Pioglitazone Enhances the Beneficial Effects of Glucocorticoids in Experimental Nephrotic Syndrome.

Glucocorticoids are the primary therapy for nephrotic syndrome (NS), but have serious side effects and are ineffective in ~20-50% of patients. Thiazolidinediones have recently been suggested to be renoprotective, and to modulate podocyte glucocorticoid-mediated nuclear receptor signaling. We hypothesized that thiazolidinediones could enhance glucocorticoid efficacy in NS. We found that puromycin aminonucleoside-induced proteinuria in rats was significantly reduced by both high-dose glucocorticoids (79%) and pioglitazone (61%), but not low-dose glucocorticoids (25%). Remarkably, pioglitazone + low-dose glucocorticoids also reduced proteinuria (63%) comparably to high-dose glucocorticoids, whereas pioglitazone + high-dose glucocorticoids reduced proteinuria to almost control levels (97%). Molecular analysis revealed that both glucocorticoids and pioglitazone enhanced glomerular synaptopodin and nephrin expression, and reduced COX-2 expression, after injury. Furthermore, the glomerular phosphorylation of glucocorticoid receptor and Akt, but not PPARγ, correlated with treatment-induced reductions in proteinuria. Notably, clinical translation of these findings to a child with refractory NS by the addition of pioglitazone to the treatment correlated with marked reductions in both proteinuria (80%) and overall immunosuppression (64%). These findings together suggest that repurposing pioglitazone could potentially enhance the proteinuria-reducing effects of glucocorticoids during NS treatment.

Dithiothreitol enhances Listeria monocytogenes mediated cell cytotoxicity.

The hybridoma Ped-2E9 based cytotoxicity assay was developed to distinguish virulent from avirulent Listeria species in 6 hr. The cytotoxicity effect on Ped-2E9 was reported to be primarily due the cytolytic action of listeriolysin O (LLO), produced by L. monocytogenes. In this study, the effect of a reducing agent, dithiothreitol (DTT, 0-2 mM) that is known to activate LLO was investigated to make the Ped-2E9 based cytotoxicity assay an even more sensitive and rapid. Also, we examined the effect of fetal bovine serum (FBS, 0-50%), a common ingredient of tissue culture media on cytotoxicity. A DTT concentration of 0.5 mM gave an optimum cytotoxicity effect, which could be measured by both alkaline phosphatase (AP) and lactate dehydrogenase (LDH) assays in just 1.5-2 hr. FBS, at levels between 10 to 50%, significantly inhibited Listeria-mediated cytotoxicity. Concentrated culture filtrates from L. monocytogenes or LLO producing recombinant L. innocua (prfA+ hlyA+) strain also caused cytotoxicity effects, which were observed by scanning electron microscopy or a cytotoxicity assay in 2-3 hr. Interestingly, addition of DTT to culture filtrates produced 100% cell cytotoxicity in just 15 min. This indicated that LLO activity, which is responsible for Ped-2E9 cytotoxicity, was augmented several folds with the addition of a reducing agent. Examination of Listeria isolates belonging to different serogroups from clinical sources or naturally contaminated meat products with DTT gave cytotoxicity results in 2 hr, which were comparable to the 5-hr assay analyzed concurrently without DTT. These results indicated that DTT, which activated the LLO, could be used in the cytotoxicity assay to enhance Listeria-mediated Ped-2E9 cell cytotoxicity. This knowledge will greatly assist us to develop a user-friendly rapid assay to screen cytopathogenic properties of Listeria species.

Education of physicians-in-training can decrease the risk for vascular catheter infection.

