Polysaccharide specific monoclonal antibodies provide passive protection against intranasal challenge with Burkholderia pseudomallei

Burkholderia pseudomallei is a Gram-negative bacillus that is the causative agent of melioidosis. The bacterium is inherently resistant to many antibiotics and mortality rates remain high in endemic areas. The lipopolysaccharide (LPS) and capsular polysaccharide (CPS) are two surface-associated antigens that contribute to pathogenesis. We previously developed two monoclonal antibodies (mAbs) specific to the CPS and LPS; the CPS mAb was shown to identify antigen in serum and urine from melioidosis patients. The goal of this study was to determine if passive immunization with CPS and LPS mAbs alone and in combination would protect mice from a lethal challenge with B. pseudomallei. Intranasal (i.n.) challenge experiments were performed with B. pseudomallei strains 1026b and K96423. Both mAbs provided significant protection when administered alone. A combination of mAbs was protective when low doses were administered. In addition, combination therapy provided a significant reduction in spleen colony forming units (cfu) compared to results when either the CPS or LPS mAbs were administered alone.     

Suppression of protein kinase C and the stimulation of glucocorticoid receptor synthesis by dexamethasone in human fibroblasts derived from tumor tissue

Exposure of fibroblasts derived from keloid tissues, desmoid and dermal tissue from individuals with Gardner's syndrome (GS) to dexamethasone resulted in the suppression of protein kinase C (PKC) activity and [3H]thymidine incorporation into DNA, and a 20-fold induction of glutamine synthetase activity. Treatment of GS and keloid fibroblasts with 0.1 microM dexamethasone for 36 h increased glucocorticoid receptor (GR) synthesis, as determined by [35S]methionine labeling and immunoprecipitation with a monoclonal antibody to the human GR. The suppression of PKC activity by dexamethasone was shown to result from a loss of protein mass as determined by immunoblotting using an antibody to PKC type III. In contrast to these results, exposure of fibroblasts isolated from normal tissues to dexamethasone did not result in the suppression PKC and [3H]thymidine incorporation, there was only a sixfold induction of glutamine synthetase, and a decrease of GR synthesis. As no primary receptor binding defect could be detected, the altered response of tumor cells to steroid-occupied receptor indicates a partial post-receptor binding defect in GS and keloid cells.     

Effects of salinity on chloride cells in the euryhaline cyprinodontid fish Rivulus marmoratus

Summary The ultrastructure and density of chloride cells in the gill, opercular epithelium, and opercular skin of the euryhaline self-fertilizing fish Rivulus marmoratus (Cyprinodontidae) were studied with electron and fluorescence microscopy. R. marmoratus raised from birth in 1, 50, 100, and 200% seawater were compared. Chloride cells from fish raised in each of the four salinities exhibited an invaginated pit structure at the apical crypt. Multicellular complexes were present in the 1% seawater group and in those fish raised in higher salinities where elaborate interdigitations were seen between cells. Chloride cells from gills of fish raised in 200% seawater had a significantly higher percentage of their cytoplasmic volume composed of mitochondria than did those from fish raised in 1% seawater (69.9% vs 37.4%). The opercular skin and opercular epithelium had the same density of chloride cells (4.2×10⁴-4.5×10⁴ chloride cells/cm²), and this number did not vary significantly with increased salinity. The opercular skin thus appears far more responsive to environmental salinity than the opercular epithelium. Chloride cells from the opercular epithelium of fish raised in 200% seawater were found to be 39% larger than those from fish raised in 1% seawater, whereas the chloride cells from the opercular skin of the 200% seawater group were 107% larger than those from the 1% seawater group.     

Critical role of angiopoietins/Tie-2 in hyperglycemic exacerbation of myocardial infarction and impaired angiogenesis

Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) are the two ligands of the Tie-2 receptor, a receptor tyrosine kinase that is expressed on the endothelium. A balanced angiopoietin/Tie-2 system is critical for the maintenance of vascular integrity. We investigated the potential role of a disrupted angiopoietin/Tie-2 system on hyperglycemic exacerbation of myocardial infarction and impaired angiogenesis. Using streptozotocin (STZ) mice subjected to myocardial ischemia, we examined the effects of shifting the Ang-2-to-Ang-1 ratio on myocardial infarction size, apoptosis, bone marrow (BM) cell-endothelial progenitor cell (EPC) differentiation, and angiogenesis. In control mice, myocardial ischemia increased expression of both Ang-2 and Tie-2. In STZ mice, Ang-2 expression was elevated, whereas Tie-2 expression was reduced, and neither was significantly altered by ischemia. Myocardial infarct size and apoptosis were increased in STZ compared with control mice. Using in vivo administration of an adenovirus containing Ang-1 or Ang-2, we found that shifting the Ang-2-to-Ang-1 ratio to favor Ang-1 reduced myocardial apoptosis and infarct size in STZ mice, while shifting the Ang-2-to-Ang-1 ratio to favor Ang-2 resulted in a significant increase in myocardial infarct size and apoptosis in control mice. Myocardial ischemia-stimulated BM cell-EPC differentiation was inhibited and myocardial angiogenesis was reduced in STZ mice. Systemic administration of Ad-Ang-1 restored BM cell-EPC differentiation and increased myocardial VEGF expression and angiogenesis in STZ mice. Our data demonstrate that disturbed angiopoietin/Tie-2 signaling contributes to the hyperglycemic exacerbation of myocardial infarction and impaired angiogenesis. Restoration of the Ang-2-to-Ang-1 ratio may be a novel therapeutic strategy for the treatment of diabetic myocardial ischemic diseases.     

Topography of short portal vessels in the rat pituitary gland: a scanning electron-microscopic and morphometric study of corrosion cast replicas

We applied scanning electron microscopy combined with imaging and morphometric techniques to analyze the dorsal topography and morphology of short portal vessels linking the capillary beds of the pituitary neural and anterior lobes in adult male albino rats. The pituitary microvasculature was replicated by intracarotid injection of Batson's No. 17 compound producing plastic casts that were advantageous for comprehensive morphometric analyses using an imaging device. The analysis revealed the existence of two types of portal vessels having quantitatively different morphological properties. The bilateral venular plexus of 3–4 vessels located at the base of the infundibular stalk (each venule measuring 300 m in length and 32 m in diameter) appears to be the major part of the short portal system in the dorsum of the rat pituitary gland. Narrower capillary-like shunt vessels (6.8 m in diameter), of about the same length as the venules, were situated throughout other subregions of the intermediate lobe cleft. The short portal vessels of both types made direct anastomoses with the capillary networks in the neural and anterior lobes. The neural lobe capillaries were twice as numerous (1324 per mm²), and only half as wide (6.2 m), as the sinusoidal capillaries in the anterior lobe (density of 637 per mm²; diameter of 13.7 m). The topographical position of the portal venular system suggests that the caudolateral subregions of the pituitary neural and anterior lobes have a functional relationship dependent on rapid interlobe transfer of neurohumoral factors such as hormones via the portal blood. This process appears to be supplemented throughout the rest of the cleft between the two lobes by a small number of capillary shunts that supply the epithelial cell lobules of the intermediate lobe in situ. The findings collectively indicate that this portal system provides a constant stream of neurohumoral information that is shared moment-by-moment between the pituitary neural and anterior lobes.     

Combined Drug Treatment of Obesity

Pharmacological treatment of obesity has been neglected as a viable therapeutic option for many years. Recent long term studies with combinations of obesity drugs gives promise that drugs may play a role in weight maintenance, which classically has been the most difficult aspect of treating obesity. Currently available obesity drugs include centrally acting adrenergic agents and serotonin agonists. Drugs still in development include a lipase inhibitor that produces fat malabsorption, a combined adrenergic-serotonergic reuptake inhibitor, various gut-central nervous system peptides, and a number of beta-3 agonists. Any of these obesity drugs given alone produces modest weight loss, and for most, weight loss continues for as long as medication is given. The most successful drug regimens to date are combinations of phentermine and fenfluramine or of ephedrine, caffeine, and/or aspirin. The former combination produces reduction in body weight and complications of obesity for 2 to almost 4 years in clinical trials to date. More research is needed to document long term efficacy and particularly the long term safety of these and other combinations.     

DACH1: Its Role as a Classifier of Long Term Good Prognosis in Luminal Breast Cancer

Background

Oestrogen receptor (ER) positive (luminal) tumours account for the largest proportion of females with breast cancer. Theirs is a heterogeneous disease presenting clinical challenges in managing their treatment. Three main biological luminal groups have been identified but clinically these can be distilled into two prognostic groups in which Luminal A are accorded good prognosis and Luminal B correlate with poor prognosis. Further biomarkers are needed to attain classification consensus. Machine learning approaches like Artificial Neural Networks (ANNs) have been used for classification and identification of biomarkers in breast cancer using high throughput data. In this study, we have used an artificial neural network (ANN) approach to identify DACH1 as a candidate luminal marker and its role in predicting clinical outcome in breast cancer is assessed.

