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Airborne microbial contents in two types of swine confinement buildings in Quebec.

Airborne microorganisms were isolated with a sampler in two types of swine confinement buildings (farrowing units and fattening units). Respirable (particles less than 5 microns) and total dust fractions were obtained. Samplings were repeated every 2 weeks for a total of 6 samplings per unit between January and April. The predominant microorganisms isolated were bacteria (up to 1.25 x 10(6) CFU/m3) with an important fraction in the respirable size range (up to 0.5 x 10(6) CFU/m3). Only small quantities of gram-negative bacteria, yeasts, and molds were found. Identification of the colonies isolated revealed a great diversity of microorganisms present in the air of the different buildings. Enterobacter agglomerans, Moraxella, Acinetobacter calcoaceticus, and Pseudomonas were the most frequently identified bacteria. Scopulariopsis, Aspergillus, Penicillium, and Candida were the most numerous fungi. Faenia rectivirgula, the causative agent of farmer's lung, was not a major contaminant. The results show some differences in airborne microbial contamination between farrowing and fattening units; the distinction, however, is not clear-cut and was observed only for the total bacteria. The level of airborne microbial contamination in swine units does not significantly vary as a function of the outside temperature. Some species of bacteria and fungi isolated in this study are known to induce extrinsic allergic alveolitis. Other fungi are known to be potentially pathogenic for man. The air of swine confinement buildings is highly contaminated with bacteria, yeasts, and molds at a level up to 1200 time higher than so-called "normal air."

Air Microbiology↗

Characterization of human T cells reactive with the Mycoplasma arthritidis-derived superantigen (MAM): generation of a monoclonal antibody against V beta 17, the T cell receptor gene product expressed by a large fraction of MAM-reactive human T cells.

While all known microbial superantigens are mitogenic for human peripheral blood lymphocytes (PBL), the functional response induced by Mycoplasma arthritidis-derived superantigen (MAM) is unique in that MAM stimulation of PBL consistently results in T cell-dependent B cell activation characterized by polyclonal IgM and IgG production. These immunostimulatory effects of MAM on the humoral arm of the human immune system warranted a more precise characterization of MAM-reactive human T cells. Using an uncloned MAM reactive human T cell line as immunogen, we have generated a monoclonal antibody (mAb) (termed C1) specific for the T cell receptor V beta gene expressed by the major fraction of MAM-reactive human T cells, V beta 17. In addition, a V beta 17- MAM-reactive T cell population exists, assessed by MAM, induced T cell proliferation and cytotoxic T cell activity. mAb C1 will be useful in characterizing the functional properties of V beta 17+ T cells and their potential role in autoimmune disease.

Antibodies, Monoclonal↗

Random mutagenesis reveals a region important for gating of the yeast K+ channel Ykc1.

YKC1 (TOK1, DUK1, YORK) encodes the outwardly rectifying K+ channel of the yeast plasma membrane. Non-targeted mutations of YKC1 were isolated by their ability to completely block proliferation when expressed in yeast. All such mutations examined occurred near the cytoplasmic ends of the transmembrane segments following either of the duplicated P loops, which we termed the 'post-P loop' (PP) regions. These PP mutations specifically caused marked defects in the 'C1' states, a set of interrelated closed states that Ykc1 enters and exits at rates of tens to hundreds of milliseconds. These results indicate that the Ykc1 PP region plays a role in determining closed state conformations and that non-targeted mutagenesis and microbial selection can be a valuable tool for probing structure-function relationships of ion channels.

Amino Acid Sequence↗

The reproductive significance of human Fallopian tube cilia.

Effective tubal transport of ova, sperm and embryos is a prerequisite for successful spontaneous pregnancy. Although there is much yet to be discovered about the mechanisms involved, it is evident that tubal transit is a far more complicated process than initially thought. Propulsion of gametes and embryos is achieved by complex interaction between muscle contractions, ciliary activity and the flow of tubal secretions. Evidence is accumulating of the important and possibly pre-eminent role of ciliary motion in this process; and this review describes current knowledge about ciliary activity and its physiological regulation. There is also a description of the effects on ciliary function of cigarette smoking and various pathological states, including endometriosis and microbial infection, with consideration given as to how altered ciliary activity may impact upon fertility.

