Adiponectin self-regulates its expression and multimerization in adipose tissue: an autocrine/paracrine mechanism?

Adiponectin, a 30-kDa peptide hormone discovered in the mid 1990s, is secreted abundantly and exclusively by adipose tissue. Adiponectin exists in three major forms: a low molecular weight (LMW) trimer, a medium molecular weight (MMW) hexamer, and a high molecular weight (HMW) 18-36 oligomer. The HMW oligomer has the most potent insulin-sensitizing activity therefore impaired adiponectin multimerization may lead to impaired glycemic control. Decreased ratio of HMW/total adiponectin has been observed in patients with obesity, type-2 diabetes mellitus, cardiovascular diseases and insulin resistance-related metabolic syndrome. Previous studies have indicated that berberine or aminoimidazole carboxamide ribonucleotide (AICAR)-induced activation of AMP-activated protein kinase (AMPK) suppresses the expression of adiponectin but promotes adiponectin multimerization in adipocytes. Since adiponectin activates AMPK through adiponectin receptors (AdipoRs) in the membranes of adipocytes, we speculate that adiponectin self-regulates its expression and multimerization in adipose tissue. The hypothesis suggests a potential drug target for treating insulin resistance and provides new interpretation of several clinical observations. In addition, we propose a rapid method for one-step detection of the distribution of adiponectin oligomers in approximately 30 min, based on the open sandwich immunoassay and fluorescence resonance energy transfer technology. With the development of this new method, the ratio of HMW/total adiponectin may be applied in clinical diagnosis as a novel biomarker for insulin resistance and metabolic disorders.     

Increased expression of peroxisome proliferator-activated receptor (PPAR)-α and PPAR-γ in human atherosclerosis

To clarify the mechanism of atherosclerosis development in humans, we studied the mRNA and protein expression of PPAR subtypes in various types of atherosclerotic lesions and their correlation with cell proliferation and macrophage invasion. Human aortas were obtained from 35 autopsied cases, and each sample was divided into halves. One half was used for the analysis of mRNA or protein expression with RT-PCR or Western blotting, respectively. The other was microscopically classified into atheromatous plaque (AP), fatty streak (FS), and diffuse intimal thickening (DIT), and was analyzed immunohistochemically. The mRNA levels of both PPARs increased significantly in atherosclerosis and tended to increase in proportion to the severity of the lesion, and the expression of PPAR-α correlated with that of PPAR-γ in FS and AP. The PPAR-γ protein increased in AP. Monocytes/macrophages, as well as endothelial and smooth muscle cells, expressed the PPAR-γ protein in plaques. This expression in the DIT was noted mainly in macrophages but was not correlated with the density of macrophages, suggesting that only certain macrophages express the PPARs in DIT. Cell proliferation did not correlate with PPARs expression in any lesion type. These findings suggest that PPARs may be associated with atheromatous plaque formation, and that PPAR-γ may be involved in the early stages of human atherosclerosis.     

What are the determinants of gene expression levels and breadths in the human genome?

In complex organisms, different tissues express different genes, which ultimately shape the function and phenotype of each tissue. An important goal of modern biology is to understand how some genes are turned on and off in specific tissues and how the numbers of different gene expression products are determined. These aspects are named 'expression breadth' (or 'tissue specificity') and 'expression level', respectively. Here, we show that we can predict substantial amount of variation in levels and breadths of gene expression using genomic information of each gene. Interestingly, many genomic traits are correlated with both aspects of gene expression in similar directions, suggesting shared molecular pathways. However, to elucidate distinctive molecular mechanisms governing gene expression levels and breadths, we need to identify the relative significance of each genomic trait on these two aspects of gene expression. To this end, we developed a novel multivariate multiple regression method. Using this new method, we show that gene compactness (in particular, the mean size of exons), codon usage bias and non-synonymous rates have a stronger influence on expression levels compared with their effects on expression breadths. In contrast, the propensity of promoter DNA methylation is a stronger indicator of expression breadths than of expression levels. Interestingly, intron DNA methylation exhibits an opposite pattern to the promoter DNA methylation in the human genome, suggesting that DNA methylation may play multiple roles depending upon its genomic targets. Furthermore, synonymous rates have stronger associations with expression breadths than with expression levels in the human genome. These findings provide clues toward distinctive molecular mechanisms regulating different aspects of gene expression.     

