Automated direct antimicrobial susceptibility testing of microscopically screened urine cultures.

Two screening methods for urine microbiology are proposed: one in which the Gram-stained smear is used to detect significant bacteriuria, and another in which Autobac antibiotic susceptibility tests are performed directly on positive urine samples. Results on 1,350 specimens indicated that an average of 18 bacteria per oil immersion field were observed in the urine of patients with significant bacteriuria, and an average of less than 1 bacterium per oil immersion field was found in the urine of patients without significant bacteriuria. Direct susceptibility testing by Autobac proved to be rapid (3 h versus 24 h) and reliable (0.5 to 1.2% discrepancies).     

Comparative evaluation of three commercial products and counterimmunoelectrophoresis for the detection of antigens in cerebrospinal fluid.

Three commercial products and counterimmunoelectrophoresis were evaluated for their ability to detect microbial antigens of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in cerebrospinal fluid from 157 patients suspected of having meningitis. Thirty-four patients were diagnosed as having bacterial meningitis by culture, microscopy, or antigen detection. The overall results showed the following detection percentages; counterimmunoelectrophoresis, 76%; Phadebact CSF, 76%; Directigen, 82%, and Bactigen, 93%. The results with purified antigen revealed that latex agglutination was more sensitive than coagglutination, which in turn was more sensitive than counterimmunoelectrophoresis.     

Growth factor antagonists for the treatment of diabetic vascular complications

Diabetic vascular disease is characterised by altered vascular reactivity and blood flow, hyperpermeability, hyperproliferative responses, and increased extracellular matrix deposition in tissues that are sites of complications. These vascular functional and structural changes have been linked to excessive glucose metabolism in target organs via at least three pathophysiological mechanisms, including increased sorbitol (polyol) pathway activity, increased nonenzymatic glycation of vascular wall proteins, and increased protein kinase C (PKC) activity. These potential mechanisms of glucose toxicity remain the subject of intense scientific investigation, and therapies targeting each of them are being evaluated in clinical trials. It is becoming increasingly clear that excessive production of growth factors provides a common denominator linking these diverse mechanisms of glucose toxicity to the functional and structural vascular alterations associated with diabetes. Increased expression of vascular endothelial growth factor (VEGF) has been linked to increased metabolism of glucose via the sorbitol pathway, to nonenzymatic glycation, and to increased PKC activity, and appears to modulate the hyperpermeability and hyperproliferative responses of diabetes. Consequently, because of the unmet medical need and market size, numerous pharmaceutical and biotechnology companies have initiated research programmes evaluating growth factor antagonists as a potential therapeutic approach for treating complications associated with diabetic vascular disease. However, before growth factor antagonists can enter clinical testing, a number of important issues must be clarified, including the physiological effect of chronic growth factor inhibition, which appears to be necessary for ameliorating chronic vascular deterioration of diabetes, and administration routes, especially for protein-based therapies.     

Longitudinal neurological follow-up of a group of HIV-seropositive and HIV-seronegative hemophiliacs: results from the hemophilia growth and development study

BACKGROUND: Boys and young men with hemophilia treated with factor infusions before 1985 had a substantial risk of acquiring the human immunodeficiency virus (HIV) and the acquired immunodeficiency syndrome. This study was designed to assess the effects of HIV and hemophilia per se on neurological function in a large cohort of subjects with hemophilia, and to investigate the relationships between neurological disease and death during follow-up. METHODS: Three hundred thirty-three boys and young men (207 HIV seropositive and 126 HIV seronegative) were evaluated longitudinally in a multicenter, multidisciplinary study. Neurological history and examination were conducted at baseline and annually for 4 years. The relationship between neurological variables, HIV serostatus, CD4+ cell counts, and vital status at the conclusion of the study was examined using logistic regression models. RESULTS: The risks of nonhemophilia-associated muscle atrophy, behavior change, and gait disturbance increased with time in immune compromised HIV-seropositive subjects compared with HIV seronegative or immunologically stable HIV-seropositive subjects. The risk of behavior change in immune compromised HIV-seropositive hemophiliacs, for example, rose to 60% by year 4 versus 10% to 17% for the other study groups. Forty-five subjects (13.5%), all of whom were HIV seropositive, died by year 4. Subjects who died had had increased risks of hyperreflexia, nonhemophilia-associated muscle atrophy, and behavior change. CONCLUSIONS: These results indicate that immune compromised, HIV-seropositive hemophiliacs have high rates of neurological abnormalities over time and that neurological abnormalities were common among subjects who later died. By contrast, immunologically stable HIV-seropositive subjects did not differ from the HIV-seronegative participants. Hemophilia per se was associated with progressive abnormalities of gait, coordination, and motor function.     

