survey of NCBI's RNA-seq archive.

Mycoplasmas are notorious contaminants of cell culture and can have profound effects on host cell biology by depriving cells of nutrients and inducing global changes in gene expression. Over the last two decades, sentinel testing has revealed wide-ranging contamination rates in mammalian culture. To obtain an unbiased assessment from hundreds of labs, we analyzed sequence data from 9395 rodent and primate samples from 884 series in the NCBI Sequence Read Archive. We found 11% of these series were contaminated (defined as ≥100 reads/million mapping to mycoplasma in one or more samples). Ninety percent of mycoplasma-mapped reads aligned to ribosomal RNA. This was unexpected given 37% of contaminated series used poly(A)-selection for mRNA enrichment. Lastly, we examined the relationship between mycoplasma contamination and host gene expression in a single cell RNA-seq dataset and found 61 host genes (P < 0.001) were significantly associated with mycoplasma-mapped read counts. In all, this study suggests mycoplasma contamination is still prevalent today and poses substantial risk to research quality.

Prescribing antibiotics in diabetic foot infection: what is the role of initial microscopy and culture of tissue samples?

The aim of this study was to evaluate the role of microscopy, Gram stain and the culture of tissue samples in the antibiotic treatment of patients with diabetic foot infection. A retrospective review of patients with a diabetic foot infection was undertaken. Data analysed included the severity of infection, antibiotic prescribing patterns, microscopy and culture results. A total of 71 patients were included, from whom 114 tissue samples were collected. Gram stain results were in agreement with final culture results in 45·8% (n = 54) of samples. Overall sensitivity and specificity of the Gram stains were low (74·5% and 69·8%, respectively), although the specificity for Gram-negative rods was high (98·5%). The presence or absence of 'pus cells' on microscopy was a poor predictor of culture results. Empirical prescribing of antibiotics was in accordance with local policy in 31·1% of patients, improving to 86·8 % following culture results. Microscopy, a skilled laboratory procedure, was generally a poor predictor of tissue culture results. However, the presence of Gram-negative rods was suggestive of isolation in the culture of such organisms and could allow the early broadening of antibiotic treatment. Despite initial poor compliance of empirical antibiotic treatment regimens, prescribing was adjusted in light of culture results, suggesting these were important for clinicians.

ERK2-dependent activation of c-Jun is required for nontypeable Haemophilus influenzae-induced CXCL2 upregulation in inner ear fibrocytes.

The inner ear, composed of the cochlea and the vestibule, is a specialized sensory organ for hearing and balance. Although the inner ear has been known as an immune-privileged organ, there is emerging evidence indicating an active immune reaction of the inner ear. Inner ear inflammation can be induced by the entry of proinflammatory molecules derived from middle ear infection. Because middle ear infection is highly prevalent in children, middle ear infection-induced inner ear inflammation can impact the normal development of language and motor coordination. Previously, we have demonstrated that the inner ear fibrocytes (spiral ligament fibrocytes) are able to recognize nontypeable Haemophilus influenzae, a major pathogen of middle ear infection, and upregulate a monocyte-attracting chemokine through TLR2-dependent NF-κB activation. In this study, we aimed to determine the molecular mechanism involved in nontypeable H. influenzae-induced cochlear infiltration of polymorphonuclear cells. The rat spiral ligament fibrocytes were found to release CXCL2 in response to nontypeable H. influenzae via activation of c-Jun, leading to the recruitment of polymorphonuclear cells to the cochlea. We also demonstrate that MEK1/ERK2 signaling pathway is required for nontypeable H. influenzae-induced CXCL2 upregulation in the rat spiral ligament fibrocytes. Two AP-1 motifs in the 5'-flanking region of CXCL2 appeared to function as a nontypeable H. influenzae-responsive element, and the proximal AP-1 motif was found to have a higher binding affinity to nontypeable H. influenzae-activated c-Jun than that of the distal one. Our results will enable us better to understand the molecular pathogenesis of middle ear infection-induced inner ear inflammation.

Aminopeptidase N facilitates entry and intracellular survival of Mycobacterium tuberculosis in monocytes.

