Erythropoietin receptor and hematological disease

This review will discuss evidence for the role of the erythropoietin (Epo) receptor in the development of erythrocytosis and other hematological disorders, The possible causative role of mutations of other genes in the pathogenesis of idiopathic erythrocytosis will be considered, Polycythemia vera (PV) is a myeloproliferative disorder that is caused by an undefined stem cell abnormality, characterized by a significant erythrocytosis, leukocytosis, and thrombocytosis. However, erythrocytosis may arise from apparent (or relative) polycythemia in which the hematocrit is raised due to a low plasma volume. In such cases the red cell mass is normal. A group of disorders with increased red cell mass caused by stimulation of erythrocyte production is known as secondary polycythemia, Investigation of such patients may reveal a congenital abnormality such as high affinity hemoglobin or an acquired abnormality caused, for example, by smoking, renal Vascular impairment, or an Epo-producing tumor. Even after thorough examination there remains a cohort of patients for whom no definite cause for the erythrocytosis can be established, A careful clinical history may reveal whether this idiopathic erythrocytosis is likely to be congenital and/or familial, in which case the term

IL-31 does not induce normal human ciliated epithelial cells to differentiate into a phenotype consistent with the pathophysiology of asthma

Abstract Background IL-31 is a novel cytokine that has been implicated in allergic diseases such as atopic dermatitis and more recently asthma. While IL-31 has been well studied in skin conditions such as atopic dermatitis, little is known about the role IL-31 plays in asthma and specifically the differentiation process of the bronchial epithelium, which is central to the pathogenesis of allergic asthma. Methods We examined the effects of IL-13 (20 ng/ml), IL-31 (20 ng/ml) and an IL-13/IL-31 combination stimulation (20 ng/ml each) on the in vitro mucociliary differentiation of paediatric bronchial epithelial cells (PBECs) from healthy patients (n=6). IL-31 receptor (IL-31-RA) expression, markers of differentiation (goblet and ciliated cells), transepithelial electrical resistance (TEER), quantification of goblet and ciliated cells, real time PCR for MUC5AC, ELISA for VEGF, EGF and MCP-1 (CCL-2) and ELISA for MUC5AC were assessed. Results We found that well-differentiated PBECs expressed IL-31-RA however it's expression did not increase upon stimulation with IL-31 or either of the other treatments. TEER indicated good formation of tight junctions which was found to be similar across all treatment groups (p=0.9). We found that IL-13 alone significantly reduced the number of ciliated cells compared with unstimulated (IL-13 stimuation: mean=4.8% (SD=2.5); unstimulated: mean=15.9%, (SD=7.4), p

New prognostic markers, determined using gene expression analyses, reveal two distinct subtypes of chronic myelomonocytic leukaemia patients

Chronic myelomonocytic leukaemia (CMML) is a heterogeneous haematopoietic disorder characterized by myeloproliferative or myelodysplastic features. At present, the pathogenesis of this malignancy is not completely understood. In this study, we sought to analyse gene expression profiles of CMML in order to characterize new molecular outcome predictors. A learning set of 32 untreated CMML patients at diagnosis was available for TaqMan low-density array gene expression analysis. From 93 selected genes related to cancer and cell cycle, we built a five-gene prognostic index after multiplicity correction. Using this index, we characterized two categories of patients with distinct overall survival (94% vs. 19% for good and poor overall survival, respectively; P = 0.007) and we successfully validated its strength on an independent cohort of 21 CMML patients with Affymetrix gene expression data. We found no specific patterns of association with traditional prognostic stratification parameters in the learning cohort. However, the poor survival group strongly correlated with high-risk treated patients and transformation to acute myeloid leukaemia. We report here a new multigene prognostic index for CMML, independent of the gene expression measurement method, which could be used as a powerful tool to predict clinical outcome and help physicians to evaluate criteria for treatments.

Protein kinases C: potential targets for intervention in diabetic nephropathy

Protein kinases C are a family of serine threonine protein kinases that play key roles in extracellular signal transduction. Inappropriate activation of protein kinase C has been implicated in the pathophysiology of many diseases, including diabetes mellitus. Indeed, protein kinase C activation may contribute not only to the pathogenesis of diabetic complications such as nephropathy and retinopathy, but also to insulin resistance. Growing awareness that protein kinase C isoforms subserve specific subcellular functions has led to the development of isoform-specific inhibitors, which may be useful investigational tools and therapeutic agents for attenuating the effects of inappropriate protein kinase C activity. Here we review the role played by protein kinases C in diabetic nephropathy and the recent progress that has been made to modulate its activity using specific inhibitors. Curr Opin Nephrol Hypertens 7:563-570. (C) 1998 Lippincott Wiiliams & Wilkins.