BACKGROUND: Procedure instruction for physicians-in-training is usually nonstandardized. The authors observed that during insertion of central venous catheters (CVCs), few physicians used full-size sterile drapes (an intervention proven to reduce the risk for CVC-related infection). OBJECTIVE: To improve standardization of infection control practices and techniques during invasive procedures. DESIGN: Nonrandomized pre-post observational trial. SETTING: Six intensive care units and one step-down unit at Wake Forest University Baptist Medical Center, Winston-Salem, North Carolina. PARTICIPANTS: Third-year medical students and physicians completing their first postgraduate year. INTERVENTION: A 1-day course on infection control practices and procedures given in June 1996 and June 1997. MEASUREMENTS: Surveys assessing physician attitudes toward use of sterile techniques during insertion of CVCs were administered during the baseline year and just before, immediately after, and 6 months after the first course. Preintervention and postintervention use of full-size sterile drapes was measured, and surveillance for vascular catheter-related infection was performed. RESULTS: The perceived need for full-size sterile drapes was 22% in the year before the course and 73% 6 months after the course (P < 0.001). The perceived need for small sterile towels at the insertion site decreased reciprocally (P < 0.001). Documented use of full-size sterile drapes increased from 44% to 65% (P < 0.001). The rate of catheter-related infection decreased from 4.51 infections per 1000 patient-days before the first course to 2.92 infections per 1000 patient-days 18 months after the first course (average decrease, 3.23 infections per 1000 patient-days; P < 0.01). The estimated cost savings of this 28% decrease was at least $63000 and may have exceeded $800000. CONCLUSIONS: Standardization of infection control practices through a course is a cost-effective way to decrease related adverse outcomes. If these findings can be reproduced, this approach may serve as a model for physicians-in-training.

Surface protein p104 is involved in adhesion of Listeria monocytogenes to human intestinal cell line, Caco-2.

Adhesion of Listeria monocytogenes to intestinal endothelial cells is an important initial event in the pathogenesis of infection which is not well understood. The suggestion has been made that some proteins, including internalin and actin polymerisation protein (ActA), and carbohydrate molecules mediate, at least in part, the adhesion of listeria to certain cultured mammalian cells. This study investigated the role of a L. monocytogenes cell-surface protein of 104 kDa (p104) in adhesion to human intestinal enterocyte-like Caco-2 cell lines by transposon (Tn916) mutagenesis and a p104-specific monoclonal antibody (MAb-H7). Genotypic and phenotypic characteristics of Tn916-transformed L. monocytogenes strains, AAMU530 and AAMU572, revealed that these strains did not express p104, and the transposon had been inserted at a single locus in the structural gene. Strains AAMU530 and AAMU572 yielded only 10 and 6.3% adhesion to Caco-2 cells. Coating of L. monocytogenes and L. innocua wild-type strains with MAb-H7 reduced adhesion to Caco-2 cells from 100% to 50 and 45%, respectively, whereas on isotype control MAb EM-7G1 had no effect. Western blot analysis with MAb-H7 indicated that p104 is present in all Listeria spp. except in L. grayi. Furthermore, p104 is also present in internalin (BUG8) and ActA (LUT12) deficient strains, suggesting that p104 is indeed different from internalin or ActA proteins. Cytotoxicity analysis of strains AAMU530 and AAMU572 demonstrated that these strains, although haemolytic and phospholipase-positive, were avirulent when tested with a hybridoma B-lymphocyte cell line. Loss of virulence could be attributed to the interruption of adhesion of mutant strains to the hybridoma cell line. These results strongly suggest that p104 is an adhesion factor in L. monocytogenes and possibly in other Listeria species and is involved in adhesion to intestinal cells.

The long-term outcome of cognitive behaviour therapy for adults in routine clinical practice.

It is recognised that there is a comparative lack of evidence on the long-term outcome of cognitive behavioural therapy for patients seen in ordinary clinical settings, as opposed to research trials. This paper presents some data on the outcome of 36 adult patients followed up an average of nearly two years after the end of therapy in a National Health Service CBT clinic. Whilst the vagaries of data collection in clinical practice mean that the generalisability of these results is still tentative, they suggest that the outcome at the end of treatment was generally well-maintained over the follow-up period, with most patients maintaining fairly stable scores on the measures used, and the rest roughly equally divided between those who deteriorated and those who improved further. A high proportion of patients at follow-up reported that they were still using various CBT strategies which they had learnt during therapy.

Alkaline phosphatase release assay to determine cytotoxicity for Listeria species.

A simple cytotoxicity assay for Listeria species was developed by assaying alkaline phosphatase (AP) release from an infected hybrid B lymphocyte (Ped-2E9) line. Eight of eight L. monocytogenes and six of 11 L. ivanovii strains induced significantly high AP release from Ped-2E9 cells compared to five other L. ivanovii strains and other Listeria spp. In contrast, all L. monocytogenes and L. ivanovii test strains showed high release of lactate dehydrogenase (LDH) activity from Ped-2E9 cells. The molecular mass of AP was estimated to be about 128-165 kDa, suggesting severe membrane damage in Ped-2E9 cells due to Listeria infection. The data presented here indicate that AP assay could be used over LDH assay to detect Listeria-induced cell cytotoxicity.