BACKGROUND AND OBJECTIVE: Aminopeptidase N (CD13) is an ectoenzyme located in the outer membrane of a variety of cells. Proteomic profiling indicates an increased expression of CD13 in phagocytes during Mycobacterium tuberculosis infection. The purpose of this study was to investigate the role of CD13 on the internalization and intracellular survival of M. tuberculosis in monocytes. METHODS: Magnetic nanoparticles and confocal microscopy were used to observe interactions between CD13 and M. tuberculosis. Mycobacterial entry and intracellular survival in monocytes were assessed with and without anti-CD13 antibody (WM15 and WM47) using flow cytometry and colony formation assay. RESULTS: By using magnetic nanoparticles and confocal microscopy, M. tuberculosis was found to be capable of binding to either soluble CD13 or membranous CD13 on monocytes. Flow cytometry showed that pretreatment of monocytes with WM15 or WM47 reduced the number of intracellular M. tuberculosis. Collectively, the data suggest that CD13 is a binding and entry receptor for M. tuberculosis on monocytes. Treatment of infected monocytes showed a greater effect of WM47 than WM15 in reducing the intracellular colonization of M. tuberculosis, suggesting that specific epitopes of CD13 may play an important role modulating intracellular M. tuberculosis survival. CONCLUSIONS: CD13 acts as a receptor for M. tuberculosis on human monocytes. The molecule facilitates internalization, and interaction of CD13 with an anti-CD13 antibody reduces intracellular M. tuberculosis survival.

Galectin-3 in cord blood of term and preterm infants.

In recent years galectin-3 has gained attention as a signalling molecule, mainly in inflammatory diseases. Data on galectin-3 expression in neonates, however, are limited, and expression of this lectin in cord blood has not yet been reported. The aim of this study was to determine galectin-3 levels in cord blood of term and preterm neonates as well as galectin-3 levels in cord blood of term neonates after stimulation with the prevalent pathogen Streptococcus agalactiae. Cord blood samples were incubated for 24 h and galectin-3 levels were assessed by enzyme-linked immunosorbent assay. There is a positive correlation between gestational age and galectin-3 levels in cord blood. Expression of galectin-3 is significantly higher in cord blood of small-for-gestational-age infants compared to appropriate-for-gestational-age infants. Stimulation with an invasive but not with a colonizing strain of S. agalactiae induced expression of galectin-3. Galectin-3 is expressed constitutively in cord blood of neonates and seems to play a role in the innate immunity of this population.

Antagonistic effect of Candida albicans and IFNγ on E-cadherin expression and production by human primary gingival epithelial cells.

Caused mainly by Candida albicans, oropharyngeal candidiasis is the most common oral complication associated with HIV disease worldwide. Host defenses against C. albicans essentially fall into two categories: specific immune mechanisms and local oral mucosal epithelial cell defenses. Since oral mucosa is the first line of defense in the form of a physical barrier against C. albicans invasion, and since epithelial cells are involved in anti-Candida innate immunity through different cytokines, we wanted to determine whether C. albicans alters E-cadherin expression and production, and whether interferon-γ (INFγ), a TH1 cytokine, is involved in the anti-Candida defense. Using primary human gingival epithelial cells, we demonstrated that C. albicans significantly decreased E-cadherin mRNA expression and protein production. This effect was basically obtained at later infective periods (24 and 48h). Interestingly, when IFNγ was added to C. albicans infected epithelial cell cultures, it prevented the side effect of C. albicans on E-cadherin mRNA expression and protein production and deposition. All together, these results suggested concomitant interactions between oral epithelial cells and IFNγ against C. albicans infection.

An overview of methods used to clarify pathogenesis mechanisms of Campylobacter jejuni.

Thermotolerant campylobacters are the most frequent cause of bacterial infection of the lower intestine worldwide. The mechanism of pathogenesis of Campylobacter jejuni comprises four main stages: adhesion to intestinal cells, colonization of the digestive tract, invasion of targeted cells, and toxin production. In response to the high number of cases of human campylobacteriosis, various virulence study models are available according to the virulence stage being analyzed. The aim of this review is to compare the different study models used to look at human disease. Molecular biology tools used to identify genes or proteins involved in virulence mechanisms are also summarized. Despite high cost and limited availability, animal models are frequently used to study digestive disease, in particular to analyze the colonization stage. Eukaryotic cell cultures have been developed because of fewer restrictions on their use and the lower cost of these cultures compared with animal models, and this ex vivo approach makes it possible to mimic the bacterial cell-host interactions observed in natural disease cases. Models are complemented by molecular biology tools, especially mutagenesis and DNA microarray methods to identify putative virulence genes or proteins and permit their characterization. No current model seems to be ideal for studying the complete range of C. jejuni virulence. However, the models available deal with different aspects of the complex pathogenic mechanisms particular to this bacterium.

The effect of Mycoplasma and mycoplasma removal agent on the hydrolase activity in fibroblasts of patients with lysosomal diseases.

