Ribotype 027 Clostridium difficile infections with measurable stool toxin have increased lactoferrin and are associated with a higher mortality.

We evaluated clinical and diagnostic indicators of severe C. difficile infection (CDI) and their association with poor clinical outcome. A total of 210 patients positive according to PCR (toxin B: tcdB) were included, with patients having a median age of 62 years and a Charlson co-morbidity index (CI) score of 5. Ninety-one percent (n = 191) were positive by toxigenic culture and 61% (n = 129) had stool toxin. Toxin-positive patients had significantly higher fecal lactoferrin (mean 316 µg/g versus 106 µg/g stool; p < 0.0001). Forty percent of patients (n = 85) were infected with ribotype 027 and significantly more of these patients had measurable stool toxin (79% vs. 50%; p < 0.0001). The mean fecal lactoferrin was significantly higher for toxin-positive 027 CDI compared with the 027 toxin-negative group (317 vs 60 µg/g; p = 0.0014). Ribotype 027 CDI with stool toxin showed a higher all-cause, 100-day mortality compared with non-027 with stool toxin (36 % vs 18%; p = 0.017). Logistic regression univariate analysis for odds ratio (OR) and p values revealed that age (OR = 1.1), intensive care unit treatment (OR = 2.7), CI (OR = 1.2), 027 CDI (OR = 2.1), white blood cell count (OR = 1.0), albumin level (OR = 0.1), and stool toxin-positive 027 CDI (OR = 2.5) were significantly associated with 100-day mortality (p < 0.05). In conclusion, CDI PCR-positive patients with 027 infection and stool toxin have increased lactoferrin and are at an increased risk of death.

Elevated lactoferrin is associated with moderate to severe Clostridium difficile disease, stool toxin, and 027 infection.

We evaluated blood and fecal biomarkers as indicators of severity in symptomatic patients with confirmed Clostridium difficile infection (CDI). Recruitment included patients with CDI based on clinical symptoms and supporting laboratory findings. Disease severity was defined by physician's assessment and blood and fecal biomarkers were measured. Toxigenic culture done using spore enrichment and toxin B detected by tissue culture were done as confirmatory tests. Polymerase chain reaction (PCR) ribotyping was performed on each isolate. There were 98 patients recruited, with 85 (87%) confirmed cases of toxigenic CDI (21 severe, 57 moderate, and seven mild), of which 68 (80%) were also stool toxin-positive. Elevated lactoferrin (p = 0.01), increased white blood cell (WBC) count (p = 0.08), and low serum albumin (p = 0.03) were all associated with the more severe cases of CDI. Ribotype 027 infection accounted for 71% of severe cases (p < 0.01) and patients with stool toxin had significantly higher lactoferrin levels and WBC counts (p < 0.05). Our findings show that elevated fecal lactoferrin, along with increased WBC count and low serum albumin, were associated with more severe CDI. In addition, patients infected with ribotype 027 and those with stool toxin had significantly higher fecal lactoferrin and WBC counts.

Glutamate dehydrogenase is highly conserved among Clostridium difficile ribotypes.

gluD was highly conserved and glutamate dehydrogenase (GDH) was readily expressed in vitro by all 77 Clostridium difficile ribotypes assayed. All ribotypes, including ARL 002, ARL 027, and ARL 106, were reactive in assays that detect C. difficile GDH.

Clostridium difficile prevalence rates in a large healthcare system stratified according to patient population, age, gender, and specimen consistency.

We evaluated Clostridium difficile prevalence rates in 2,807 clinically indicated stool specimens stratified by inpatient (IP), nursing home patient (NH), outpatient (OP), age, gender, and specimen consistency using bacterial culture, toxin detection, and polymerase chain reaction (PCR) ribotyping. Rates were determined based on the detection of toxigenic C. difficile isolates. We identified significant differences in the rates between patient populations and with age. Specimens from NH had a higher rate (46%) for toxigenic C. difficile than specimens from IP (18%) and OP (17%). There were no gender-related differences in the rates. Liquid specimens had a lower rate (15%) than partially formed and soft specimens (25%) and formed specimens (18%) for the isolation of toxigenic C. difficile. The nontoxigenic rate was lowest for NH (4%) and highest for patients<20 years of age (23%). We identified 31 different toxigenic ribotypes from a sampling of 190 isolates that showed the lowest diversity in NH. Fluoroquinolone resistance was observed in 93% of the 027 isolates, all of the 053 isolates, and in four other ribotypes. We observed different rates for toxigenic C. difficile in stratified patient populations, with the highest rate for NH, a low overall nontoxigenic rate, and fluoroquinolone resistance.