Effects of neutralizing on intrusive thoughts: an experiment investigating the etiology of obsessive-compulsive disorder.

A large sample of non-clinical subjects were screened and those who reported experiencing relatively frequent intrusive thoughts with associated neutralizing were selected. These subjects were randomly allocated to one of two conditions: both groups listened to repeated recorded presentations of one of their intrusive thoughts and were then required either to (a) neutralize it, or (b) distract themselves for a similar period. Ratings of discomfort were taken during this procedure (first phase), and during identical presentations of the same thought without neutralizing or distracting (second phase). Results showed that the group who neutralized during the first phase experienced significantly more discomfort during the second phase and significantly stronger urges to neutralize and distract. There was also evidence that engaging in neutralizing responses during the first phase made it difficult to stop neutralizing during the second phase. The results are considered in the context of the cognitive-behavioural hypothesis that obsessional disorders develop as a consequence of neutralizing normal intrusive thoughts.

Frozen stored murine hybridoma cells can be used to determine the virulence of Listeria monocytogenes.

Murine hybridoma cells, designated Ped-2E9, when stored up to 60 days at -196 degrees C or up to 48 days at -80 degrees C, gave results equivalent to those for freshly grown murine hybridoma cells in an in vitro pathogenicity assay of Listeria species. Thus, laboratories do not need to have their own tissue culture facilities to maintain the hybridoma cells for the assay described.

A six-hour in vitro virulence assay for Listeria monocytogenes using myeloma and hybridoma cells from murine and human sources.

An in vitro cell culture assay using myeloma cells and hybrid lymphocytes was developed which detected pathogenic Listeria strains in just 6 h. Three separate hybridoma cell lines, murine Ped-2E9 and EM-7G1 and human RI.37 and murine myeloma NS1 cells, proved equally sensitive in responding to virulent Listeria species. Listeria monocytogenes along with other Listeria spp., collected from food and clinical sources, were inoculated at 10(8) cfu/ml into a suspension of Ped-2E9 (10(6)/ml). Pathogenic Listeria spp. killed 80% of hybridoma cells by 4 h, as determined by trypan blue exclusion test. Conversely, none of all nonpathogenic Listeria spp. killed the hybridoma cells. Ped-2E9 cells exposed to three strains of L. monocytogenes strains showed 96-97.5% death in 6 h measured by trypan blue staining and release of 91-97% of lactate dehydrogenase (LDH) enzyme. RI.37 cells showed similar results. A multiplicity of exposure (MOE) of 100 L. monocytogenes to 1 hybridoma cell or of 10:1 killed about 80% of the hybridoma cells in 4 or 6 h respectively. The in vitro virulence assay of L. monocytogenes with hybridoma cells compared favorably with the immunocompromised mouse model, yielding results in 6 h instead of 3 days. Intracellular L. monocytogenes and L. innocua were not recovered from Ped-2E9 hybridoma cells after 2 or 4 h of exposure. However, attachment of both L. monocytogenes and L. innocua cells on Ped-2E9 cell surfaces were observed under epifluorescence microscopy. Direct contact of hemolysin positive L. monocytogenes with hybridoma cells is essential to cause death, since hybridoma cells were not killed when they were separated from the growing bacteria by a 0.45 microns filter.

Do psychosocial factors influence outcome in severely depressed female psychiatric in-patients?

Fifty-nine women admitted to hospital because of severe depression were studied prospectively during hospital admission and nine months following hospital discharge in order to identify psychosocial and illness factors associated with prognosis. Outcome was measured in terms of both depression scores and recovery at the time of follow-up. In keeping with the findings of other studies, the outcome was often poor, with only 54% having recovered nine months after discharge, poorer outcome being associated with more negative self-esteem measured when the women were depressed and with suicidal ideas. The findings indicate that in severely depressed women likely to be admitted to hospital, psychosocial factors may have less relevance to outcome, at least in the short term, than in less severely depressed patients studied in community or out-patient samples.

Glutathione S-transferase in intestine, liver and hepatic lesions of mummichog (Fundulus heteroclitus) from a creosote-contaminated environment.