Chlamydia↗

Contributions of mucosal immune cells to methotrexate-induced mucositis.

The use of high doses of the anti-cancer drug methotrexate (MTX) is associated with intestinal damage. As a result, mucosal immune cells become increasingly exposed to a vast amount of microbial stimuli. We aimed at determining whether these cells are still functional during MTX treatment. Furthermore, we assessed if activation of the mucosal immune system would play a role in the pathogenesis of mucositis. A contributive role to mucositis for the adaptive immune system was established by showing that mucosal lymphocytes from MTX-treated mice secreted enhanced amounts of cytokines upon ex vivo polyclonal stimulation. Next, in vitro experiments revealed that macrophages were not affected by MTX in the capacity to produce tumor necrosis factor-alpha (TNF-alpha) and IL-10 after LPS exposure. Moreover, peritoneal macrophages from MTX-treated mice produced more IL-10 and TNF-alpha upon LPS stimulation, compared with cells derived from control mice. These data indicate a persistence of both innate and adaptive immune responses in this model. The clinical relevance of these findings was further established by the fact that LPS exposure prior to MTX treatment aggravated the course of mucositis. Furthermore, LPS-responsive mice recovered more slowly compared with LPS-unresponsive mice from MTX treatment. Finally, we found an increase in weight loss and intestinal damage upon MTX treatment in IL-10-deficient mice in comparison to wild-type controls, suggesting a protective role for IL-10 in mucositis. We conclude that mucosal immune responses remain resilient during MTX-induced mucositis. Whereas TNF-alpha production may contribute to mucosal damage, IL-10 may regulate by restricting excessive mucositis.

Animals↗

The influence of T cell depletion on recovery of T cell proliferation to herpesviruses and Candida after allogeneic bone marrow transplantation.

To test the influence of T cell depletion of the marrow in allogeneic bone marrow transplantation on functional T cell recovery, in vitro lymphocyte proliferation tests (LPTs) to microbial antigens were regularly performed in 23 recipients of normal BM and in 25 patients receiving BM with a fixed low number of T cells (1 x 10(5) T cells/kg body weight; recipients of T-depleted BM). The long-term recovery of positive LPT to at least 1 of the 4 tested microbial antigens--Candida, herpes simplex virus (HSV), varicella-zoster virus (VZV), and cytomegalovirus--was nearly similar in both groups: 16/23 versus 18/25. Recovery of LPT to Candida and HSV in the first 3 months appeared to be greatly influenced by prophylactic measures; only 2/23 recipients of normal BM, receiving amphotericin B, showed a positive LPT to Candida versus 13/25 recipients of T-depleted BM (P less than 0.01). In contrast, only 1/23 seropositive recipients of T-depleted BM, receiving acyclovir, showed a positive LPT to HSV versus 9/22 recipients of normal BM (P less than 0.05). A positive LPT to CMV in the first 3 months was found in 9/9 seropositive recipients of normal BM, versus in 5/11 seropositive recipients of T-depleted BM (P less than 0.05). Five of the 6 patients with a negative LPT died of CMV-interstitial pneumonia versus 1/14 with positive LPT (P less than 0.01). We conclude that in CMV-seropositive recipients of allogeneic BM, T cell depletion of the graft affects the early recovery of T cell proliferation to CMV, which is associated with a higher risk of fatal CMV-interstitial pneumonia.

Antigens, Fungal↗

The suicidal DNA repair methyltransferases of microbes.

Virtually every organism so far tested has been found to possess an extremely efficient DNA repair mechanism to ensure that certain alkylated oxygens do not accumulate in the genome. The repair is executed by DNA methyltransferases (MTases) which repair DNA O6-methylguanine (O6MeG), O4-methylthymine (O4MeT) and methylphosphotriesters (MePT). The mechanism is rather extravagant because an entire protein molecule is expended for the repair of just one, or sometimes two, O-alkyl DNA adduct(s). Cells profit from such an expensive transaction by earning protection against death and mutation by alkylating agents. This review considers the structure, function and biological roles of a number of well-characterized microbial DNA repair MTases.