Glucose-induced oxidative stress in vascular contractile cells - Comparison of aortic smooth muscle cells and retinal pericytes

Free radical-mediated damage to vascular cells may be involved in the pathogenesis of diabetic vasculopathy. The aim of this study was to compare the extent of glucose-induced oxidative stress in both vascular smooth muscle cells (VSMCs) and pericytes and the effect on antioxidant enzyme gene expression and activities. Porcine aortic VSMC and retinal pericytes were cultured in either 5 or 25 mmol/l glucose for 10 days. Intracellular malondialdehyde (MDA) was measured as a marker of peroxidative damage, and mRNA expression of CuZn-SOD, MnSOD, catalase, and glutathione peroxidase (GPX) were measured by Northern analysis. Glutathione (GSH) was also measured. There was a significant increase in MDA in VSMCs in 25 mmol/l glucose (1.34 +/- 0.11 vs. 1.88 +/- 0.24 nmol/mg protein, 5 vs. 25 mmol/l D-glucose, mean +/- SE, n = 15, P

Activation of extracellular signal-regulated kinase in proliferative glomerulonephritis in rats

Multiple extracellular mitogens are involved in the pathogenesis of proliferative forms of glomerulonephritis (GN), In vitro studies demonstrate the pivotal role of extracellular signal-regulated kinase (ERK) in the regulation of cellular proliferation in response to extracellular mitogens. In this study, we examined whether this kinase, as a convergence point of mitogenic stimuli, is activated in proliferative GN in vivo, Two different proliferative forms of anti-glomerular basal membrane (GEM) GN in rats were induced and whole cortical tissue as well as isolated glomeruli examined using kinase activity assays and Western blot analysis, Administration of rabbit anti-rat GEM serum to rats, preimmunized with rabbit IgG, induced an accelerated crescentic anti-GEM GN. A significant increase in cortical, and more dramatically glomerular ERK activity was detected at 1, 3, and 7 d after induction of GN, Immunization of Wistar-Kyoto rats with bovine GEM also induced a crescentic anti-GBM GN with an increase of renal cortical ERK activity after 4, 6, and 8 wk, ERK is phosphorylated and activated by the MAP kinase/ERK kinase (MEK), We detected a significant increase in the expression of glomerular MEK in the accelerated form of anti-GEM CN, providing a possible mechanism of long-term activation of ERK in this disease model, In contrast to ERK, activation of stress-activated protein kinase was only detectable at early stages of proliferative GN, indicating these related kinases to serve distinct roles in the pathogenesis of GN, Our observations point to ERK as a putative mediator of the proliferative response to immune injury in GN and suggest that upregulation of MEK is involved in the long-term regulation of ERK in vivo.

Type IVB pili are required for invasion but not for adhesion of Salmonella enterica serovar Typhi into BHK epithelial cells in a cystic fibrosis transmembrane conductance regulator-independent manner

The cystic fibrosis transmembrane conductance regulator (CFTR) has been proposed as an epithelial cell receptor for the entry of Salmonella Typhi but not Salmonella Typhimurium. The bacterial ligand recognized by CM is thought to reside either in the S. Typhi lipopolysaccharide core region or in the type IV pili. Here, we assessed the ability of virulent strains of S. Typhi and S. Typhimurium to adhere to and invade BHK epithelial cells expressing either the wild-type CFTR protein or the Delta F508 CFTR mutant. Both S. Typhi and S. Typhimurium invaded the epithelial cells in a CFTR-independent fashion. Furthermore and also in a CFTR-independent manner, a S. Typhi pilS mutant adhered normally to BHK cells but displayed a 50% reduction in invasion as compared to wild-type bacteria. Immunofluorescence microscopy revealed that bacteria and CFTR do not colocalize at the epithelial cell surface. Together, our results strongly argue against the established dogma that CFTR is a receptor for entry of Salmonella to epithelial cells. (C) 2011 Elsevier Ltd. All rights reserved.

