Occupational hazards associated with endoscope high-level disinfection: case vignettes, review of literature and recommendations for mitigation.

BACKGROUND: High-level disinfection is crucial in preventing instrument-related infections. However, inadequate process and practice may expose technicians to chemicals and other hazards. OBJECTIVE: The aim of this article is to describe the health effects related to high-level disinfection, the process of identifying hazards and safer practice recommendations. PARTICIPANTS: Two endoscope technicians with different clinical presentations were evaluated for workplace exposures. METHODS: In addition to acute clinical care, corroborative information was obtained through walkthrough and observing patients performing their daily tasks, interview of co-workers, environmental assessment and review of published literature. Recommendations for improvement and clinical follow up were made. RESULTS: Clinical evaluation and workplace assessment identified potential exposures to: denatonium benzoate, ortho-phthalaldehyde, proteinase subtilisin, and isopropyl alcohol. Environmental monitoring showed adequate ventilation but with potential for acute high-level exposure to high-level disinfectants. Ergonomic stressors and noise were addressed. Following work restrictions and work practice changes, both patients were able to return to work without recurrence of symptoms. CONCLUSION: The occupational hazards of working in an endoscopy disinfection unit include chemicals that are irritants and/or allergenic. In addition to bioengineering controls, administrative controls and proper respiratory and dermal protections may mitigate exposure and allow workers to continue working safely.

Glutamyl-gamma-boronate inhibitors of bacterial Glu-tRNA(Gln) amidotransferase.

Analogues of glutamyl-gamma-boronate (1) were synthesized as mechanism-based inhibitors of bacterial Glu-tRNA(Gln) amidotransferase (Glu-AdT) and were designed to engage a putative catalytic serine nucleophile required for the glutaminase activity of the enzyme. Although 1 provides potent enzyme inhibition, structure-activity studies revealed a narrow range of tolerated chemical changes that maintained activity. Nonetheless, growth inhibition of organisms that require Glu-AdT by the most potent enzyme inhibitors appears to validate mechanism-based inhibitor design of Glu-AdT as an approach to antimicrobial development.

Azole-antifungal binding to a novel cytochrome P450 from Mycobacterium tuberculosis: implications for treatment of tuberculosis.

Although antibiotics against Mycobacterium tuberculosis have decreased the incidence of tuberculosis infections significantly, the emergence of drug-resistant strains of this deadly pathogen renders current treatments ineffective. Therefore, it is imperative to identify biochemical pathways in M. tuberculosis that can serve as targets for new anti-mycobacterial drugs. We recently cloned, expressed, and purified MT CYP51, a soluble protein from M. tuberculosis that is similar in sequence to CYP51 (lanosterol-14alpha-demethylase) isozymes, pharmacological targets for several anti-mycotic compounds. Its striking amino acid sequence similarity to that of mammalian and fungal CYP51s led to the hypothesis that MT CYP51 plays an important role in mycobacterial biology that can be targeted for drug action. In this manuscript, we established through spectral analysis that several azole antifungals bind MT CYP51 with high affinity. The effects of several azole compounds on the growth of M. bovis and M. smegmatis, two mycobacterial species that closely resemble M. tuberculosis were examined. We established a correlation between the affinity of azole compounds to MT CYP51 and their ability to impair the growth of M. bovis and M. smegmatis. These results suggest that the metabolic functions of MT CYP51 may be comparable to those of CYP51 in yeast and fungi and may lead to the development of a new generation of anti-mycobacterial agents.

Oral administration of chitin down-regulates serum IgE levels and lung eosinophilia in the allergic mouse.