Cytotoxicity of Clostridium difficile toxin A for human colonic and pancreatic carcinoma cell lines.

The use of bacterial exotoxins may constitute novel adjuncts to treatment of gastrointestinal tract malignancies. Clostridium difficile toxin A was evaluated for its cytotoxic effect in vitro on 24 human cell lines and strains including carcinomas of the colon, pancreas, prostate, lung, breast, and lymphoid malignancies, as well as nonmalignant tissues. All nine colon and five pancreas cell lines were extraordinarily sensitive to the cytotoxic effect of Clostridium difficile toxin A at very low concentrations. This effect, which occurred rapidly and was dose dependent, was observed in all cells of seven colon and two pancreas cell lines at concentrations as low as 1-5 ng/ml (10(-12) to 10(-11) M), whereas cells derived from other sites required 60 to greater than 500 ng/ml to achieve an equivalent effect. The data suggest that Clostridium difficile toxin A may have potential therapeutic value in the treatment of some gastrointestinal tract cancers.

Inactivation of human plasma alpha 1-proteinase inhibitor by a metalloproteinase from Serratia marcescens.

The interaction of a Serratia marcescens metalloproteinase with human plasma alpha 1-proteinase inhibitor has been investigated. The enzyme was not inactivated by this inhibitor but, instead, converted the native plasma protein into an inactive form of decreased molecular weight. Amino terminal sequence analysis indicated that the interaction of the inhibitor and enzyme was at the reactive site of the inhibitor, with peptide-bond cleavage resulting in the inactivation. This process may be important in necrotic processes occurring during bacterial infiltration of host tissues.

Preclinical neurochemical and electrophysiological profile of 1192U90, a potential antipsychotic.

11192U90 was submitted to receptor binding and monoamine uptake assays. It bound potently at serotonin 5-HT2, dopaminergic D2, serotonin 5-HT1A, and adrenergic alpha 1 and alpha 2 receptors. It also bound to dopaminergic D1, serotonin 5-HT3, serotonin 5-HT4, and sigma sites, albeit with lower affinity. It was essentially inactive at 22 other sites, including those for cholinergic M1 and M2. It weakly inhibited uptake of 3H-norepinephrine, 3H-serotonin and 3H-dopamine. Acute doses of 1192U90 (5 and 20 mg/kg P.O.) increased whole-brain levels of dopamine metabolites but did not affect levels of norepinephrine, dopamine, and serotonin. Subcutaneous injection of 1192U90 (0.8 mg/kg/day) and clozapine (20 mg/kg/day) for 28 days preferentially decreased the number of spontaneously active dopamine cells in the ventral tegmental area (VTA) but not the substantia nigra (SN) of rats, as measured by population sampling. This outcome is characteristics of atypical antipsychotics like clozapine. Acute injections of 1192U90 reversed the rate-inhibiting effects of microiontophoretically applied dopamine and intravenously injected apomorphine and d-amphetamine on dopamine cell firing. Intravenous injection or iontophoretic application of 1192U90 or the 5-HT1A agonist (+/-)8-OH-DPAT inhibited the firing rates of dorsal raphe nucleus (DRN) neurons in rats, and the effects of both compounds were blocked by iontophoretically applied S(-) propranolol, a 5-HT1A antagonist. The results suggest that 1192U90 is a preferential dopamine D2 antagonist as well as a 5-HT1A agonist that may prove to be an atypical antipsychotic.

Immunization against experimental Pseudomonas aeruginosa and Serratia marcescens keratitis. Vaccination with lipopolysaccharide endotoxins and proteases.