Different sugar residues of the lipopolysaccharide outer core are required for early interactions of Salmonella enterica serovars Typhi and Typhimurium with epithelial cells

The role of lipopolysaccharide (LPS) in entry of Salmonella Typhimurium into epithelial cells remains unclear. In this study, we tested the ability of a series of mutants with deletions in genes for the synthesis and assembly of the O antigen and the outer core of LPS to adhere to and invade HeLa, BHK, and IB3 epithelial cells lines. Mutants devoid of O antigen, or that synthesized only one O antigen unit, or with altered O antigen chain lengths were as able as the wild type to enter epithelial cells, indicating that this polysaccharide is not required for invasion of epithelial cells in vitro. In contrast, the LPS core plays a role in the interaction of S. Typhimurium with epithelial cells. The minimal core structure required for adherence and invasion comprised the inner core and residues Glc I Gal I of the outer core. A mutant of S. Typhimurium that produced a truncated LPS core lacking the terminal galactose residue had a significant lower level of adherence to and ingestion by the three epithelial cell lines than did strains with this characteristic. Complementation of the LPS production defect recovered invasion to parental levels. Heat-killed bacteria with a core composed of Glc 1 Gal I. but not bacteria with a core composed of Glc 1, inhibited uptake of the wild type by HeLa cells. A comparison of the chemical structure of the S. Typhi core with the published chemical structure of that of S. Typhimurium indicated that the Glc I Gal 1 Glc 11 backbone is conserved in both serovars. However, S. Typhi requires a terminal glucose for maximal invasion. Therefore, our data indicate that critical saccharide residues of the outer core play different roles in the early interactions of serovars Typhi and Typhimurium with epithelial cells. (C) 2010 Elsevier Ltd. All rights reserved.

A Decade of Burkholderia cenocepacia Virulence Determinant Research

The Burkholderia cepacia complex (Bcc) is a group of genetically related environmental bacteria that can cause chronic opportunistic infections in patients with cystic fibrosis (CF) and other underlying diseases. These infections are difficult to treat due to the inherent resistance of the bacteria to antibiotics. Bacteria can spread between CF patients through social contact and sometimes cause cepacia syndrome, a fatal pneumonia accompanied by septicemia. Burkholderia cenocepacia has been the focus of attention because initially it was the most common Bcc species isolated from patients with CF in North America and Europe. Today, B. cenocepacia, along with Burkholderia multivorans, is the most prevalent Bcc species in patients with CF. Given the progress that has been made in our understanding of B. cenocepacia over the past decade, we thought that it was an appropriate time to review our knowledge of the pathogenesis of B. cenocepacia, paying particular attention to the characterization of virulence determinants and the new tools that have been developed to study them. A common theme emerging from these studies is that B. cenocepacia establishes chronic infections in immuno-compromised patients, which depend more on determinants mediating host niche adaptation than those involved directly in host cells and tissue damage.

Burkholderia cenocepacia O antigen lipopolysaccharide prevents phagocytosis by macrophages and adhesion to epithelial cells

Chronic respiratory infections by the Burkholderia cepacia complex (Bcc) are of great concern to patients with cystic fibrosis. Bcc isolates may survive intracellularly within amoebae, respiratory epithelial cells and macrophages. The molecular mechanisms facilitating colonization and pathogenesis remain unclear. Given the importance of bacterial adhesion to host surfaces in microbial pathogenesis, we investigated the role of the O antigen LPS in the interaction of Burkholderia cenocepacia, a member of the Bcc, with macrophages and epithelial cells. Our results demonstrated that the O antigen modulates phagocytosis but does not affect intracellular survival of B. cenocepacia. Internalization of strains that lack O antigen was significantly increased compared to that of their isogenic smooth counterparts. However, no differences between rough and smooth strains were found in their ability to delay phagosomal maturation. We also found that the O antigen interfered with the ability of B. cenocepacia to adhere to bronchial epithelial cells, suggesting that this polysaccharide may mask one or more bacterial surface adhesins.

RpoS and RpoN are involved in the growth-dependent regulation of rfaH transcription and O antigen expression in Salmonella enterica serovar Typhi

We reported earlier that the production of O antigen lipopolysaccharide (LPS) by Salmonella enterica serovar Typhi (Salmonella typhi) increases at the onset of stationary phase and correlates with a growth-regulated expression of the rfaH gene under the control of the alternative sigma factor RpoN (Microbiology 148 (2002) 3789). In this study, we demonstrate that RpoS also modulates rfaH promoter activity as revealed by the absence of growth-dependent regulation of an rfaH-lacZ transcriptional fusion and O antigen production in a S. typhi rpoS mutant. Introduction of a constitutively expressed rpoN gene into the rpoS mutant restored increased production of O antigen during stationary phase, suggesting that constitutive production of RpoN could overcome the RpoS defect. Similar results were observed when an rpoS rpoN double mutant was transformed with the intact rpoN gene. Thus, we conclude that both RpoS and RpoN control the rfaH promoter activity and concomitantly, the production of O-specific LPS in S. typhi.