Previous studies showed that local macrophages phagocytose nonantigenic chitin particles (1-10 micrometer polymers of N-acetyl-d -glucosamine) through mannose receptors and produce IL-12, IL-18, and TNF-alpha. These cytokines lead to the production of IFN-gamma by NK cells. To determine whether chitin could down-regulate Th2 responses, chitin was given orally (8 mg/day for 3 days before and 13 days during ragweed allergen immunization) in BALB/c and C57BL/6 mice. These ragweed-immunized mice were given ragweed intratracheally on day 11. Three days after the challenge, the immunized mice with saline (controls) showed increases in serum IgE levels and lung eosinophil numbers. The chitin treatment resulted in decreases of these events in both strains. To dissect the inhibitory mechanisms of Th2 responses, spleen cells (4 x 106 cells/ml) isolated from the ragweed-immunized mice (controls) were cultured in the presence of ragweed and/or chitin for 3 days (recall responses). Ragweed alone stimulated the production of IL-4 (0.6 ng/ml), IL-5 (20 U/ml), and IL-10 (3.2 ng/ml), but not IFN-gamma. Ragweed/chitin stimulation resulted in significant decreases of IL-4, IL-5, and IL-10 levels and the production of IFN-gamma (48 U/ml). Moreover, spleen cells isolated from the chitin-treated mice showed ragweed-stimulated IFN-gamma production (15 U/ml) and significantly lower levels of the Th2 cytokines, suggesting that the immune responses were redirected toward a Th1 response. Collectively, these results indicate that chitin-induced innate immune responses down-regulate Th2-facilitated IgE production and lung eosinophilia in the allergic mouse.

Immunoregulatory roles of IL-10 in innate immunity: IL-10 inhibits macrophage production of IFN-gamma-inducing factors but enhances NK cell production of IFN-gamma.

In our study of the immunoregulatory roles of IL-10 in innate immunity, nonantigenic phagocytosable chitin particles were administered i.v. to IL-10-deficient (knockout (KO)) mice or KO mice pretreated with anti-NK1.1 or anti-IFN-gamma Abs. The results established that chitin treatment of KO mice increased superoxide anion release from alveolar macrophages (Mphi) to a level much higher than that in wild-type (WT) mice. The results also suggested that the NK cell is the source of IFN-gamma that is primarily responsible for this alveolar Mphi priming. To further study the roles of IL-10-inhibiting chitin-induced IFN-gamma production, we used spleen cell cultures. The experiments showed that IL-12, IL-18, and TNF-alpha, which were produced by chitin-stimulated Mphi, contributed to the IFN-gamma-inducing activity of chitin. Our results established that exogenous IL-10 inhibited chitin-induced IFN-gamma production in spleen cell cultures from both KO and WT mice. Exogenous IL-10 also inhibited IL-12 and TNF-alpha production by chitin-stimulated Mphi. Exogenous IL-10 decreased IL-12- or IL-18-induced IFN-gamma levels in KO but not in WT NK cell cultures. However, exogenous IL-10 enhanced IFN-gamma levels when NK cells were stimulated simultaneously with both IL-12 and IL-18 in KO and WT cultures. Our in vitro data indicate that IL-10 has differential effects on chitin-induced IFN-gamma production. However, the inhibitory effects of endogenous IL-10 appear to be dominant in the chitin-induced alveolar Mphi priming response in vivo.

Intestinal fat suppressed intake of fat longer than intestinal sucrose.

Although animals eventually stop eating when only experiencing the oro-sensory stimuli from a food, they stop eating much more rapidly if they also receive postgastric stimuli simultaneously. This suggests that the postgastric effects of a nutrient influence the hedonic value of food or motivation to consume that food, and thus, can influence food selection within the time frame of a meal. In this experiment, rats were equipped with a gastric fistula and duodenal cannula. This combination allowed them to receive the same oro-sensory stimuli, but different postgastric nutrients. While ingesting either a fat (Intralipid) or carbohydrate (sucrose) solution, both of which drained from the gastric fistula, the rats received a duodenal infusion of either sucrose (10 mLs, 0.24 kcals/mL), fat (10 mLs, 0.25 kcals/mL) or saline (10 mLs, 0 kcals/mL). While ingesting the Intralipid, a duodenal infusion of fat suppressed intake quicker and longer than an infusion of sucrose. While the animals ingested sucrose, sucrose and fat suppressed intake equivalently.