Amino Acid Sequence↗

Eubacteria show their true colors: genetics of carotenoid pigment biosynthesis from microbes to plants.

The opportunities to understand eubacterial carotenoid biosynthesis and apply the lessons learned in this field to eukaryotes have improved dramatically in the last several years. On the other hand, many questions remain. Although the pigments illustrated in Fig. 2 represent only a small fraction of the carotenoids found in nature, the characterization of eubacterial genes required for their biosynthesis has not yet been completed. Identifying those eukaryotic carotenoid biosynthetic mutants, genes, and enzymes that have no eubacterial counterparts will also prove essential for a full description of the biochemical pathways (81). Eubacterial crt gene regulation has not been studied in detail, with the notable exceptions of M. xanthus and R. capsulatus (5, 33, 39, 45, 46, 84). Determination of the rate-limiting reaction(s) in carotenoid biosynthesis has thus far yielded species-specific results (12, 27, 47, 69), and the mechanisms of many of the biochemical conversions remain obscure. Predicted characteristics of some carotenoid biosynthesis gene products await confirmation by studying the purified proteins. Despite these challenges, (over)expression of eubacterial or eukaryotic carotenoid genes in heterologous hosts has already created exciting possibilities for the directed manipulation of carotenoid levels and content. Such efforts could, for example, enhance the nutritional value of crop plants or yield microbial production of novel and desirable pigments. In the future, the functional compatibility of enzymes from different organisms will form a central theme in the genetic engineering of carotenoid pigment biosynthetic pathways.

Bacteria↗

SadB is required for the transition from reversible to irreversible attachment during biofilm formation by Pseudomonas aeruginosa PA14.

Current models of biofilm formation by Pseudomonas aeruginosa propose that (i) planktonic cells become surface associated in a monolayer, (ii) surface-associated cells form microcolonies by clonal growth and/or aggregation, (iii) microcolonies transition to a mature biofilm comprised of exopolysaccharide-encased macrocolonies, and (iv) cells exit the mature biofilm and reenter the planktonic state. Here we report a new class of P. aeruginosa biofilm mutant that defines the transition from reversible to irreversible attachment and is thus required for monolayer formation. The transposon insertion carried by the sadB199 mutant was mapped to open reading frame PA5346 of P. aeruginosa PA14 and encodes a protein of unknown function. Complementation analysis and phage-mediated transduction demonstrated that the transposon insertion in PA5346 was the cause of the biofilm-defective phenotype. Examination of flow cell-grown biofilms showed that the sadB199 mutant could initiate surface attachment but failed to form microcolonies despite being proficient in both twitching and swimming motility. Closer examination of early attachment revealed an increased number of the sadB199 mutant cells arrested at reversible attachment, functionally defined as adherence via the cell pole. A positive correlation among biofilm formation, irreversible attachment, and SadB level was demonstrated, and furthermore, RpoN and FleR appear to negatively affect SadB levels. Fractionation studies showed that the SadB protein is localized to the cytoplasm, and with the use of GPS-linker scanning mutagenesis, the C-terminal portion of SadB was shown to be dispensable for function, whereas the two putative domains of unknown function and the linker region spanning these domains were required for function. We discuss the results presented here in the context of microbial development as it applies to biofilm formation.

Bacterial Adhesion↗

Microbial type I fatty acid synthases (FAS): major players in a network of cellular FAS systems.