This study was designed to evaluate the effect of mycoplasma contamination on acid hydrolase activity and the action of the mycoplasma removal agent (MRA), in cultures of human fibroblasts from individuals with lysosomal diseases. For this purpose, we measured the activity of the b-galactosidase, arylsulphatase B (ASB), hexosaminidase A and a-glucosidase enzymes. The activity of the above mentioned enzymes in fibroblasts contaminated by mycoplasma was measured before and after the addition of the MRA. The results were then compared to the enzymatic activity in contamination-free cultures. Only the ASB enzyme showed significant alteration in activity both in the presence of mycoplasma and MRA. The remaining enzymes did not suffer significant interference by the presence of the two agents. Of the four enzymes tested, three did not suffer significant alterations by the presence of the mycoplasma nor from the MRA. However, the activity measured in the ASB enzyme increased significantly in the presence of mycoplasma and MRA and could lead to a doubtful diagnosis. Therefore, we suggest that contamination should be prevented by using aseptic techniques as well as the MRA in those fibroblast cultures that cannot be discarded.

Potentials of Actinomycetes from Reserved Environments as Antibacterial Agents Against Drug-Resistant Clinical Bacterial Strains.

BACKGROUND: Increased antibiotic resistant properties of pathogens has led to a pronounced search for new effective antibiotics from microbes in diverse ecological niches. This study focused on isolating actinomycetes from soil of reserved areas and profiling them for antibacterial potentials. METHODS: The isolates (IS-2, IS-4, IS-6, IS-10, IS-14) were assessed for antagonistic activity against ten multi-drug resistant bacterial strains (Gram positive and negative) by cross streak and well diffusion methods. RESULTS: During the primary screening, four of the isolates showed good antagonistic activity against the test strains. Notably, all the bacterial strains except Pseudomonas aeruginosa responded sensitively to at least one of the actinomycetes screened. The crude extracts of the secondary metabolites of the four actinomycetes (IS-2, IS-4, IS-6, IS-10) with considerably significant antagonistic activities inhibited the growth of all the bacterial strains efficiently. All the test bacterial strains were sensitive to at least one of the extracts at a concentration of 100µg/ml. The minimum inhibitory concentration of the extracts against the isolates ranged from 12.5 - 25µg/ml. The crude extracts of IS-4 and IS-6 identified as Streptomyces glauciniger NBRC 100913 and Streptomyces griseoplanus NRRL-ISP 5009 by I6s rRNA sequencing, showed higher antibacterial activities against the bacterial strains. Significantly, the ethyl acetate crude extract of the actinomycetes demonstrated better antibacterial activities than the standard antibiotics (ofloxacin, amoxicillin/clavulanate, cefuroxime and ceftriaxone). CONCLUSION: This study reports remarkable anti-MRSA activities as well as broad spectrum antibacterial potentials of extracts of Streptomyces spp. worthy of further exploration.

Characterization of a new human diploid cell strain, IMR-90.

A new human diploid fibroblast-like cell line has been established from lung tissue of a female fetus. This has been frozen away in large quantity and characterized for use in research and related purposes. This is the first of a planned series of human cell lines to be established, characterized, and banked in large quantity in support of the National Institute on Aging research and general cell biology.

Activation of spontaneous murine leukemia virus-related antigen by lymphocytic choriomeningitis virus.

Persistent infection with lymphocytic choriomeningitis (LCM) virus activates a phenotypic expression of murine leukemia viruis-related antigen. NZB and (NZB x NZW)F(1) mice, which normally carry large amounts of Gross virus, and C57BL/6 and NZW mice, which normally carry little virus, were infected with LCM virus. All had Gross soluble antigen in their plasmas at 3 months of age, while noninfected matched controls of all strains did not. This effect was seen after infection with LCM virus that was tissue passed or plaque purified. Similarly, cultures of mouse-embryo fibroblasts produced Gross soluble antigen when infected with LCM virus, but noninfected cultures failed to do so.

Nitrosative stress inhibits production of the virulence factor alginate in mucoid Pseudomonas aeruginosa.

Alginate is a critical virulence factor contributing to the poor clinical prognosis associated with the conversion of Pseudomonas aeruginosa to mucoid phenotypes in cystic fibrosis (CF). An important mechanism of action is its ability to scavenge host innate-immune reactive species. We have previously analyzed the bacterial response to nitrosative stress by S-nitrosoglutathione (GSNO), a physiological NO radical donor with diminished levels in the CF lung. GSNO substantially increased bacterial nitrosative and oxidative defenses and so we hypothesized a similar increase in alginate production would occur. However, in mucoid P. aeruginosa, there was decreased expression of the majority of alginate synthetic genes. This microarray data was confirmed both by RT-PCR and at the functional level by direct measurements of alginate production. Our data suggest that the lowered levels of innate-immune nitrosative mediators (such as GSNO) in the CF lung exacerbate the effects of mucoid P. aeruginosa, by failing to suppress alginate biosynthesis.