Scintigraphic assessment of gastric emptying of canned and dry diets in healthy cats.

OBJECTIVE: To characterize factors that affect solid-phase gastric emptying in healthy cats by use of nuclear scintigraphy and to assess differences in emptying patterns of dry and canned diets. ANIMALS: 20 healthy cats. PROCEDURE: 2 groups of 10 cats each were fed dry or canned diet for at least 2 weeks before scintigraphy was done. Diets were labeled with 99mTc-disofenin. After ingestion of labeled meals, scintigraphic images were obtained at 0, 15, 30, 45, and 60 minutes, then every 30 minutes to 6 hours. Gastric emptying scans were obtained 3 times for each cat for each diet, in a complete crossover design. The T90, T50, and T20 (times when 90, 50, and 20% of initial meal activity remained in the stomach, respectively) were derived from gastric emptying curves fit to nonlinear models. A mixed models approach was used for data analysis. RESULTS: Gastric emptying was well described by a nonlinear model. Meal size, water intake, and diet type significantly (P < 0.05) effected gastric emptying. The T90, T50, and T20 increased with meal size, regardless of diet type or water intake. Gastric emptying of a dry diet meal took significantly (P < 0.05) longer than that of an isocaloric meal of canned diet, except when meal size was small. Differences in gastric emptying of dry and canned diets varied with the phase (T90 vs T50 vs T20) of emptying. CONCLUSION: Water intake, meal size, and diet type significantly influence gastric emptying in healthy cats, and these factors must be considered in analysis of gastric emptying data.

Analysis of the alcABC operon encoding alcaligin biosynthesis enzymes in Bordetella bronchiseptica.

We previously cloned a B. bronchiseptica (Bb) genomic DNA fragment that complements a Bb alcaligin biosynthesis mutant, and reported the identification of a gene, alcA, with predicted protein sequence similarity to siderophore biosynthesis enzymes from other organisms. In the present study we show that further nt sequencing of this region revealed two open reading frames (ORFs) 3' to alcA that encode putative proteins AlcB and AlcC, with significant sequence similarity to the aerobactin biosynthesis enzymes IucB and IucC, respectively. RT-PCR analysis indicated that the three ORFs are encoded on a single transcript, and that this operon is repressed at the transcriptional level by Fe. Primer extension analysis placed the transcriptional start point (tsp) 35 nt from the 5' end of the Fur consensus sequence and 188 nt from the putative start of translation of AlcA.

Alveolar macrophage priming by intravenous administration of chitin particles, polymers of N-acetyl-D-glucosamine, in mice.

Intravenous (i.v.) administration of phagocytosable chitin particles (1 to 10 microm) in C57BL/6 mice and SCID mice primed alveolar macrophages (Mphi) within 3 days to yield up to a 50-fold increase in their oxidative burst when elicited in vitro with phorbol myristate acetate (PMA). C57BL/6 mice pretreated with monoclonal antibodies (MAbs) against mouse gamma interferon (IFN-gamma) or NK1.1 showed a markedly decreased level of alveolar Mphi priming following injection of chitin particles. To confirm IFN-gamma production in vitro, spleen cells isolated from normal C57BL/6 mice and SCID mice were cultured with chitin particles. Significant IFN-gamma production was observed following stimulation with chitin but not with chitosan or latex beads. When spleen cells were treated with anti-NK1.1 MAb, IFN-gamma production was significantly inhibited. Another set of experiments showed that when C57BL/6 mice were pretreated i.v. with a small dose IFN-gamma, a higher level of priming was induced with not only phagocytosable chitin particles but also phagocytosable chitosan and even latex beads. Likewise, the spleen cell cultures preconditioned with IFN-gamma provided an up-regulation of IFN-gamma production by these phagocytosable particles. Taken together, the in vivo and in vitro results suggest that (i) the alveolar Mphi priming mechanism is due, at least in part, to direct activation of Mphi by IFN-gamma, which is produced by NK1.1+ CD4- cells; (ii) IFN-gamma would have an autocrine-like effect on Mphi and make them more responsive to particle priming; and (iii) phagocytosis of particulates, probably by a postmembrane event such as interiorization, appears to be important for the up-regulation of alveolar Mphi priming and IFN-gamma production.