AMD and micronutrient antioxidants

Age-related maculopathy (ARM) is a common clinical entity. The late-stage manifestations of ARM, which are known as age-related macular degeneration (AMD), have devastating consequences for vision. Various risk factors have been identified in the development of the condition, which are consistent with the premise that oxidative stress plays an important role in its pathogenesis. Thus, the possibility that antioxidant balance can be manipulated through diet or supplementation has created much interest. Associations between diet and nutrition and the clinical features of ARM have been described. Scrutiny of the literature shows consistency in the report of notable reductions in serum micronutrients in wet AMD, however, the evidence for causation is still circumstantial. In this comprehensive review of the clinical literature, we have assessed the evidence for a link between diet and nutrition as risk factors for the development of ARM and AMD. All published case control, population-based, and interventional studies on ARM were examined. Although initial support appeared to be moderate and somewhat contradictory, the evidence that lifetime oxidative stress plays an important role in the development of ARM is now compelling. The positive outcomes in the Age-Related Eye Diseases Study, a major controlled clinical trial, have given hope that modulation of the antioxidant balance through supplementation can help prevent progression of ARM to AMD.

Identification of a general secretory pathway in a human isolate of Burkholderia vietnamiensis (formerly B. cepacia complex genomovar V) that is required for the secretion of hemolysin and phospholipase C activities

Members of the Burkholderia cepacia complex can secrete proteases, lipases, and hemolysins. We report in this study the identification of a general secretory pathway present in a B. vietnamiensis (formerly genomovar V) clinical isolate, which is required for the efficient secretion of phospholipase C and hemolysin activities. Southern blot hybridization experiments revealed that this general secretion pathway is highly conserved among the different genomovars of the B. cepacia complex and is homologous to a similar system described in B. pseudomallei. We also show that this pathway appears not to be necessary for intracellular survival of B. vietnamiensis within Acanthamoeba polyphaga.

Intracellular survival of Burkholderia cepacia complex isolates in the presence of macrophage cell activation

Strains of the Burkholderia cepacia complex have emerged as a serious threat to patients with cystic fibrosis due to their ability to infect the lung and cause, in some patients, a necrotizing pneumonia that is often lethal. It has recently been shown that several strains of the B. cepacia complex can escape intracellular killing by free-living amoebae following phagocytosis. In this work, the ability of two B. cepacia complex strains to resist killing by macrophages was explored. Using fluorescence microscopy, electron microscopy and a modified version of the gentamicin-protection assay, we demonstrate that B. cepacia CEP021 (genomovar VI), and Burkholderia vietnamiensis (previously B. cepacia genomovar V) CEP040 can survive in PU5-1.8 murine macrophages for a period of at least 5 d without significant bacterial replication. Furthermore, bacterial entry into macrophages stimulated production of tumour necrosis factor and primed them to release toxic oxygen radicals following treatment with phorbol myristoyl acetate. These effects were probably caused by bacterial LPS, as they were blocked by polymyxin B. Infected macrophages primed with interferon gamma produced less nitric oxide than interferon-gamma-primed uninfected cells. We propose that the ability of B. cepacia to resist intracellular killing by phagocytic cells may play a role in the pathogenesis of cystic fibrosis lung infection. Our data are consistent with a model where repeated cycles of phagocytosis and cellular activation without bacterial killing may promote a deleterious inflammatory response causing tissue destruction and decay of lung function.

Pathogenicity and molecular genetics of O-specific side-chain lipopolysaccharides of Escherichia coli

Lipopolysaccharide (LPS), a glycolipid molecule found on the outer leaflet of outer membranes of gram-negative bacteria, consists of three moieties: lipid A, core oligosaccharide, and the O-specific polysaccharide chain. The O-specific side chain, which extends to the extracellular milieu, plays an important role in pathogenicity, especially during the initial stages of infection, because of its ability to interact with serum complement. In recent years, several laboratories have used recombinant DNA tools to determine, at the molecular level, the organization, expression, and regulation of genes involved in LPS biosynthesis in Salmonella and Escherichia coli. An increased understanding of the molecular aspects of the O-specific side-chain genes will shed light on the intimate details related with the formation of the O-specific side chain, its assembly onto the lipid A--core, and the translocation and insertion of the complete LPS molecule into the outer membrane. It will also contribute to the understanding of the evolution of these genes and the correlation of chemical diversity of O-specific side chains with the genetic diversity of O-specific side-chain genes. In addition, since the O-specific side chains are involved in the pathogenicity of medically important gram-negative bacteria, a basic understanding of the regulation and expression of O-specific side chain LPS genes will contribute to the field of molecular pathogenesis. This article provides an overview of the role of O-specific side chains in septicemic infections and also discusses the current status of molecular genetic studies on O-specific side-chain genes from E. coli.