Developmental toxicity of the dithiocarbamate pesticide sodium metam in zebrafish.

Sodium metam (NaM), a dithiocarbamate, is a general agricultural biocide applied prior to planting for the elimination of nematodes, soil pathogens, and weeds. There is a remarkable paucity of information about the mechanism of action and the risk that dithiocarbamates may pose to developing vertebrates. We have characterized NaM toxicity during early life stage exposure in zebrafish. Zebrafish embryos are most sensitive to NaM exposure during gastrulation and early segmentation (4-14 hours post fertilization, hpf). For mortality, the dose response curve is steep with an LC(50) estimate of 1.95 microM (248 ppb) at 48 hpf. The most notable malformation among surviving embryos was a severely twisted notochord, which became evident by 24 hpf. Surprisingly, this notochord defect was not immediately lethal and the animals continued to grow despite delays in hatching, apparent paralysis, and an inability to feed. We have characterized the notochord malformation using histological and in situ hybridization techniques. collagen 2a1 mRNA expression is normally localized to the notochord sheath cells at 24 hpf, whereas in NaM-exposed embryos it is misexpressed in the notochord cells. Histological staining and myoD expression indicate that the myotomes of the NaM-exposed embryos are less defined, compacted and block-shaped compared to controls. The degradation product of NaM, methyl isothiocyanate (MITC), causes similar malformations at similar concentrations as NaM, suggesting that MITC or another common product may be the active toxicant. Our results indicate that developing zebrafish are sensitive to NaM and MITC and we believe that this model is ideal to elucidate the molecular mechanism(s) and etiology of NaM toxicity in vertebrates.     

Vascular dysfunction induced by AGE is mediated by VEGF via mechanisms involving reactive oxygen species, guanylate cyclase, and protein kinase C

OBJECTIVE: These experiments were designed to elucidate mechanisms mediating vascular dysfunction induced by advanced glycation end products (AGEs). METHODS: Skin chambers were mounted on the backs of Sprague-Dawley rats and 1 week later, granulation tissue that formed in the bottom of the chamber was exposed twice daily for 7 days to glycated rat serum albumin in the presence and absence of inhibitors of reactive oxygen intermediates, nitric oxide synthase and guanylate cyclase, protein kinase C (PKC), and a neutralizing vascular endothelial growth factor (VEGF) antibody. Vascular (125)I-albumin clearance and blood flow were quantified by use of a double isotope-dilution technique and radiolabeled microspheres, respectively. RESULTS: Albumin permeation and blood flow were increased dose-dependently to a maximum of 2 to 3 times controls by increasing the extent of glucose modification, the concentration, or the duration of exposure to glycated albumin. These increases were significantly attenuated by probucol and superoxide dismutase; N(G)-nitro-L-arginine-methyl ester (L-NAME), a nitric oxide synthase inhibitor; LY83583, a guanylate cyclase inhibitor; and LY333531, a beta-isoform-selective protein kinase C inhibitor. A neutralizing VEGF monoclonal antibody also markedly attenuated the permeability and blood flow increases induced by glycated albumin. CONCLUSIONS: These observations indicate potentially important roles for oxygen free-radicals and nitric oxide in mediating permeability and blood flow changes induced by glycated proteins via mechanisms involving increased protein kinase C activity and VEGF production. Striking similarities in the mechanism by which hyperglycemia and glycated proteins induce vascular dysfunction suggest that a common pathway mediates effects of these different metabolic imbalances on vascular dysfunction.     