Myelodysplasia associated with Turner syndrome.

PURPOSE: This study reports the association of myelodysplasia with Turner syndrome. PATIENT AND METHODS: An 11-year-old girl with Turner syndrome was found to have mild macrocytic anemia that persisted during 2 years. RESULTS: Examination of the bone marrow revealed dyserythropoietic features with multinucleation consistent with refractory anemia. Levels of hemoglobin F were also markedly elevated (57%). She also had transient neutropenia and thrombocytopenia, as well as abnormal platelet function studies. The hematopoietic abnormalities were mild and may have been missed were she not followed for her hypertension and aortic coarctation. CONCLUSIONS: Myelodysplastic syndromes in children are frequently associated with chromosomal abnormalities, but an association with Turner syndrome has not been previously described. This could be due to the fact that mild hematopoietic abnormalities in these patients may not be investigated.

Intestinal fat differentially suppresses sham feeding of different gustatory stimuli.

To determine the intestinal contribution to short-term satiety for solutions of varying palatability, 10 ml of either 0.15 M NaCl or lipid (Intralipid: 0.125, 0.25, 0.5, and 1.0 kcal/ml) was infused at a rate of 0.5 ml/min into the duodenum of rats that were sham feeding either a liquid diet (0.5 kcal/ml), 0.3 M sucrose (0.4 kcal/ml), or a 0.1 M solution of glucose polymers (Polycose 0.4 kcal/ml). Differences in palatability were estimated by the total consumption of each solution over 90 min in a one-bottle test. The intake of solutions maximally ingested during the saline infusions (Polycose > Sucrose > liquid diet) was the most sensitive to the lipid infusions. All four lipid concentrations suppressed intake of Polycose, the solution consumed the most; the three highest concentrations suppressed intake of sucrose (intermediate consumption), and only the two highest concentrations suppressed intake of the complete diet, the solution consumed the least. Nevertheless, the duration of suppression was shorter for the solutions the rats drank the most. For the solution the rats drank the least (liquid diet), the two high concentrations of lipid that suppressed intake did so for the entire experimental period, whereas for Polycose, al lipid infusions suppressed intake, but it recovered to control levels for all but the highest concentration. Other studies have reported that increasing diet palatability shortens the duration of satiety. The current results suggest that this effect may reflect the duration of intake suppression elicited by nutrients in the intestine.

Identification of alcA, a Bordetella bronchiseptica gene necessary for alcaligin production.

The alcA gene, essential for the production of the dihydroxamate siderophore, alcaligin, by Bordetella bronchiseptica, was cloned and sequenced. The alcA gene was identified on a 4.7-kb EcoRI genomic fragment adjacent to a Tn5lac transposon insertion that inactivated alcaligin production in strain MBORD846. Analysis of the alcA nucleotide sequence revealed a putative Fur-binding site, suggesting that expression of this gene is repressed by iron. The deduced amino-acid sequence of this open reading frame had significant homology with the Escherichia coli iucD gene product, an enzyme required for biosynthesis of the dihydroxamate siderophore aerobactin.

bvg Repression of alcaligin synthesis in Bordetella bronchiseptica is associated with phylogenetic lineage.