The present review focuses on microbial type I fatty acid synthases (FASs), demonstrating their structural and functional diversity. Depending on their origin and biochemical function, multifunctional type I FAS proteins form dimers or hexamers with characteristic organization of their catalytic domains. A single polypeptide may contain one or more sets of the eight FAS component functions. Alternatively, these functions may split up into two different and mutually complementing subunits. Targeted inactivation of the individual yeast FAS acylation sites allowed us to define their roles during the overall catalytic process. In particular, their pronounced negative cooperativity is presumed to coordinate the FAS initiation and chain elongation reactions. Expression of the unlinked genes, FAS1 and FAS2, is in part constitutive and in part subject to repression by the phospholipid precursors inositol and choline. The interplay of the involved regulatory proteins, Rap1, Reb1, Abf1, Ino2/Ino4, Opi1, Sin3 and TFIIB, has been elucidated in considerable detail. Balanced levels of subunits alpha and beta are ensured by an autoregulatory effect of FAS1 on FAS2 expression and by posttranslational degradation of excess FAS subunits. The functional specificity of type I FAS multienzymes usually requires the presence of multiple FAS systems within the same cell. De novo synthesis of long-chain fatty acids, mitochondrial fatty acid synthesis, acylation of certain secondary metabolites and coenzymes, fatty acid elongation, and the vast diversity of mycobacterial lipids each result from specific FAS activities. The microcompartmentalization of FAS activities in type I multienzymes may thus allow for both the controlled and concerted action of multiple FAS systems within the same cell.

Corynebacterium↗

Microbial population dynamics on leaves.

Microbial population dynamics on leaves in time and space are a function of immigration, emigration, growth, and death. Insight into the relative significance of each population process to the generation of specific dynamics for individual microorganisms is necessary to understanding the ecology and life history strategy of the microorganism and to developing effective control strategies. Additionally, information on the significance of within-leaf versus extra-leaf processes to the generation of phyllosphere dynamics is important to determining the range of spatial scales over which a population should be studied. Unfortunately, such information is difficult to obtain due to the lack of effective methodologies for distinguishing these processes within phyllosphere populations. Future research efforts should focus on the quantification of immigration, emigration, growth, and death relative to the population dynamics of phyllosphere microorganisms.

Journal Article↗

Interactions of surfactant protein D with fatty acids.

Surfactant Protein D (SP-D) plays important roles in antimicrobial host defense, inflammatory and immune regulation, and pulmonary surfactant homeostasis. The best-characterized endogenous ligand is phosphatidylinositol; however, this lipid interaction at least in part involves the carbohydrate moiety. In this study we observed that SP-D binds specifically to saturated, unsaturated, and hydroxylated fatty acids (FA). Binding of biotinylated-SP-D to FAs or biotinylated FA to SP-D was dose-dependent, saturable, and specifically competed by the corresponding unlabeled probe. Specific binding to FA chains was also demonstrated by solution phase competition for FA binding to acrylodan-labeled FA binding protein (ADIFAB), and by overlay of thin layer chromatograms with SP-D. Maximal binding to FA was dependent on calcium, and binding was localized to the neck and carbohydrate recognition domains (CRD) using recombinant trimeric neck+CRDs. Saccharide ligands showed complex, dose-dependent effects on FA binding, and FAs showed dose- and physical state-dependent effects on the binding of SP-D to mannan. In addition, CD spectroscopy suggested alterations in SP-D structure associated with binding to monomeric FA. Together, the findings indicate specific binding of FA to one or more sites in the CRD. We speculate that the binding of SP-D to the fatty acyl chains of surfactant lipids, microbial ligands, or other complex lipids contributes to the diverse biological functions of SP-D in vivo.

Animals↗

Upregulation of alpha 9 integrin and tenascin during epithelial regeneration after debridement in the cornea.

Stratified epithelia are exposed to abrasive forces and are required to respond rapidly to injury to minimize fluid loss and the risk for microbial infection. Healing involves a cell migratory phase to reestablish barrier function and cell proliferation to restratify the epithelium. Cell migration during re-epithelialization involves cell sliding, termed sheet movement, during which cells retain their cell-cell junctions while dynamically altering their shape and cell-substrate interactions to permit movement across the exposed wound bed. Proteins of the integrin family of receptor molecules modulate cell shape, cell migration, and signal transduction in many cell types. In epithelial cells, integrins of the beta 1 family have been implicated in regulating cell proliferation and differentiation, alpha 9 beta b1 is one of the newer members of the integrin beta family and has been recently shown to function as a tenascin receptor. Although little is known about its function in vivo, studies in developing mouse cornea and eyelid suggest that it may play a role in epithelial differentiation. Using a debridement wound model in the mouse cornea, we show in this study that (a) in response to small debridement wounds that close without cell proliferation, alpha 9 integrin protein and mRNA are not induced during migration but are induced during restratification, (b) larger debridement wounds that require cell proliferation to generate the cells necessary for sheet movement result in a dramatic induction of alpha 9 protein and its mRNA during both migration and restratification, and (c) tenascin, an alpha 9 beta 1 ligand, accumulates beneath epithelial cells during restratification but not during cell migration. Therefore, alpha 9 integrin protein production and tenascin accumulation are dynamically regulated in response to corneal epithelial injury.

