Flow cytometric method for quantifying viable Mycoplasma agassizii,an agent of upper respiratory tract disease in the desert tortoise (Gopherus agassizii)

Aims: Mycoplasma agassizii can cause upper respiratory tract disease in the threatened desert tortoise of the Southwestern United States. Two technical challenges have impeded critical microbiological studies of this microorganism: (i) its small size limits the use of light microscopy for cell counting and (ii) its extremely slow growth in broth and agar cultures impedes colony counting. Our aim was to develop a rapid and sensitive flow cytometric method using a vital fluorescent dye to enumerate viable M. agassizii cells. Methods and Results: Here, we demonstrate that the nonfluorescent molecule 5‐carboxyfluorescein (5‐CF) diacetate acetoxymethyl ester penetrates M. agassizii cell membranes and it is converted in the cytoplasm to the fluorescent molecule 5‐CF by the action of intracellular esterases. Labelled mycoplasma cells can be easily detected by flow cytometry, and cultures with as few as 100 viable mycoplasma cells ml^?1 can be labelled and counted in less than 1 h. Experiments using temperature‐induced cell death demonstrated that only viable M. agassizii cells are labelled with this procedure. Conclusions: A rapid and sensitive flow cytometric technique has been developed for enumerating viable M. agassizii cells. Significance and Impact of the Study: This technique should facilitate basic immunological, biochemical and pharmacological studies of this important pathogen which may lead to new diagnostic and therapeutic methods.     

Profiling bacterial survival through a water treatment process and subsequent distribution system

AIMS: To profile fractions of active bacteria and of bacteria culturable with routine heterotrophic plate count (HPC) methods through a typical water treatment process and subsequent distribution system. In doing so, investigate how water treatment affects both bacterial abundance and diversity, and reveal the identities of active bacteria not detected by traditional HPC culture. METHODS AND RESULTS: Profiling active fractions was performed by flow cytometric cell sorting of either membrane-intact (BacLight kit) or enzymatically active (carboxyfluorescein diacetate, CFDA) bacteria, followed by eubacterial 16S rDNA-directed PCR and denaturing gradient gel electrophoresis (DGGE). Water treatment significantly reduced active bacterial numbers detected by the BacLight kit and CFDA assay by 2.89 and 2.81 log respectively. Bacterial diversity was also reduced from > 20 DGGE bands in the active fractions of reservoir water to only two bands in the active fractions of finished water. These two bands represented Stenotrophomonas maltophila, initially culturable by HPC, and a Burkholderia-related species. Both species maintained measurable traits of physiological activity in distribution system bulk water but were undetected by HPC. CONCLUSIONS: Flow cytometric cell sorting with PCR-DGGE, to assess water treatment efficacy, identified active bacteria from a variety of major phylogenetic groups undetected by routine HPC. Following treatment S. maltophila and a Burkholderia-related species retained activity and entered distribution undetected by HPC. SIGNIFICANCE AND IMPACT OF THE STUDY: Methods used here demonstrate how water treatment operators can better monitor water treatment plant efficacy and assess distribution system instability by the detection and identification of active bacteria recalcitrant to routine HPC culture.     

Comparative suitability of CFDA‐SE and rhodamine 6G for in vivo assessment of leukocyte‐endothelium interactions

Intravital fluorescence microscopy (IVM) is a predestined tool for investigating the fate of leukocytes during the process of leukocyte recruitment. In the present study, the commonly used dye for this purpose, rhodamine 6G, and carboxyfluorescein diacetate succinimidyl ester (CFDA‐SE) were compared for leukocytes labelling with respect to suitability for IVM studies. Their potential in labelling different leukocytes subpopulations as well as their fluorescence intensities were assessed by flow cytometry revealing distinct differences between both dyes. These differences had a profound impact on their application for in vivo imaging of leukocyte‐endothelium interactions. In summary, CFDA‐SE revealed superior in labelling leukocytes for in vivo microscopy with respect to image quality. In addition, we could show the efficiency of CFDA‐SE also under disease condition in an animal model of sepsis. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Extracellular calcium does not contribute to cryopreservation-induced cytotoxicity