Rabbits vaccinated with lipopolysaccharide endotoxins or with purified protease preparations from Pseudomonas aeruginosa and Serratia marcescens before corneal challenge with the viable bacteria exhibited significantly less corneal damage than rabbits not vaccinated with the bacterial products. However, the rabbits vaccinated with the lipopolysaccharide endotoxin preparations were significantly better protected than rabbits vaccinated with the bacterial proteases. Rabbits vaccinated with antisera raised against the proteases showed significantly less corneal damage than rabbits vaccinated with normal rabbit serum, and the passive protection was not significantly different than that elicited by active immunization against the bacterial proteases. The ability of the antiserum raised against the pseudomonas elastolytic protease to passively protect against severe corneal damage produced by experimentally induced pseudomonas keratitis was confirmed in mice. These findings support the idea that the bacterial endotoxins and proteases are virulence factors during the development of pseudomonas and serratia keratitis.

Clostridium difficile toxins A and B can alter epithelial permeability and promote bacterial paracellular migration through HT-29 enterocytes.

Clostridium difficile toxins A and B are the widely recognized etiologic agents of antibiotic-associated diseases ranging from diarrhea to pseudomembranous colitis. We hypothesized that C. difficile toxins may alter intestinal epithelial permeability and facilitate bacterial penetration of the intestinal epithelial barrier. Experiments were designed to clarify the effects of C. difficile toxins A and B on the flux of inert particles across HT-29 enterocyte monolayers, and to correlate these results with bacteria-enterocyte interactions. In all experiments, mature, confluent HT-29 cultures were preincubated 16 h with toxin A or B (1-100 ng/mL). To study alterations in epithelial permeability, toxin-treated enterocytes were incubated with 5 pM solutions of 10- and 40-kD inert dextran particles. Toxin A, but not toxin B, was associated with increased dextran flux through enterocyte monolayers. To study bacteria-enterocyte interactions, toxin-treated enterocytes were incubated with 10(8) Salmonella typhimurium, Proteus mirabilis, or Escherichia coli. Although numbers of internalized bacteria were generally unaffected, both toxins were associated with increased bacterial adherence, as well as increased bacterial transmigration through enterocyte monolayers. Bacterial transmigration was significantly greater using toxin A- compared to toxin B-treated enterocytes, consistent with the observation that dextran flux was significantly greater using toxin A- compared to toxin B-treated enterocytes. Thus intestinal colonization with toxigenic C. difficile may facilitate bacterial penetration of the intestinal epithelium by a mechanism involving increased permeability of the intestinal epithelial barrier.

Proteolysis of human fixed, washed platelets by gram-negative bacterial metalloproteases: effect on von Willebrand factor-human platelet interactions.

Fixed, washed platelets (FWP) are usually stable to aggregation with von Willebrand factor (vWF) from human and certain animal plasmas over several months of storage. When one lot of FWP lost its stability in less than 1 week, studies demonstrated contamination with Serratia marcescens. Extracellular proteases produced by S. marcescens, as well as by Pseudomonas aeruginosa and Escherichia coli, were found to cause loss of FWP aggregability. Purified proteases were prepared from cell-free culture filtrates of S. marcescens and two different strains of P. aeruginosa. They were used to study the effect on the interaction of FWP and vWF. All three purified proteases destroyed FWP aggregability in a time- and concentration-dependent fashion. The protease produced by S. marcescens (SP) was found to be at least eight times more potent against FWP as a substrate than either of the two P. aeruginosa enzymes. The ability of SP to destroy FWP aggregability was prevented by EDTA and could be restored by the addition of Zn2+ in slight molar excess. When compared with trypsin and chymotrypsin, SP was found to be highly selective in digesting the FWP membrane, even at concentrations greater than that established to give a similar loss of FWP aggregability. SP does not induce aggregation of fresh, washed platelets or PRP, but renders them unaggregable with vWF. These proteases may be useful research tools for studying membranes and vWF-platelet interactions.

Clostridium difficile toxins may augment bacterial penetration of intestinal epithelium.