Animals↗

Innate immunity of the human newborn: distinct cytokine responses to LPS and other Toll-like receptor agonists.

Human newborns are at increased risk of microbial invasion and display diminished responses to many vaccines indicating a functional immaturity of the immune system at birth. Such altered immune reactivity may reflect the demands of in utero existence, including the need to avoid potentially harmful inflammatory immune reactions. Despite normal basal expression of Toll-like receptors and membrane CD14, innate immune responses of neonatal mononuclear cells to lipopolysaccharide are characterized by markedly reduced release of the pro-inflammatory Th1-polarizing cytokines TNF-alpha and interferon-gamma with relative preservation of anti-inflammatory Th2-polarizing cytokines. Differences between newborns and adults with respect to TLR-induced TNF-alpha release extend to a range of TLR agonists, including bacterial lipopeptides, and are due to differences in soluble factors present in blood plasma. Soluble factors in neonatal blood plasma suppress TLR-induced TNF-alpha release from monocytes and efforts to identify and characterize these inhibitors are on-going. Such altered immunity to TLR agonists is likely to alter both innate and adaptive immune responses in newborns profoundly. Definition of the mechanisms underlying distinct neonatal immunity promises to identify novel ways to prevent and treat infection in this relatively high-risk population.

Cytokines↗

Cytochrome P450 proteins and potential utilization in biodegradation.

The cytochrome P450 enzymes are major catalysts involved in the oxidations of xenobiotic chemicals in microorganisms as well as higher animals and plants. Because of their functional roles, they offer potential in biodegradation technology. A number of microbial P450s have already been characterized and offer advantages in terms of their high catalytic rates and facile expression in microorganisms. One approach to extending the catalytic selectivity to more compounds in the environment is rational design. In three cases, the three-dimensional structures of bacterial cytochrome P450 enzymes are available and can be further understood through studies with molecular dynamics. Many mammalian cytochrome P450 enzymes have been studied extensively and have potential for biodegradation because of their broad catalytic selectivities (e.g., P450 2E1). Several advances have been made in the heterologous expression of these proteins in microorganisms. Improvements under development include electron transfer from flavodoxin and the use of cytochrome P450:NADPH-cytochrome P450 reductase fusion proteins. Random mutagenesis offers the potential of improving the catalytic activities of some of these proteins. Future challenges include the use of cytochrome P450 expression vectors in microorganisms capable of thriving in the environment; recent success in expression of vectors in Salmonella genotoxicity tester strains may be encouraging in this regard.

Bacteria↗

Quantification of mast cells and microflora in the vaginal cul-de-sac of the brushtail possum (Trichosurus vulpecula).

Mast cells are typically associated with biological responses to intracellular parasitic or bacterial invasion in gut and pulmonary tissues. Large numbers of these cells are present in the vaginal cul-de-sac, a component of the reproductive tract in female brushtail possums (Trichosurus vulpecula). The presence of mast cells in this area may reflect microbial presence or indicate a role for mast cells in reproductive function in this marsupial. In this study, the number and spatial location of mast cells in vaginal cul-de-sac tissues at different reproductive stages was determined, and related to the presence of microflora. Vaginal cul-de-sac tissue was collected from juvenile (n = 5), lactationally anoestrous and cyclic adult brushtail possums (n = 6 per group). Oestrous cyclicity was induced by removal of pouch young and reproductive status was confirmed by laparoscopy. Tissue was collected aseptically for microbiological procedures. The fractionator and optical disector stereological methods were used to quantify mast cell populations. In all groups, microflora populations were low (< 5 x 10(5) organisms g(-1)). Mast cell density in epithelial tissue was highest in lactationally anoestrous and luteal phase brushtail possums and lowest in follicular phase animals. No correlation between the numbers of bacteria isolated and mast cell density was apparent. Gram stained tissue showed no evidence of bacterial invasion. Mast cell density was significantly lower in epithelial tissue from follicular phase animals, corroborating previous studies in which an increase in mast cell degranulation coincided with high oestrogen concentrations.