Endothelin-mediated remodeling in aortas of diabetic rats

BACKGROUND: Smooth muscle cells proliferation and extracellular matrix (ECM) protein deposition are key features of diabetic macroangiopathy. In the present study, we have studied the role of endothelin(A) (ET(A)) receptor, the predominant receptor on smooth muscle cells, in diabetes-induced vascular hypertrophy and remodeling. METHODS: Streptozotocin-induced diabetic rats were administrated a selective ET(A) receptor antagonist, TBC3214, for 26 weeks. Following treatment, aortas were harvested and subjected to gene expression and morphometric analyses. We quantified fibronectin (FN) and plasminogen activator inhibitor-1 (PAI-1) expression as indicators of increased ECM protein synthesis. ET-1, ET-3, transforming growth factor-beta1 (TGF-beta1) and angiotensinogen mRNA levels were measured to elucidate genes involved in FN expression. We have investigated an embryonic splice variant of FN, oncofetal FN, and nonmuscle myosin heavy chain (SMemb) as vascular remodeling indicators. RESULTS: Our results show that diabetes leads to upregulation of FN, PAI-1, ET-1, ET-3, TGF-beta1 and angiotensinogen mRNA levels in association with increased medial thickness. Immunohistochemical analyses revealed concurrent protein level changes. Diabetes also upregulated oncofetal FN and SMemb mRNA levels. Treatment with TBC3214 attenuated the mRNA levels of several genes and prevented increased medial thickness. CONCLUSIONS: These results indicate that diabetes-induced vascular hypertrophy and remodeling is associated with reexpression of embryonic forms of FN and myosin heavy chain. Such changes are ET-dependent and may be mediated via TGF-beta1 and angiotensin.     

Sj?gren-Larsson syndrome: inherited defect in the fatty alcohol cycle

We investigated fatty alcohol metabolism in eight patients with Sj?gren-Larsson syndrome, and in nine obligate heterozygotes. Fatty alcohol: nicotinamide-adenine dinucleotide oxidoreductase (FAO) activity was deficient in cultured skin fibroblasts (mean 18% of normal, n = 8) and peripheral blood leukocytes (mean 22% of normal, n = 3) from patients with Sj?gren-Larsson syndrome. The palmitoyl coenzyme A-inhibitable component of FAO activity was decreased to 10% and 15% of normal in fibroblasts and leukocytes, respectively, of patients with Sj?gren-Larsson syndrome. Most affected patients accumulated long-chain fatty alcohol in plasma, with a greater relative accumulation of octadecanol (mean threefold greater than normal) than hexadecanol (mean twofold greater than normal). Erythrocyte lipid alkyl ether linkages derived from hexadecanol were slightly increased in three of four patients. Fibroblasts and leukocytes from heterozygotes with Sj?gren-Larsson syndrome showed mean FAO activities that were intermediate between those seen in homozygotes and in normal control subjects. The heterozygotes had normal fatty alcohol concentrations in plasma. These studies demonstrate FAO deficiency in patients with Sj?gren-Larsson syndrome, and suggest that accumulation of fatty alcohol or its metabolic products may be important in the pathogenesis of this disorder.     

Evaluation of estrogenic activity from a municipal wastewater treatment plant with predominantly domestic input

The purpose of this study was to survey estrogenic releases from two primarily domestic wastewater treatment plants over three seasons (1996-1999). Mature male channel catfish were maintained at two sites within each WWTP and a reference site for 21 days. Estrogenic activity of effluent was assessed by the Yeast Estrogen Screen (YES) assay (in 1999) and the expression of the female egg yolk precursor protein, vitellogenin (Vtg) in caged male channel catfish (1996-1998). Serum Vtg of animals exposed at WWTP-A was induced 220% above reference values in the Fall of 1996 and 480% in Spring of 1997. In animals exposed to effluent of WWTP-B, serum Vtg was elevated 370% in Spring of 1997 and 480% in Fall of 1997 relative to fish held in a reference location. Serum 17-beta-estradiol (E2) levels were also significantly elevated 13 and 16-fold in the Fall 1997 and Summer 1998 in the fish exposed to WWTP-A effluent. A 13.5-fold increase in serum E2 was observed in fish exposed to WWTP-B during Fall 1997. Utilizing an E2 concentration-Vtg response curve generated in the laboratory, effluent from both plants (in 1997 and 1998) had estrogen equivalent values ranging from 23 to 123 ng/l E2 equivalents. These values were comparable with YES values obtained from 1999, which indicated the presence of 21 to 147 ng/l E2 equivalents. E2 was responsible for 3 (fall) to 100% (summer) of the YES activity. Glucuronides of E2 were also observed in the treated effluent. These studies indicate that variable estrogenic activity is present in municipal wastewater resulting from domestic activities and that this activity may be significantly altered by environmental factors.