Clostridium difficile infection.

Infection of the colon with the Gram-positive bacterium Clostridium difficile is potentially life threatening, especially in elderly people and in patients who have dysbiosis of the gut microbiota following antimicrobial drug exposure. C. difficile is the leading cause of health-care-associated infective diarrhoea. The life cycle of C. difficile is influenced by antimicrobial agents, the host immune system, and the host microbiota and its associated metabolites. The primary mediators of inflammation in C. difficile infection (CDI) are large clostridial toxins, toxin A (TcdA) and toxin B (TcdB), and, in some bacterial strains, the binary toxin CDT. The toxins trigger a complex cascade of host cellular responses to cause diarrhoea, inflammation and tissue necrosis - the major symptoms of CDI. The factors responsible for the epidemic of some C. difficile strains are poorly understood. Recurrent infections are common and can be debilitating. Toxin detection for diagnosis is important for accurate epidemiological study, and for optimal management and prevention strategies. Infections are commonly treated with specific antimicrobial agents, but faecal microbiota transplants have shown promise for recurrent infections. Future biotherapies for C. difficile infections are likely to involve defined combinations of key gut microbiota.

New Commercially Available IgG Kits and Time-Resolved Fluorometric IgE Assay for Diagnosis of Allergic Bronchopulmonary Aspergillosis in Patients with Cystic Fibrosis.

Allergic bronchopulmonary aspergillosis (ABPA) is difficult to diagnose; diagnosis relies on clinical, radiological, pathological, and serological criteria. Our aim was to assess the performance of two new commercially available kits and a new in-house assay: an Aspergillus fumigatus enzyme-linked immunosorbent assay (ELISA) IgG kit (Bordier Affinity Products), an Aspergillus Western blotting IgG kit (LDBio Diagnostics), and a new in-house time-resolved fluorometric IgE assay (dissociation-enhanced lanthanide fluorescent immunoassay, or DELFIA) using recombinant proteins from an Aspergillus sp. recently developed by our laboratory for ABPA diagnosis in a retrospective study that included 26 cystic fibrosis patients. Aspergillus fumigatus-specific IgG levels measured by a commercial ELISA kit were in accordance with the level of precipitins currently used in our lab. The ELISA kit could accelerate and help standardize ABPA diagnosis. Aspergillus fumigatus-specific IgE levels measured by ImmunoCAP (Phadia) with A. fumigatus M3 antigen and by DELFIA with a purified protein extract of A. fumigatus were significantly correlated (P < 10(-6)). The results with recombinant antigens glucose-6-phosphate isomerase and mannitol-1-phosphate dehydrogenase were encouraging but must be confirmed with sera from more patients. The DELFIA is an effective tool that can detect specific IgE against more fungal allergens than can be detected with other commercially available tests.

[Erythrocyte sorbitol levels in patients with thyrotoxicosis].

Hyperglycemia and impaired glucose tolerance are frequently observed in patients with hyperthyroidism. However, little is known about whether altered polyol metabolism in hyperthyroidism is present or not. To examine changes in polyol metabolism in hyperthyroidism, we investigated changes in erythrocyte sorbitol, glyceraldehyde reductase (GAR) and sorbitol dehydrogenase (SDH) activities during hyperthyroid and euthyroid states in patients with thyrotoxicosis. Mean levels of erythrocyte sorbitol and GAR were 32.0 +/- 1.6nM/g.Hb and 147.1 +/- 0.3mU/g.Hb, respectively. In thyrotoxic patients in a hyperthyroid state, these values were significantly higher than those in euthyroid controls. Mean level of erythrocyte SDH in thyrotoxic patients was weak but was significantly increased in comparison with that of euthyroid controls. However, mean levels of erythrocyte sorbitol and GAR were remarkably reduced to 23.6 +/- 1.4nM/g.Hb and 125.3 +/- 4.6mU/g.Hb in thyrotoxic patients in a euthyroid state after treatment with anti-thyroid drugs or by subtotal thyroidectomy. Mean level of SDH, on the other hand, was increased after the treatment. In addition, positive correlations were observed between the level of erythrocyte sorbitol or GAR, and the level of free thyroxine(FT4) or free triiodothyronine(FT3). A negative correlation was observed between the level of erythrocyte SDH and the level of FT4 or FT3. These results suggest that the level of erythrocyte sorbitol may be increased through direct acceleration of erythrocyte GAR activity by increased thyroid hormone levels in patients with thyrotoxicosis.

Studies on clinical markers of diabetes mellitus. 6. Red blood cell sorbitol and diabetic complications.