Detection and characterization of OX40 ligand expression in human airway smooth muscle cells: a possible role in asthma?

BACKGROUND: The airway smooth muscle (ASM) cell, originally thought of as a passive structural cell, is now well recognized as an active participant in the pathologic events that occur during persistent asthma. Cell-surface molecules play an important role in the development of an immune response. A number of cell-surface molecules are expressed on ASM cells, and these might contribute to the inflammatory reaction. OBJECTIVE: The purpose of this study was to determine whether OX40 ligand (OX40L), a molecule known to be involved in T-cell activation, was present on the ASM cell surface. METHODS: We used real-time RT-PCR to detect mRNA expression and flow cytometry, ELISA, and immunoprecipitation to detect the presence of cell-surface protein on ASM cells isolated from asthmatic and nonasthmatic individuals. ELISAs and Western blotting were used to determine the functional outcomes of engagement of OX40L. RESULTS: OX40L was present on both asthmatic and nonasthmatic ASM cells. Engagement of OX40L with recombinant OX40:Fc resulted in a significantly greater increase in release of IL-6 from ASM cells of asthmatic patients than from ASM cells of nonasthmatic patients (P<.01). Ligation of OX40L resulted in a rapid translocation of protein kinase C beta2 to the cell membrane. Conclusion: Because the receptor for OX40L, OX40, is expressed on CD4+ T cells within 48 hours of stimulation through the T-cell receptor, elucidation of the cross-talk between OX40 and OX40L could be very important in understanding the interaction of cells present in the inflamed airways of an asthmatic patient.     

Aberrant alteration of vascular endothelial growth factor-family signaling in human tubal ectopic pregnancy: what is known and unknown?

More than 98% of ectopic pregnancies occur in the Fallopian tube. Because many facets of tubal ectopic pregnancy remain unclear, prediction, prevention and treatment of tubal ectopic pregnancy are still a major clinical challenge. Compelling evidence suggests that angiogenic growth factors are involved in normal and abnormal implantation. While acknowledging the importance of an intrauterine pregnancy requires the development of a local blood supply and angiogenesis, we hypothesize that the hypoxic- and estrogen-dependent regulation of vascular endothelial growth factor/placental growth factor expression, secretion, and signaling pathways that are possibly involved in the pathophysiology of tubal ectopic pregnancy. Our hypothesis may also lead to a new therapeutic strategy for women with tubal ectopic pregnancy.     

Retrograde embolism and atherosclerosis development in the human thoracic aorta: are the fluid dynamics explanations valid?

The mechanisms of atherosclerosis development in the thoracic aorta is miscellaneous and still only partially understood. The marked variability of the sites of deposition of atherosclerotic plaques in the aorta could not be clarified based solely on the risk-factors theory of atherosclerosis. The sites of deposition of atherosclerotic plaques are considered to be affected by blood-flow patterns that cause areas of altered shear stress on the aortic wall. Close relations between protruding aortic plaques (PAP), stroke and peripheral emboli were established. The analysis of PAP distribution and motion to characterize atherogenesis in the human thoracic aorta and the pathogeneses of embolic events was performed. We concluded that protruding aortic plaques and markers of relative aortic flow instability (occurrences of vortices) are predominantly noticed in the human arch and in the descending aorta, whereas the ascending aorta showed lesser prevalence of atheromatosis. Reversal and rotational blood-flow in the thoracic aorta most likely exist in all patients with systemic emboli and mobile protruding aortic atheromas. Therefore, retrograde cerebral embolism from distal aortic plaques is conceivable.     

SGCE isoform characterization and expression in human brain: implications for myoclonus–dystonia pathogenesis?

Myoclonus–dystonia (M–D) is a neurological movement disorder with involuntary jerky and dystonic movements as major symptoms. About 50% of M–D patients have a mutation in ɛ-sarcoglycan (SGCE), a maternally imprinted gene that is widely expressed. As little is known about SGCE function, one can only speculate about the pathomechanisms of the exclusively neurological phenotype in M–D. We characterized different SGCE isoforms in the human brain using ultra-deep sequencing. We show that a major brain-specific isoform is differentially expressed in the human brain with a notably high expression in the cerebellum, namely in the Purkinje cells and neurons of the dentate nucleus. Its expression was low in the globus pallidus and moderate to low in caudate nucleus, putamen and substantia nigra. Our data are compatible with a model in which dysfunction of the cerebellum is involved in the pathogenesis of M–D.     