Comparative activities of antibiotics against intracellular non-typeable Haemophilus influenzae.

INTRODUCTION: Non-typeable Haemophilus influenzae (NTHi) is a major bacterial pathogen of community-acquired respiratory tract infection and is usually found extracellularly, although studies have revealed that NTHi may possess the ability to invade human epithelial cells where it is then protected against attack by the local immune system and partly against the effect of antibiotics. The aim of the present study was to assess the ability of ampicillin, azithromycin, telithromycin, ciprofloxacin and moxifloxacin, five antibiotics in common clinical use, to kill NTHi within bronchial epithelial cells. METHODS: Confluent human bronchial epithelial cells were infected with NTHi 77, a particularly invasive clinical strain. Extracellular bacterial cells were killed with gentamicin and the intracellular bacteria were incubated with antibiotics at concentrations of 1 mg/l or 10 mg/l for 4 h or 8 h. Viable intracellular bacteria were counted after lysis of the epithelial cells. RESULTS: With the exception of ampicillin, all the antibiotics caused significant reduction of intracellular bacteria at concentrations of 10 mg/l and exposure for 4 h or at 1 mg/l for 8 h. At 1 mg/l, moxifloxacin eliminated 94% of intracellular NTHi after 4 h and 98% after 8 h; ciprofloxacin, azithromycin and telithromycin only achieved killing indices below 75 after 4 h but 86-90% killing after 8 h. At 10 mg/l, moxifloxacin, ciprofloxacin, telithromycin and azithromycin were able to achieve 99.7%, 96.3%, 86.7% and 74.7% eradication of intracellular bacteria, respectively, after exposure for 4 h. CONCLUSION: These results demonstrate the rapid antibacterial efficacy of moxifloxacin against intracellular NTHi in vitro. Moxifloxacin, which combines high extracellular and intracellular activities, could be an important tool in the treatment of recurrent respiratory tract infections.

Molecular Identification of Fungal Contamination in Date Palm Tissue Cultures.

Fungal contamination of in vitro cultures of date palm (Phoenix dactylifera L.) is the major constraint to their initiation and maintenance. Different molecular approaches have been applied successfully to analyze both inter- and intraspecific variation among fungal species as well as determine their identity. This chapter describes step-by-step procedures of molecular identification of fungal contaminants by internal transcribed spacer (ITS) products of the most common fungal contaminants of date palm tissue culture. To begin with, samples of genera Alternaria, Aspergillus, Cladosporium, Epicoccum, and Penicillium were collected to isolate each fungal genus and extraction of genomic DNA. Polymerase chain reactions were accomplished by ITS primers (ITS1 and ITS4) for each fungal contaminant as well as for sequencing. Subsequently, they are analyzed by Basic Local Alignment Search Tool (BLAST) search of ITS sequence to reveal the identity of each individual fungal contaminant species. The molecular identification herein is a rapid and reliable procedure to identify date palm fungal contaminants which is very important in their control and treatment.

Best practices for detecting and mitigating the risk of cell culture contaminants.

A variety of biological and chemical contaminants can adversely impact cells in culture, ranging from outright destruction of the culture, mutation, phenotypic changes to relatively minor changes in morphology, or growth rate. There are various approaches to detecting and mitigating the risk of biological or microbial contaminants in cell cultures, and these are discussed in this article. Chemical contaminants typically arise from improper handling or sourcing of cell culture reagents, glassware, or other types of consumables. These and other sources of chemical contaminants of cell cultures are discussed. The occurrence of chemical contamination is mitigated through adherence to best practices in sourcing and handling of such materials and by avoiding the use of volatile solvents within incubators that are used for maintaining cell cultures.

Hijacking the ERK signaling pathway: Mycobacterium leprae shuns MEK to drive the proliferation of infected Schwann cells.

Schwann cells are the target of Mycobacterium leprae, the pathogen responsible for leprosy. Once inside the cell, M. leprae activates the host's proliferative machinery, thereby increasing the number of cells susceptible to infection. This astonishing manipulation of the mammalian cell cycle is the subject of recent work by Tapinos and Rambukkana, who show that M. leprae drives proliferation through a novel route to extracellular signal-regulated kinase (ERK). In this Perspective, we discuss this important piece of work and highlight the noncanonical pathway used by M. leprae to induce proliferation.