BACKGROUND: Clostridium difficile can be recovered from many high-risk hospitalized patients receiving broad-spectrum antibiotic therapy. Clostridium difficile toxins A and B have been associated with increased intestinal permeability in vitro and there is growing evidence that increased intestinal permeability may be a common mechanism whereby enteric bacteria penetrate the intestinal epithelium. HYPOTHESIS: Clostridium difficile-induced alterations in the intestinal barrier facilitate microbial penetration of the intestinal epithelium, which in turn facilitates the translocation of intestinal bacteria. DESIGN: Mature Caco-2 enterocytes were pretreated with varying concentrations of toxin A or toxin B followed by 1 hour of incubation with pure cultures of either Salmonella typhimurium, Escherichia coli, or Proteus mirabilis. The effects of toxins A and B on enterocyte viability, cytoskeletal actin, and ultrastructural topography were assessed using vital dyes, fluorescein-labeled phalloidin, and scanning electron microscopy, respectively. The toxins' effects on bacterial adherence and bacterial internalization by cultured enterocytes were assessed using enzyme-linked immunosorbent assay and quantitative culture, respectively. Epithelial permeability was assessed by changes in transepithelial electrical resistance and by quantifying paracellular bacterial movement through Caco-2 enterocytes cultivated on permeable supports. RESULTS: Neither toxin A nor toxin B had a measurable effect on the numbers of enteric bacteria internalized by Caco-2 enterocytes; however, both toxins were associated with alterations in enterocyte actin, decreased transepithelial electrical resistance, and increased bacterial adherence and paracellular transmigration. CONCLUSION: Clostridium difficile toxins A or B may facilitate bacterial adherence and penetration of the intestinal epithelial barrier.

Comparison of culture, cytotoxicity assays, and enzyme-linked immunosorbent assay for toxin A and toxin B in the diagnosis of Clostridium difficile-related enteric disease.

Clostridium difficile culture, test tube, and microtiter cytotoxicity assays, and enzyme-linked immunosorbent assays (ELISAs) for toxin A and toxin B, were simultaneously performed on 113 fresh diarrheal stool specimens randomly selected from those submitted to our clinical laboratory for routine C. difficile testing. The performance of these tests in diagnosing C. difficile-related enteric disease (CDRED) was based on a clinical assessment of the likelihood of CDRED as determined by a systematic review of case histories blinded from the test results. Among 61 antibiotic recipients, both the microtiter cytotoxicity assay and the toxin A ELISA were highly specific for CDRED (95% and 100%, respectively). Specificities for the other procedures were much lower (tube cytotoxicity assay, 79%; culture, 74%; and toxin B ELISA, 56%). The high sensitivities of the culture (89%) and toxin B ELISA (83%) were somewhat negated by their low specificities. The only test that was both specific and had acceptable sensitivity (78%) was the microtiter cytotoxicity assay. This study indicates that ELISAs for detection of C. difficile toxins are not as reliable as the cytotoxicity assay in the laboratory diagnosis of CDRED, and that clinical correlation is essential in the evaluation of any new test for CDRED.

Deletions in the repeating sequences of the toxin A gene of toxin A-negative, toxin B-positive Clostridium difficile strains.

The repeating sequences of the toxin A gene from toxin A-negative, toxin B-positive (toxin A-, toxin B+) strains of Clostridium difficile which were isolated in geographically separated facilities in Japan and Indonesia were determined. All six strains tested had identical repeating sequences with two deletions (1548 and 273 nucleotides in size) in the toxin A gene. A PCR method was designed to detect the deletions and the deletions were confirmed in all 50 toxin A-, toxin B+ strains examined by this method. Western immunoblot analysis revealed that polyclonal antiserum against native toxin A did not react with the concentrated culture filtrates of the toxin A-, toxin B+ strains. These results may suggest that toxin A-, toxin B+ strains have deletions of the two thirds of the repeating regions of the toxin A gene, which encodes the epitopes fully responsible for the reaction with the polyclonal antiserum.

Pharmacological characterization and selectivity of the NPY antagonist GR231118 (1229U91) for different NPY receptors.

Neuropeptide Y (NPY) is widely distributed throughout the central and peripheral nervous system and exerts a wide range of physiological responses by activating specific receptors. In this study we have characterized the potency of the high affinity peptide dimer antagonist, GR231118, to displace radiolabeled NPY/PYY from different tissues and cell lines expressing Y1 or Y2 receptors and from CHO cells stably transfected with human cDNA encoding for Y1, Y2 and Y4 receptors. GR231118 displays high affinity for Y1 and Y4 receptors, equal or better than that of NPY itself, while its activity is several fold weaker for Y2 receptors. Displacement of radiolabeled PYY from rat hypothalamic membranes by GR231118, reveals the existence of high and low affinity binding sites which may be equated to Y1 and Y2 receptors respectively suggesting that the compound maybe used as a tool to dissect central NPY receptors.