The role of C/EBP-α expression in human liver and liver fibrosis and its relationship with autophagy

Aim: To investigate the expression of CCAAT enhancer binding protein-α (C/EBP-α) in normal human liver and liver fibrosis and its probable association with autophagy. Methods: Double label immunohistochemistry was used to detect the location of C/EBP-α in hepatocytes and hepatic stellate cells (HSCs). The expression of C/EBP-α, Atg5, and Atg6 was also evaluated by immunohistochemistry in paraffin sections of human liver. HSC-T6 cells were treated with rapamycin and 3-methyladenine (3MA) to induce or inhibit autophagy, and the expression of C/EBP-α protein was detected by Western blotting. Results: Double label immunohistochemistry showed that C/EBP-α was predominantly located in hepatocytes and that its expression was significantly decreased in fibrosis compared with normal liver. Atg5 expression was increased in fibrosis but was located primarily in liver septa and peri-vascular areas, which was consistent with the distribution of HSCs. In contrast, Atg6 was not expressed in normal or fibrotic liver. Treatment of HSC-T6 cells in culture with rapamycin or 3MA decreased or increased C/EBP-α expression, respectively, as shown by Western blotting. Conclusion: C/EBP-α was primarily expressed in hepatocytes in normal liver, but its expression decreased significantly in liver fibrosis. Autophagy might play a role in liver fibrosis through its association with C/EBP-α, but this hypothesis warrants further investigation.     

The differential expression of TGF-β1, ILK and wnt signaling inducing epithelial to mesenchymal transition in human renal fibrogenesis: an immunohistochemical study

Epithelial-to-mesenchymal transition (EMT) is a process for fully differentiated epithelial cells to undergo a phenotypic change to fibroblasts via diverse intracellular signaling pathways. While the pivotal role of fibroblasts in renal fibrosis is widely accepted, their origin remains undefined. In addition, although a large number of studies have provided evidence of EMT in human kidney diseases, specific signaling pathways leading to EMT have not yet been discovered in humans. To evaluate the origin of interstitial fibroblasts and signaling pathways involved in the EMT process, we analyzed the differential expression of EMT-related molecules in paraffin-fixed sections from 19 human fibrotic kidneys and 4 control kidneys. In human fibrotic kidneys, tubular epithelial cells (TECs) with intact tubular basement membrane (TBM) showed loss or down-regulation of an epithelial marker (E-cadherin), de novo expression of mesenchymal markers (vimentin and fibronectin), and significant up-regulation of inducers and mediators controlling the EMT process (transforming growth factor-β1 (TGF-β1), p-Smad2/3, β1-integrin, p38 mitogen-activated protein kinase (MAPK), WNT5B and β-catenin) in the areas of interstitial inflammation and fibrosis, compared with their expression in control kidneys. In conclusion, the type II EMT process in humans is thought to be an adaptive response of TECs to chronic injury and is regulated by interconnections of TGF-β/Smad, integrin/integrin-linked kinase (ILK) and wnt/β-catenin signaling pathways.     

Myeloperoxidase and lactoferrin expression in semen fluid: Novel markers of male infertility risk?

Research QuestionInfections and/or inflammation processes of male genital tract are highly prevalent and often associated with risk of infertility. These conditions represent a possible cause of leukocytospermia, which is still under debate. Leukocytes are key-factors to reactive oxygen species (ROS) production and the increase of ROS in semen fluid is associated with the worsening of semen parameters. At present, there are not appropriate andrological tests to identify asymptomatic inflammatory conditions when the amount of leukocytes is in the normal range.DesignWe studied the innate immunity profile of myeloperoxidase and lactoferrin (MPO/LAC) proteins expressed in the semen fluid of 39 men evaluated for couple infertility, in the absence of leukocytospermia.ResultsThe presence of both MPO and LAC proteins was associated with a decrease of sperm concentration and of progressive/total motility, whereas the increase of MPO-/LAC + indicated a worse sperm morphology. It is worth to report the predictive potential of MPO+/LAC + pattern (above 4.36 %) as a biological marker to distinguish normozoospermic from pathological patients.ConclusionOur findings indicate MPO/LAC analysis as a potential diagnostic tool to identify asymptomatic conditions eventually related to male infertility, even when the number of leukocytes in semen fluid is below 1 million/mL.     