The relationship between red blood cell sorbitol content and diabetic complications (cataract, retinopathy, neuropathy, and nephropathy) was examined in 23 non-insulin-dependent diabetic (NIDD) patients. Sorbitol content was abnormally high in 21 cases out of 23 NIDD patients. Sorbitol content in the non-neuropathy group and neuropathy group was 47.3 +/- 11.9 and 59.6 +/- 23.6 nmol/gHb, respectively. In the non-cataract group and cataract group, it was 49.0 +/- 17.6 and 66.0 +/- 23.5 nmol/gHb, respectively. The contents in the Scott I group and Scott II + III group were 54.9 +/- 20.7 and 58.7 +/- 24.0 nmol/gHb, respectively. Sorbitol content in the non-nephropathy group and nephropathy group was 52.8 +/- 19.8 and 61.1 +/- 21.9 nmol/gHb, respectively. The possibility that glyceraldehyde reductase (GAR) and sorbitol dehydrogenase (SDH) levels in red blood cells are also useful indicators of the presence of diabetic complications is strongly suggested.

Variations in serum sorbitol dehydrogenase, aspartate transaminase, and isoenzyme 5 of lactate dehydrogenase activities in horses given carbon tetrachloride.

Seven horses were given 0.5 mg of carbon tetrachloride/kg of body weight via a nasogastric tube. Subsequent hepatocellular damage was monitored by serum enzyme determinations of sorbitol dehydrogenase, isoenzyme 5 of lactate dehydrogenase, and aspartate transaminase activities. Creatinine kinase activity was evaluated as an indicator of muscle cell damage. Sorbitol dehydrogenase, isoenzyme 5 of lactate dehydrogenase, and aspartate transaminase activities were significantly (P less than 0.05) increased by 24 hours after carbon tetrachloride administration. Isoenzyme 5 of lactate dehydrogenase and sorbitol dehydrogenase activities returned to baseline several days before aspartate transaminase activity returned to baseline. Creatine kinase activity remained unchanged.

Plasma sorbitol dehydrogenase determination in experimental hepatotoxicity using the Abbott Bichromatic Analyzer.

An automated single reagent micro-assay for the determination of sorbitol dehydrogenase (EC 1.1.1.14) catalytic activity concentration in mouse plasma was developed for the Abbott Bichromatic Analyzer (ABA-100). The kinetic rate determination was linear up to 250 U/l, had a CV of 2.65%, and required only 25 microliters of sample. Experimentally induced hepatotoxicity in the mouse by acetaminophen produced a dose dependent increase in blood sorbitol dehydrogenase activity.

Human erythrocyte sorbitol metabolism and the role of sorbitol dehydrogenase.

Rapid fluctuation of erythrocyte sorbitol in response to the changes in plasma glucose concentration has been reported from clinical evidence. We performed more extensive in vitro and in vivo studies focussing on how fast sorbitol was accumulated and how fast the accumulated sorbitol was oxidised in response to the changes in ambient glucose concentration. Incubation studies of intact erythrocytes from healthy subjects and diabetic patients showed that erythrocyte sorbitol increased rapidly in response to increased ambient glucose concentration and the accumulated sorbitol easily decreased according to the rapid reduction of ambient glucose concentration. In addition, the higher the glucose concentration in the medium, the more erythrocytes could accumulate sorbitol. The rapid response of sorbitol levels to ambient glucose concentration was further confirmed by the results of a 75 g oral glucose tolerance test in non-diabetic subjects and diabetic patients with gastrectomy, who showed marked early hyperglycaemia caused by rapid absorption of ingested glucose and subsequent rapid reduction of plasma glucose concentration (erythrocyte sorbitol levels changed concomitantly). These findings strongly indicate that the measurement of erythrocyte sorbitol is not useful as an index of medium or long term glycaemic control.

[Diagnostic and prognostic role of organ-specific plasma enzymes in the acute period of cerebral circulatory disorders]. / Diagnosticheskaia i prognosticheskaia rol' organospetsificheskikh fermentov plazmy krovi v ostrom periode narusheniia mozgovogo krovoobrashcheniia.

Sixty-two patients with ischemic stroke were studied over time (1st-12th day) for the activity of enzymes and isoenzymes of energy metabolism (CPK, LDH, SDH). It has been shown that the brain-specific CPK BB-isoenzyme may serve as a marker of the severity and extension of brain damage while the aerobic LDH fractions may be useful in evaluation of the reversibility of hypoxic changes in the brain. The authors consider the employment of the studied enzymes for the prognosis of vascular and tissue damage.

Effect of cyclopiazonic acid on delayed hypersensitivity to Mycobacterium tuberculosis, complement activity, serum enzymes, and bilirubin in guinea pigs.