Recent studies have shown that Bordetella bronchiseptica utilizes a siderophore-mediated transport system for acquisition of iron from the host iron-binding proteins lactoferrin and transferrin. We recently identified the B. bronchiseptica siderophore as alcaligin, which is also produced by B. pertussis. Alcaligin production by B. bronchiseptica is repressed by exogenous iron, a phenotype of other microbes that produce siderophores. In this study, we report that alcaligin production by B. bronchiseptica RB50 and GP1SN was repressed by the Bordetella global virulence regulator, bvg, in addition to being Fe repressed. Modulation of bvg locus expression with 50 mM MgSO4 or inactivation of bvg by deletion allowed strain RB50 to produce alcaligin. In modulated organisms, siderophore production remained Fe repressed. These observations contrasted with our previous data indicating that alcaligin production by B. bronchiseptica MBORD846 and B. pertussis was repressed by Fe but bvg independent. Despite bvg repression of alcaligin production, strain RB50 was still able to acquire Fe from purified alcaligin, suggesting that expression of the bacterial alcaligin receptor was not repressed by bvg. We tested 114 B. bronchiseptica strains and found that bvg repression of alcaligin production was strongly associated with Bordetella phylogenetic lineage and with host species from which the organisms were isolated.

Identification of alcaligin as the siderophore produced by Bordetella pertussis and B. bronchiseptica.

The siderophores produced by iron-starved Bordetella pertussis and B. bronchiseptica were purified and were found to be identical. Using mass spectrometry and proton nuclear magnetic resonance, we determined that the siderophore produced by these organisms was identical to alcaligin, a siderophore produced by Alcaligenes denitrificans.

Simple technique for the preparation of silicone gel particles: the effect of silicone gel particles on oxidative responses of macrophages.

A simple technique was developed to prepare phagocytosable-size particles from the silicone gel used in breast implants. Sonication of silicone gel (1 g) in 5 ml of 20 mM sodium phosphate buffer (pH 7.2) containing 1% (wt/vol) polyoxypropylene-polyethylene block surfactant (F-68 or F-108) produced silicone gel particles ranging from 1-50 microns in diameter. Passage of the suspension through a series of filters yielded phagocytosable particles (1-5 microns in diameter) at a concentration of ca. 2 x 10(9) particles/ml. The particles remained as individual particles, did not coalesce to form large clumps, and were not pelleted by centrifugation (2000 x g, 20 min). They were not toxic for rabbit alveolar macrophages (AM) during 24 h of incubation at 37 degrees C, did not elicit an oxidative burst from AM in vitro in a luminol-enhanced chemiluminescent assay, and did not significantly increase the phorbol myristate acetate (PMA)-elicited oxidative burst by AM. AM isolated from rabbits 2 days after the intravenous injection of silicone particles were not primed or activated (i.e., the AM did not show an enhanced oxidative burst when elicited with PMA in vitro). However, AM isolated from rabbits 2 days after intratracheal injection of the particles were primed but only exhibited a 4-6-fold increase in the oxidative burst elicited with PMA.

A siderophore production mutant of Bordetella bronchiseptica cannot use lactoferrin as an iron source.

Bordetella bronchiseptica secreted a hydroxamate siderophore when grown in Fe-depleted medium. A Tn5lac insertion mutant of B. bronchiseptica, DBB22, did not produce this hydroxamate siderophore and was incapable of using lactoferrin as an Fe source. Our data suggest that B. bronchiseptica uses a siderophore for removal of Fe from lactoferrin and transferrin rather than relying upon a receptor for these host Fe-binding proteins.

Glutamine-supported motility of adult filarial parasites in vitro and the effect of glutamine antimetabolites.

The survival in culture of adult female Brugia pahangi, Acanthocheilonema viteae, and Onchocerca volvulus and adult male Onchocerca gibsoni was assessed by measuring parasite motility. Survival of all species was maximal in a nutritionally complex medium (RPMI-1640). All species survived for up to 48 hr in a simpler medium in which the only energy source was 10 mM glutamine; motility in this medium was dependent upon pH. For the species of Onchocerca, motility was maintained better in the presence of glutamine as the sole energy source than in glucose-only medium. Motility of B. pahangi incubated in 10 mM succinate was equivalent to that seen with 10 mM glutamine, but no other tricarboxylic acid intermediate supported this parasite in vitro. Antimycin A (1 microM) and potassium cyanide (KCN, 100 microM) paralyzed B. pahangi incubated in 10 mM glutamine, an effect antagonized by glucose. KCN at 10 or 100 microM was effective also against Onchocerca gutturosa in glutamine-only medium. Several glutamine antimetabolites reduced motility of B. pahangi by 72 hr. This inhibition was prevented by 2 mM glutamine. However, the inhibition of motility in the species of Onchocerca caused by these compounds was attenuated only partially by glutamine. These data demonstrate that, under certain conditions, filarial nematodes can utilize non-sugar substrates as energy sources. The differential sensitivity seen among these organisms to mitochondrial toxins and glutamine antimetabolites may be related to the extent to which they can use these alternative substrates to generate energy.