Elevated expression of transforming growth factor β3 in carbon tetrachloride-treated rat liver and involvement of retinoid signaling

Transforming growth factor (TGF) β is a pro-fibrotic cytokine. While three isoforms (TGF-β1, 2 and 3) are known, the functional differences between them are obscure. To investigate the roles of TGF-β isoforms during liver fibrogenesis, male Wistar rats were administrated carbon tetrachloride (CCl4) subcutaneously twice a week for two months. Livers were excised and sectioned for histochemical examinations. These livers were also used to quantitate the expression of genes associated with fibrogenesis, including TGF-β isoforms, as well as those associated with retinoid metabolism. Expression levels of Tgfb1 and Tgfb3 were up-regulated in CCl4-treated rat livers while that of Tgfb2 was not changed. The mRNAs for lecithin-retinol acyltransferase (Lrat) and retinoic acid hydroxylase, Cyp26a1, were also elevated. By immunohistochemical staining, TGF-β3 protein was found to be localized mainly in liver parenchymal cells (hepatocytes). These results indicate that retinoid mobilization likely takes place within the rat's liver following CCl4 treatment, and suggest the possibility that the expression of Tgfb mRNA is regulated by retinoic acid receptors. Reporter analyses of a region of the Tgfb3 gene were performed using the rat liver parenchymal cell line, RLC-16, and a positively responsive region was identified within its intron.     

Allelic exclusion of immunoglobulin gene rearrangement and expression: why and how?

Since the discovery of the allelic exclusion of immunoglobulin (Ig) gene expression by Pervis in the 1960s [J. Exp. Med. 122 (1965) 853], much attention has been focused on its mechanism. Much less attention has been paid, however, to the question of why B cells demonstrate such unusual genetic regulation of antigen receptor gene expression. A large body of literature implicates the Ig gene products as feedback regulators of their own genetic rearrangement [Adv. Immunol.78 (2001)169; Science 236 (1987)816]. While a role for Ig gene products in the regulation of V(D)J recombination is beyond debate, it is extremely unlikely that such a feedback mechanism would be fast enough to avoid occasional near-simultaneous rearrangement of allelic loci leading to dual receptor gene expression. This review will suggest an hypothesis to answer the 'why bother' aspect of allelic exclusion and then go on to propose a mechanism, distinct from feedback regulation, which may contribute to the allelic exclusion of Ig gene expression.     

Comparison of RANTES expression in Crohn's disease and ulcerative colitis: an aid in the differential diagnosis?

BACKGROUND: RANTES (regulated on activation, normal T cell expressed and secreted) expression is increased in inflammatory bowel disease (IBD). RANTES is produced at higher levels in granulomatous conditions, so increased RANTES expression can be expected in Crohn's disease compared with ulcerative colitis. AIM: To compare RANTES expression between intestinal biopsy specimens of patients with Crohn's disease and those with ulcerative colitis. MATERIALS AND METHODS: A prospective study of patients presenting with lower gastrointestinal symptoms at the Bahrain Specialist Hospital from July 2004 to April 2005 was carried out. Endoscopic colonic biopsy specimens were taken from every patient and subjected to (a) routine haematoxylin and eosin staining examination by light microscopy, (b) immunohistochemistry for examination of RANTES protein expression by light microscopy and (c) in situ hybridisation for examination of RANTES mRNA expression by light microscopy. RANTES expression was assessed and quantified. RESULTS: 58 patients were enrolled to the study. Of them, 40 had IBD (21 had Crohn's disease and 19 had ulcerative colitis), 15 were controls with normal colonic biopsy results or non-inflammatory lesions and 3 had colonic inflammatory lesions other than IBD. RANTES expression in lymphocytes or histiocytes was significantly higher (p = 0.04) in new patients with ulcerative colitis than in those with Crohn's disease analysed by immunohistochemistry (IHC). CONCLUSION: RANTES expression in lymphocytes or histiocytes is significantly higher in patients with ulcerative colitis than in those with Crohn's disease. Hence, RANTES IHC can be an effective method for distinguishing between biopsy specimens of patients with ulcerative colitis from those of patients with Crohn's disease, where routine histological features are indeterminate. RANTES IHC may prove to be a useful technique for identifying early or equivocal granulomas.     