Role of tumor necrosis factor and nitric oxide in the cytotoxic effects of Clostridium difficile toxin A and toxin B on macrophages.

Clostridium difficile, the bacterium involved in antibiotic-associated colitis, produces two exotoxins, toxin A (TxA) and toxin B (TxB). Although these toxins are well recognized as being cytotoxic to several mammalian cell types, the mechanisms involved are not fully understood. The aim of the present investigation was to examine the cytotoxicity of TxA and TxB to peritoneal macrophages in culture and to investigate whether tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) are involved in the process. As a control, the effect of E. coli LPS was also investigated. TxA, TxB and LPS were dose-dependently cytotoxic to macrophage monolayers, with TxB being the most potent. All of the toxins stimulated the release of TNF-alpha from macrophages. TxB was again the most active in inducing this response. The TNF-alpha released appears to be involved in the action of LPS and TxA, but not of TxB, since a mAb against TNF-alpha inhibited the cytotoxicity of the former two but had no effect on the latter. NO is not involved in the effects of TxA and TxB since these toxins did not induce the production of this mediator in macrophages, even in the presence of IFN-gamma. In addition, L-imino-ethyl-L-ornithine (L-NIO), a NO synthase inhibitor, did not modify the macrophage death caused by TxA or TxB. Although LPS was able to induce the production of high amounts of NO, NO did not mediate the LPS cytotoxicity since L-NIO did not influence the degree of macrophage death caused by LPS. TxA and TxB therefore appear to exert cytotoxic effects on cultured macrophages by different mechanisms. TNF-alpha is involved in TxA and LPS-mediated cytotoxicity but not in the toxicity caused by TxB. NO is not involved in the killing action of any of these toxins.

Production of monoclonal antibodies against Clostridium difficile cytotoxin using immunosorbent binding bioassay procedure.

In the following study, a novel screening approach was used to develop monoclonal antibodies specific for toxin B of Clostridium difficile. The approach, which consisted of an immunosorbent binding bioassay (ISBBA), is based on antigen immunocapture by monoclonal antibodies and detection of biological activity. Our results showed ISBBA, which uses unpurified antigen, to be more sensitive than the neutralization assay and ELISA for the detection of toxin B antibody.

Subacute ethanol consumption reverses p-xylene-induced decreases in axonal transport.

Human exposure to organic solvents is often complicated by ethanol ingestion and the literature is replete with demonstrations of metabolic interactions between ethanol and organic solvents at a pharmacokinetic level. Because of the possible modulation of xylene toxicity by ethanol consumption, the present group of studies characterizes the effect of ethanol on the p-xylene-induced decrease in axonal transport in the rat optic system previously reported by our laboratory. Long-Evans, hooded, male rats were divided randomly into two groups: those receiving 10% ethanol in their drinking water and those receiving water only. These two groups were further subdivided into two groups which were either exposed by inhalation to 1600 ppm p-xylene for 6 h/day, 5 days/week for 8 exposure-days or were treated identically except that they were exposed to air while in the inhalation chambers. The ethanol-drinking rats were given ethanol 6 days prior to and on the days of the inhalation exposure. Immediately after removal from the inhalation chambers on the last exposure day, the animals were injected intraocularly with [35S]methionine and [3H]fucose to measure the synthesis and rapid axonal transport of proteins and glycoproteins, respectively, in the retinal ganglion cells. The animals were sacrificed 20 h later, and the amount of radioactivity in different areas of the retinal ganglion cells was determined by liquid scintillation counting. As in previous experiments, the xylene exposure group showed a significant reduction in axonal transport of proteins and glycoproteins, whereas the ethanol exposure alone produced no significant reductions in the transport of either proteins or glycoproteins. In the animals receiving both ethanol and xylene, however, the ethanol treatment prevented the decreased transport characteristic of the xylene only animals, i.e. in all areas of the optic projections the level of transport were similar to the level present in the control groups. These data suggest that the xylene-induced reduction in rapid axonal transport was reversed (or prevented) by subacute ethanol consumption.

The correlation between neurotoxic esterase inhibition and mipafox-induced neuropathic damage in rats.