Cytosolic glutathione S-transferase (GSH transferase) activity towards 1-chloro-2,4-dinitrobenzene (CDNB) was elevated approximately three to four-fold in intestine and liver of mummichog (Fundulus heteroclitus) collected from a creosote-contaminated site in the Elizabeth River, Virginia. Intestinal GSH transferase activity at the most heavily contaminated site, at a moderately contaminated site and at a relatively clean site averaged 3.64, 2.83 and 1.11µmoles/min/mg respectively, while values for liver at these sites averaged 2.84, 1.75 and 0.93µmoles/min/mg. In addition, densitometric tracings of sodium dodecylsulfate-polyacrylamide gels of intestine and liver cytosol revealed a similar trend in the staining intensity of a 25.8 kD protein band, which lies within the molecular weight range of GSH transferase subunits. Activity in putative preneoplastic and neoplastic hepatic lesions of fish collected from the creosote-contaminated site was not significantly different from that of adjacent normal tissue. In the laboratory, dietary betanaphthoflavone (ßNF) treatment resulted in a three-fold increase in intestinal GSH transferase. Hepatic GSH transferase activity in the same fish was not affected by dietary ßNF although hepatic monooxygenase activity, measured as ethoxyresorufin O-deethylase (EROD), was. The results of this study indicate a response of the intestinal detoxification system to environmental contaminants and supports previous studies on the importance of intestinal metabolism of foreign compounds. Further, our results indicate the trend towards elevated GSH transferase in liver of feral fish could not be attributed to a cancerous disease state in these fish but indicates chemical induction in this organ as well.

Behaviour therapy and obsessional ruminations: can failure be turned into success?

This paper considers the possible reasons for the relative failure of behavioural treatment for 'ruminations' (obsessional thoughts without overt compulsive behaviour). It is argued that this problem is due to an inadequate conceptualisation of the nature of ruminations and that as a result, exposure and response prevention have not been applied effectively. Effective treatment of ruminations requires systematically applied exposure in conjunction with the identification and prevention of covert compulsive behaviours. The practical problems of implementing 'covert exposure and response prevention' are considered and a form of habituation training is described, which is designed to overcome these inadequacies. A series of single case studies using this technique is presented.

Ultrasound artifacts affecting the diagnosis of breast masses.

An investigation of the outcome of over 1600 breast ultrasound examinations revealed a number of scanning artifacts. Artifactual echoes in cysts resulted from inappropriate scanning factors, including focal zone placement, gain, TGC, and gray scale, and from partial volume effect. The absence of a sign suspicious for carcinoma, posterior shadowing to a solid mass, resulted from faulty focal zone placement, or from the mass resting on chest wall tissue. Pathology was sometimes simulated by postsurgical scars, ducts, and benign calcifications. Corrective procedures to alleviate the various scanning artifacts are recommended.

Dual effects of calcium on the oxidation of fatty acids to ketone bodies in liver mitochondria.

The addition of calcium ions (Ca2+) to rat liver mitochondria, under conditions of rapid accumulation of 10-40 nmol Ca2+/mg protein, inhibited the oxidation of long and medium chain fatty acids to ketone bodies, whereas higher quantities of Ca2+ activated the process. The mitochondrial NADH:NAD ratio exhibited corresponding depression and elevation. Both inhibitory and stimulatory actions of Ca2+ were operative in liver mitochondria from fed and fasted rats and appear to be localized in the mitochondrial inner membrane-matrix region. These observations may signify involvement of Ca2+ in the regulation of fatty acid oxidation and ketogenesis.

Genetic aspects of canine mixed leukocyte cultures.

Recent data stress the importance of matching donor and recipient of an organ graft for both the serologically defined (SD) and lymphocyte-defined (LD) determinants. To allow experimental evaluation of the effect of these SD and LD structures in a noninbred experimental animal, mixed leukocyte culture tests were performed between SD identical and nonidentical dogs to clarify the LD system in these animals. The results of these experiments can be summarized as follows: (a) In the dog there is a LD locus distinct from the known SD loci, which in all probability is localized outside the first (SD-1) series locus on the chromosome. (b) The crossing-over frequency between the SD and LD loci on the chromosome is low. (c) Studies in SD identical unrelated dogs and random unrelated dogs show an apparent high linkage disequilibrium between SD and LD loci. (d) The LD system in dogs is polymorphic.