Th1/Th2 subsets: distinct differences in homing and chemokine receptor expression?

The functional specialization of T effector cells according to cytokine secretion patterns has been recognized as an important parameter shaping local immune responses. Here we discuss evidence that T cell subsets might also develop distinctive properties related to homing and trafficking into inflamed sites. First, ligands for the inflammation-induced endothelial selectins were found to be induced by IL-12, and hence selectively expressed on Th1 cells generated in vitro. However, their expression on effector cells occuring in vivo is less well correlated with the Th subset. Second, a variety of receptors for and responses towards chemokines have been found to be differentially associated with Th subsets. Notably CCR5 and, to a lesser degree CXCR3 were preferentially found on Th1 cells, CCR4, CCR8 and, more controversial, CCR3 and CXCR4 on Th2 cells. Although many points, such as stability of the phenotype versus dependency on inducing cytokines and activation stages remain to be clarified, it appears that this field provides new insights into the regulation of locally balanced activities of Th subsets and might constitute a promising field for the development of new immunosuppressive drugs.     

Detection of partial and complete acrosome reaction in human spermatozoa: which inducers and probes to use?

The acrosome reaction (AR), an essential step for achieving mammalian fertilization, was recently introduced as a means of clinical evaluation of male fertility. However, most of the available techniques for acrosomal status assessment (except those employing electron microscopy) do not define whether the measurements represent partial or complete AR. We, therefore, performed a crossover investigation of the types of inducers and probes required for detecting partial or complete AR in human spermatozoa. The acrosomal status before and after stimulation with four AR inducers was evaluated after incubation for 3 h in capacitating conditions. We used a fluorescence-activated cell sorter with fluorescein isothiocyanate-conjugated monoclonal antibody CD46 (FITC-CD46) targeting the inner acrosomal membrane for detecting a complete AR, and fluorescein isothiocyanate-Pisum sativum agglutinin (FITC-PSA) targeting the acrosomal content for detection of both partial and complete AR. Without stimulation or following stimulation with progesterone, follicular fluid (FF) or phorbol myristate ester (PMA), the AR could be detected with FITC-PSA but not with FITC-CD46. Following stimulation with the calcium ionophore A23187, the AR could be detected by both FITC-PSA and FITC-CD46. These results suggest that spontaneous AR as well as AR induced by progesterone, PMA and FF are partial. In contrast, the AR induced by A23187 is total, i.e. both partial and complete. These findings are valuable for both research and clinical purposes and are a step towards an international agreement on a standard test for human sperm AR, for which there is an urgent need.     

Expression patterns of PSA-NCAM in the human ganglionic eminence and its vicinity: role of PSA-NCAM in neuronal migration and axonal growth?

The ganglionic eminence (GE) is a transient but conspicuous structure of the developing forebrain which not only gives rise to a large number of precursor neurons and glial cells for various structures of the forebrain, but in addition serves as an intermediate target for growing axons to the cerebral cortex. To investigate the roles of the highly polysialylated isoform of the neural cell adhesion molecule (PSA-NCAM) in cell migration and axonal growth within the GE and its neighbouring structures, the spatio-temporal expression pattern of PSA-NCAM was examined in human fetal forebrains between 14 and 36 weeks of gestation with a specific immunohistochemical method. Scattered PSA-NCAM-positive cells were found in the centre but more frequently in the marginal zone of the GE. Intensely labelled cells were also identified in the gangliothalamic body, basolateral nuclei of the amygdala and the subventricular and intermediate zone adjacent to the GE. This cellular immunoreactivity started to appear in various structures during the period from 14 to 19 weeks and gradually diminished after 25-28 weeks. Strong immunoreactivity was also detected in fibres running from the intermediate zone of the neocortex to the internal capsule from 16 weeks onwards, and after 24 weeks, the immunoreactivity was gradually decreased. In the vicinity of the GE, between 16 and 22 weeks, short fibre bundles were observed to leave the longitudinally oriented axons of the internal capsule to reach the marginal zone of the GE. Our results suggest a close relationship between PSA-NCAM expression and neuronal migration (over short distances) and transitory axonal projections (target recognition and axonal fasciculation) in the region of the GE.