Materials and methods

A reiterative ANN approach incorporating a network inferencing algorithm was used to identify ER-associated biomarkers in a publically available cDNA microarray dataset. DACH1 was identified in having a strong influence on ER associated markers and a positive association with ER. Its clinical relevance in predicting breast cancer specific survival was investigated by statistically assessing protein expression levels after immunohistochemistry in a series of unselected breast cancers, formatted as a tissue microarray.

Results

Strong nuclear DACH1 staining is more prevalent in tubular and lobular breast cancer. Its expression correlated with ER-alpha positive tumours expressing PgR, epithelial cytokeratins (CK)18/19 and ‘luminal-like’ markers of good prognosis including FOXA1 and RERG (p<0.05). DACH1 is increased in patients showing longer cancer specific survival and disease free interval and reduced metastasis formation (p<0.001). Nuclear DACH1 showed a negative association with markers of aggressive growth and poor prognosis.

Conclusion

Nuclear DACH1 expression appears to be a Luminal A biomarker predictive of good prognosis, but is not independent of clinical stage, tumour size, NPI status or systemic therapy.     

Utilization of renewable agricultural residues for the production of extracellular halostable cellulase from newly isolated Halomonas sp. strain PS47

A newly isolated biopolymer-degrading halophilic bacterium, Halomonas sp. strain PS47, yielded higher cellulase activity (0.0076 U/ml) in mineral salt medium (MM63). Activity increased to 0.029 U/ml when carboxymethyl cellulose (0.5 % w/v) was used as carbon source and further to 0.138 U/ml when a combination of yeast extract and peptone was used as nitrogen source. Enzyme secretion was maximal during late exponential and stationary phases (0.15 U/ml, 48 h). Among different agro-residues (1 % w/v), wheat bran gave the highest activity (0.12 U/ml) at pH 7.5, 30 °C and 6 % (w/v) NaCl. The cellulase exhibited higher activity at pH 7.1 and 50 °C. The enzyme exhibited activity over a wide range of NaCl concentrations (0–4 M). Optimum activity was at 0–1 M NaCl. At 4 M NaCl, activity was reduced to 65 % of the initial value. The present investigation thus contributes to the limited information available on halostable cellulases.     

Rub1p processing by Yuh1p is required for wild-type levels of Rub1p conjugation to Cdc53p

In Saccharomyces cerevisiae, Rub1p, like ubiquitin, is conjugated to proteins. Before protein conjugation, the carboxyl-terminal asparagine residue of Rub1p is removed. Rub1p conjugation is dependent on the carboxyl-terminal processing enzyme Yuh1p, whereas Rub1p lacking the asparagine residue is conjugated without Yuh1p. Thus, Yuh1p is the major processing enzyme for Rub1p.     

Fibrinogen binding sites P336 and Y338 of clumping factor A are crucial for Staphylococcus aureus virulence

We have earlier shown that clumping factor A (ClfA), a fibrinogen binding surface protein of Staphylococcus aureus, is an important virulence factor in septic arthritis. When two amino acids in the ClfA molecule, P(336) and Y(338), were changed to serine and alanine, respectively, the fibrinogen binding property was lost. ClfAP(336)Y(338) mutants have been constructed in two virulent S. aureus strains Newman and LS-1. The aim of this study was to analyze if these two amino acids which are vital for the fibrinogen binding of ClfA are of importance for the ability of S. aureus to generate disease. Septic arthritis or sepsis were induced in mice by intravenous inoculation of bacteria. The clfAP(336)Y(338) mutant induced significantly less arthritis than the wild type strain, both with respect to severity and frequency. The mutant infected mice developed also a much milder systemic inflammation, measured as lower mortality, weight loss, bacterial growth in kidneys and lower IL-6 levels. The data were verified with a second mutant where clfAP(336) and Y(338) were changed to alanine and serine respectively. When sepsis was induced by a larger bacterial inoculum, the clfAP(336)Y(338) mutants induced significantly less septic death. Importantly, immunization with the recombinant A domain of ClfAP(336)SY(338)A mutant but not with recombinant ClfA, protected against septic death. Our data strongly suggest that the fibrinogen binding activity of ClfA is crucial for the ability of S. aureus to provoke disease manifestations, and that the vaccine potential of recombinant ClfA is improved by removing its ability to bind fibrinogen.