Anestrus↗

Microbiota of successful osseointegrated dental implants.

BACKGROUND: The long-term survival of dental implants depends, in part, on control of bacterial infection in the peri-implant region. Periodontal pathogens colonized implants symptomatic through infection, whereas the microbiota of successful implants was similar to that of periodontal health. This study examined the impact on the peri-implant microbiota of crown restorations; implant type; length of time of loading; history of implant or periodontal infections; and whether implants replaced single or multiple teeth. It was of particular interest to evaluate implant colonization by species in a newly described red complex of periodontal pathogens, Porphyromonas gingivalis and Bacteroides forsythus. METHODS: This study sampled 43 partially edentulous subjects with successfully osseointegrated titanium root-form dental implants. Eighty-one (81) non-submerged and 20 submerged asymptomatic implants, 83 crowned, and 36 uncrowned teeth were sampled from peri-implant or subgingival sites. The microbiota of samples was evaluated using whole genomic DNA probes in a checkerboard assay to 23 subgingival species. RESULTS: Implants were colonized principally by oral streptococci, capnocytophagae, Veillonella parvula, Peptostreptococcus micros, and Fusobacterium nucleatum. The periodontal species, P. gingivalis, B. forsythus, Prevotella intermedia, Prevotella nigrescens, and Campylobacter rectus were detected in a few subjects. The microbiota around crowned implants and crowned teeth was similar. Streptococcus oralis, P. intermedia, and Selenomonas noxia were elevated in samples from uncrowned teeth compared to crowned teeth and implants. Microbial complexity increased as loading time increased, but colonization by periodontal pathogens, including red complex species, was higher in subjects with previous periodontal disease. No differences were observed in the microbiota of 1- and 2-stage implants, or between implants supporting single or multiple restorations. CONCLUSIONS: While presence of crowns had only a minor impact on the peri-implant microbiota, microbial changes were observed the longer the implants had been in function and in those patients with a history of periodontal or peri-implant infections. A history of periodontitis had a greater impact on the peri-implant microbiota than implant loading time. The major influence on the peri-implant microbiota was, however, the microbiota on remaining teeth. P. gingivalis and B. forsythus, red complex periodontal pathogens, colonized several implants, although all implants were successfully osseointegrated.

Adult↗

[Bacterial infection and production of immunoglobulin in the tonsil].

Immunoglobulin (Ig) isotype switching is the process whereby B cells initially expressing IgM and/or IgD on their surface switch to other Ig heavy-chain loci. We demonstrated that TGF-beta, IL-10 and VIP in the presence of CD40 mAb, can induce isotype switching for IgA in human tonsillar B cells by the generation of switch circular DNA (S alpha/S mu), the induction of alpha germ-line transcripts and a significant amount of IgA production. IgA is the predominant immunoglobulin effector molecule of mucosal immunity that functions as the first line of specific immunologic defense against many microbial pathogens. However, IgA causes IgA nephropathy. We investigated here, whether tonsillar mononuclear cells from patients with IgA nephropathy produce HP-specific IgA and/or IgA-related cytokines by stimulation with components of HP outer membranes (OMHP). HP-specific IgA was predominately induced by tonsillar mononuclear cells of IgA nephropathy, compared to those from chronic tonsillitis. Production of IL-10 and TGF-beta was enhanced by stimulation with OMHP in tonsillar mononuclear cells from IgA nephropathy. These results suggested that local infection of HP and HP-specific IgA induction in the tonsil are associated with pathogens in IgA nephropathy.

Bacterial Infections↗