Food intake and serum insulin responses to intraventricular infusions of insulin and IGF-I.

Previous studies reported that intracerebroventricular (ICV) infusion of insulin decreased food intake in rats and baboons. Insulin can bind to insulin-like growth factor I (IGF-I) receptors and mimic the response of IGF-I. Our objective was to determine the effects of ICV infused-insulin or IGF-I on food intake in sheep. In the present study, a 6-day ICV infusion of insulin (123 ng/kg of body weight/day) but not of IGF-I (123 ng/kg of body weight/day) decreased food intake by 40% (p less than 0.003) and body weight (p less than 0.015) compared with control sheep. In addition, sheep that received ICV insulin or IGF-I had only half the concentration of insulin in serum as compared with controls. Our results support the hypothesis that ICV insulin does not decrease food intake through IGF-I receptors. Nevertheless, apparently both insulin and IGF-I in the brain can influence the concentration of insulin in blood.

The gene family encoding eggshell proteins of Schistosoma japonicum.

The four closely related genes encoding eggshell proteins in the human parasite Schistosoma japonicum are described. A cDNA and a genomic DNA library were constructed and members of the eggshell protein gene family isolated. The four genes in this family do not contain introns, and differ in organization and nucleotide sequence from the related set of genes in Schistosoma mansoni and Schistosoma haematobium. The coding sequences of two of the S. japonicum genes and their flanking regions were determined. Transcription start sites for these genes were shown by primer extension analysis to occur 47 and 50 nucleotides in front of the start codon. A female-specific component in nuclear extracts binds to a DNA fragment containing conserved sequences upstream of the transcription start sites. The deduced protein sequences of 207 and 212 amino acids are composed of 50% glycine with continuous glycine regions as long as 11 residues. In vitro translations of male and female RNAs revealed female-specific translation products, the sizes of which were consistent with the eggshell proteins.

Physiological role of HMG-CoA reductase in regulating egg production by Schistosoma mansoni.

Pathological lesions observed in humans infected with Schistosoma mansoni are due to the eggs produced by the female parasite. Mevinolin, a potent inhibitor of the enzyme hydroxymethylglutaryl-CoA (HMG-CoA) reductase, blocks egg production by this parasite. In this report, we demonstrate that cholesterol precursors, mevalonate and farnesol, stimulate egg production by the female parasite and that these precursors can reverse the mevinolin-induced inhibition of egg production. Because the parasite cannot synthesize cholesterol, we incubated parasites in a culture media containing radiolabeled acetate with and without mevinolin. We isolated nonsterol lipids from the parasite and observed that mevinolin dramatically reduced the conversion of acetate into the polyisoprenoid (dolichols) lipids of the parasite. Dolichols and other nonsterol lipids did not stimulate egg production. HMG-CoA reductase activity was observed in homogenates of the parasite and was inhibited by mevinolin (Ki = 52 nM), but its activity was tripled when the parasite was chronically exposed to low doses of the drug. Parasites with increased reductase activity produced five to six times more eggs. Lastly, chronic administration of large doses of mevinolin to infected mice resulted in a marked reduction of the pathology associated with the infection. These results suggest that egg production in S. mansoni is associated with the parasite's HMG-CoA reductase activity and that a nonsterol lipid produced in the biochemical pathway regulated by this enzyme stimulates egg production.