Summary The possible role of extracellular calcium ([Ca⁺²]e) in cryopreservation-induced cytotoxicity was tested using Madin-Darby canine kidney (MDCK) cells and a fluorescent multiple endpoint assay. MDCK cells maintained in 2 mM [Ca⁺²]e and treated with the calcium ionophore, ionomycin, increased their intracellular calcium ([Ca⁺²]i) as revealed by the calcium indicator dye, Fluo3 and the bottom-reading spectrofluorometer, CytoFluor 2300. The addition of 10 mM [ethylene bis (oxyethylenenitrilo)]-tetraacetic acid (EGTA) to the extracellular medium before treatment with ionomycin blocked this ionomycin-dependent increase in [Ca⁺²]i. A number of site and activity-specific fluorescent probes were surveyed to determine which indicator dye might best reveal the ionomycin-induced cytotoxic events during this increase in [Ca⁺²]i. Although most dyes changed their emission profiles in response to calcium, neutral red was found to best reflect the loss of [Ca⁺²]i homeostasis. The NR50 for a 15-min exposure to ionomycin in the presence of 2 mM [Ca⁺²]e was approximately 2μM ionomycin, but ionomycin had little apparent effect on neutral red retention when 10 mM EGTA was added to the extracellular medium. Thus it was clear that an increase in [Ca⁺²]i could be cytotoxic to MDCK cells and that neutral red could monitor this cytotoxic episode. To test if [Ca⁺²]e was similarly cytotoxic during cryopreservation, MDCK cells were subjected to cryopreservation in the presence of dimethylsulfoxide (DMSO). In contrast to previous studies, plasma membrane integrity, not lysosomal function, seemed to best correlate with cell survival subsequent to cryopreservation. In addition, decreasing [Ca⁺²]e had no discernable effect on the retention of plasma membrane indicator dyes, neutral red, or cell survival. It is concluded that a) plasma membrane indicator dyes, not neutral red, might be better indicators of cytotoxicity occurring during cryopreservation; b) DMSO might be toxic to lysosomes during cryopreservation of cultured cells; and c) although [Ca⁺²]e can contribute to cytotoxicity, the presence of [Ca⁺²]e might not influence cryopreservation-induced cytotoxicity.     

Adult human CD133/1 kidney cells isolated from papilla integrate into developing kidney tubules

Approximately 60,000 patients in the United States are waiting for a kidney transplant due to genetic, immunologic and environmentally caused kidney failure. Adult human renal stem cells could offer opportunities for autologous transplant and repair of damaged organs. Current data suggest that there are multiple progenitor types in the kidney with distinct localizations. In the present study, we characterize cells derived from human kidney papilla and show their capacity for tubulogenesis. In situ, nestin⁺ and CD133/1⁺ cells were found extensively intercalated between tubular epithelia in the loops of Henle of renal papilla, but not of the cortex. Populations of primary cells from the renal cortex and renal papilla were isolated by enzymatic digestion from human kidneys unsuited for transplant and immuno-enriched for CD133/1⁺ cells. Isolated CD133/1⁺ papillary cells were positive for nestin, as well as several human embryonic stem cell markers (SSEA4, Nanog, SOX2, and OCT4/POU5F1) and could be triggered to adopt tubular epithelial and neuronal-like phenotypes. Isolated papillary cells exhibited morphologic plasticity upon modulation of culture conditions and inhibition of asymmetric cell division. Labeled papillary cells readily associated with cortical tubular epithelia in co-culture and 3-dimensional collagen gel cultures. Heterologous organ culture demonstrated that CD133/1⁺ progenitors from the papilla and cortex became integrated into developing kidney tubules. Tubular epithelia did not participate in tubulogenesis. Human renal papilla harbor cells with the hallmarks of adult kidney stem/progenitor cells that can be amplified and phenotypically modulated in culture while retaining the capacity to form new kidney tubules. This article is part of a Special Issue entitled: Polycystic Kidney Disease.     

Expression of S100A8/A9 in HaCaT keratinocytes alters the rate of cell proliferation and differentiation

S100A8/A9 promotes NADPH oxidase in HaCaT keratinocytes and subsequently increases NFκB activation, which plays important roles in the balance between epidermal growth and differentiation. S100A8/A9-positive HaCaT cells present with a significantly reduced rate of cell division and greater expression of two keratinocyte differentiation markers, involucrin and filaggrin, than control cells. S100A8/A9 mutants fail to enhance NFκB activation, TNFα-induced IL-8 gene expression and NFκB p65 phosphorylation, and S100A8/A9-positive cells demonstrate better cell survival in forced suspension culture than mutant cells. S100A8/A9 is induced in epithelial cells in response to stress. Therefore, S100A8/A9-mediated growth arrest could have implications for tissue remodeling and repair.