Cyclopiazonic acid (CPA) was given daily to groups of guinea pigs at doses of 0.00625, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, and 1.95 mg/day for 30 days. All guinea pigs were sensitized and survivors were skin tested twenty-five days later with Mycobacterium tuberculosis. Mortalities occurred only in the two greatest dose groups. Signs of disease included anorexia, roughened hair coat, diarrhea and incoordination. The major histopathologic changes occurring in these two groups included hepatocellular vacuolar degeneration and necrosis of the gastric mucosa with infiltration of neutrophils in the deep gastric mucosa. CPA did not affect cutaneous hypersensitivity to M. tuberculosis, complement activity, serum glycocholic acid concentrations or weight gains. There were increases in aspartate aminotransferase, alanine aminotransferase, and sorbitol dehydrogenase concentrations in the serum of guinea pigs in the two greater dose groups, but no changes were found in serum concentrations of SAP. There was a slight increase in the serum bilirubin concentrations in the greater dose groups.

Concentration-related changes in blood and tissue parameters of hepatotoxicity and their interdependence in rats exposed to bromobenzene and 1,2-dichlorobenzene.

Liver damage resulting from 4 h exposure to bromobenzene (BB) (146-957 ppm) and 1,2-dichlorobenzene (DCB) (245-739 ppm) as model toxicants was evaluated in rats. The modifications considered were the increases in serum glutamate dehydrogenase (GLDH) and sorbitol dehydrogenase (SDH) activities and the decreases in centrolobular liver-cell glucose-6-phosphatase (G6-Pase) staining intensity. A linear inverse relationship was established between the logarithmic values of blood enzyme activities and liver G6-Pase staining intensity. In addition, the levels of exposure to each test chemical were found to be linearly related to liver G6-Pase staining intensity and to the logarithmic values of blood enzyme activities.

Hyperglycemia-induced activation of human erythrocyte aldose reductase and alterations in kinetic properties.

Incubation of human erythrocytes with varying concentrations of glucose resulted in a several-fold increase in aldose reductase (alditol:NADP+ 1-oxidoreductase, EC 1.1.1.21) activity as determined by the rate of NADPH oxidation and the rate of sorbitol formation. As compared to aldose reductase from human erythrocytes not incubated with glucose (native enzyme), aldose reductase from 30 mM glucose-incubated erythrocytes (activated enzyme) exhibited altered kinetic and inhibition properties. Native enzyme showed biphasic kinetics with substrates (glucose and glyceraldehyde), was strongly inhibited by 15 microM ADP, 1,3-diphosphoglycerate, 2,3-diphosphoglycerate and 3-phosphoglycerate, and aldose reductase inhibitors such as sorbinil and alrestatin. The activated enzyme, on the other hand, exhibited monophasic kinetics, low Km for substrates, was not inhibited by the phosphorylated intermediates, and was less susceptible to inhibition by aldose reductase inhibitors. In erythrocytes of the diabetic subjects, we have found an excellent correlation between aldose reductase activity and plasma glucose levels and have observed that whenever the blood glucose level was higher than 15 mM, all of the erythrocyte aldose reductase was present in the activated form and exhibited properties similar to those observed with aldose reductase obtained from 30 mM glucose-incubated erythrocytes.

Purification and properties of aldose reductase and aldehyde reductase II from human erythrocyte.

Aldose reductase (EC 1.1.1.21) and aldehyde reductase II (L-hexonate dehydrogenase, EC 1.1.1.2) have been purified to homogeneity from human erythrocytes by using ion-exchange chromatography, chromatofocusing, affinity chromatography, and Sephadex gel filtration. Both enzymes are monomeric, Mr 32,500, by the criteria of the Sephadex gel filtration and polyacrylamide slab gel electrophoresis under denaturing conditions. The isoelectric pH's for aldose reductase and aldehyde reductase II were determined to be 5.47 and 5.06, respectively. Substrate specificity studies showed that aldose reductase, besides catalyzing the reduction of various aldehydes such as propionaldehyde, pyridine-3-aldehyde and glyceraldehyde, utilizes aldo-sugars such as glucose and galactose. Aldehyde reductase II, however, did not use aldo-sugars as substrate. Aldose reductase activity is expressed with either NADH or NADPH as cofactors, whereas aldehyde reductase II can utilize only NADPH. The pH optima for aldose reductase and aldehyde reductase II are 6.2 and 7.0, respectively. Both enzymes are susceptible to the inhibition by p-hydroxymercuribenzoate and N-ethylmaleimide. They are also inhibited to varying degrees by aldose reductase inhibitors such as sorbinil, alrestatin, quercetrin, tetramethylene glutaric acid, and sodium phenobarbital. The presence of 0.4 M lithium sulfate in the assay mixture is essential for the full expression of aldose reductase activity whereas it completely inhibits aldehyde reductase II. Amino acid compositions and immunological studies further show that erythrocyte aldose reductase is similar to human and bovine lens aldose reductase, and that aldehyde reductase II is similar to human liver and brain aldehyde reductase II.