The correlation between neuropathic damage and inhibition of neurotoxic esterase or neuropathy target enzyme (NTE) was examined in rats acutely exposed to Mipafox (N, N'-diisopropylphosphorodiamidofluoridate), a neurotoxic organophosphate. Brain and spinal cord NTE activities were measured in Long-Evans male rats 1 hr post-exposure to various dosages of Mipafox (ip, 1-15 mg/kg). These data were correlated with histologically scored cervical cord damage in a separate group of similarly dosed rats sampled 14-21 days post-exposure. Those dosages (greater than or equal to 10 mg/kg) that inhibited mean NTE activity in the spinal cord greater than or equal to 73% and brain greater than or equal to 67% of control values produced severe (greater than or equal to 3) cervical cord pathology in 85% of the rats. In contrast, dosages of Mipafox (less than or equal to 5 mg/kg) which inhibited mean NTE activity in spinal cord less than or equal to 61% and brain less than or equal to 60% produced this degree of cord damage in only 9% of the animals. These data indicate that a critical percentage of NTE inhibition in brain and spinal cord sampled shortly after Mipafox exposure can predict neuropathic damage in rats several weeks later.

Stimulation of enzyme secretion from isolated pancreatic acini by Clostridium difficile toxin B.

Exposure of isolated rat pancreatic acini to increasing concentrations (10 ng - 800 ng/ml) of toxin B from Clostridium difficile produced a biphasic effect on the rate of secretion of amylase, trypsinogen, and chymotrypsinogen. Whereas doses of toxin B from 10-30 ng/ml increased enzyme secretion by 15-20%, doses between 30 ng and 60 ng/ml showed a regression of this effect, whereafter the rate of secretion of amylase, trypsinogen, and chymotrypsinogen increased with increasing concentrations of the toxin. Toxin B concentration of 800 ng/ml enhanced amylase, trypsinogen and chymotrypsinogen secretion by 119%, 185% and 195%, respectively, when compared with the basal level. Stimulation of enzyme secretion by toxin B was not affected by the presence of either actinomycin-D or cycloheximide, at a concentration which inhibited acinar RNA or protein synthesis by 80-90%. Although toxin B as well as CCK8, carbachol and secretin by themselves caused significant stimulation in amylase, trypsinogen and chymotrypsinogen secretion from isolated pancreatic acini, toxin B together with either CCK8, carbachol or secretin produced no further augmentation in enzyme secretion than what was observed with the secretagogues alone. It is concluded that toxin B of Cl. difficile exerts a direct effect on pancreatic acinar cells as evidenced by stimulation of enzyme secretion.

Vascular and glomerular effects of Clostridium difficile toxin A peptide on the isolated rat kidney.

Toxin A peptide from Clostridium difficile caused damage and secretion in the intestinal mucosa. These effects are mediated in part by pro-inflammatory substances. In order to evaluate and compare the biologic effect of toxin A on renal vascular, glomerular and tubular functions, we studied this toxin in isolated rat kidneys. Isolated kidneys from adult male Wistar rats (260-320 g) were perfused with Krebs-Henseleit solution containing 60 mg/ml dialyzed bovine serum albumin. We studied the effect of toxin A peptide (3.2 x 10(-6) M, injected into perfusate) on glomerular filtration rate (GFR), urinary flow rate (UF) and total sodium reabsorption (TNa+, %). All experiments were preceded by a 30-min basal period, and in another group of kidneys the time course of the variables was followed without toxin infusion for unpaired control. Toxin A (TxA) reduced the perfusion pressure (PP), from PPcontrol/30min = 124.89 +/- 1.91 to PPTxA/120min = 88.13 +/- 5.1 mmHg (N = 6, P < 0.01) with a maximal effect at 120 min after toxin infusion. TxA also caused a significant decrease in GFR with maximal effect at 90 min after toxin infusion (GFRcontrol/30min = 0.53 +/- 0.05 to GFRTxA/90min = 0.30 + 0.05 ml min-1g-1; N = 6, P < 0.01). TxA did not alter renal tubular sodium transport when compared with a control without toxin infusion. In addition, toxin-treated kidneys caused a time-dependent increase in urinary flow from UFcontrol/30min = 0.16 +/- 0.08 to UFTxA/120min = 0.35 +/- 0.1 ml min-1g-1 (N = 6, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)