Showing papers in "Journal of Molecular Medicine in 2006"

Toll-like receptors and innate immunity

Abstract: The innate immune system is an evolutionally conserved host defense mechanism against pathogens. Innate immune responses are initiated by pattern recognition receptors (PRRs), which recognize specific structures of microorganisms. Among them, Toll-like receptors (TLRs) are capable of sensing organisms ranging from bacteria to fungi, protozoa, and viruses, and play a major role in innate immunity. However, TLRs recognize pathogens either on the cell surface or in the lysosome/endosome compartment. Recently, cytoplasmic PRRs have been identified to detect pathogens that have invaded cytosols. In this review, we focus on the functions of PRRs in innate immunity and their downstream signaling cascades.

Quantitative real-time RT-PCR data analysis: current concepts and the novel “gene expression’s CT difference” formula

Abstract: For quantification of gene-specific mRNA, quan- titative real-time RT-PCR has become one of the most frequently used methods over the last few years. This article focuses on the issue of real-time PCR data analysis and its mathematical background, offering a general concept for efficient, fast and precise data analysis superior to the commonly used comparative CT (ΔΔCT) and the standard curve method, as it considers individual amplification efficiencies for every PCR. This concept is based on a novel formula for the calculation of relative gene expres- sion ratios, termed GED (Gene Expression's CT Difference) formula. Prerequisites for this formula, such as real-time PCR kinetics, the concept of PCR efficiency and its determination, are discussed. Additionally, this article offers some technical considerations and information on statistical analysis of real-time PCR data.

Role of apoA-I, ABCA1, LCAT, and SR-BI in the biogenesis of HDL.

Abstract: The concentration, composition, shape, and size of plasma high-density lipoprotein (HDL) are determined by numerous proteins that influence its biogenesis, remodeling, and catabolism. The discoveries of the HDL receptor (scavenger receptor class B type I, SR-BI) and the ABCA1 (ATP-binding cassette transporter A1) lipid transporter provided two missing links that were necessary to understand the biogenesis and some of the functions of HDL. Existing data indicate that functional interactions between apoA-I and ABCA1 are necessary for the initial lipidation of apoA-I. Through a series of intermediate steps, lipidated apoA-I proceeds to form discoidal HDL particles that can be converted to spherical particles by the action of lecithin:cholesterol acyltransferase (LCAT). Discoidal and spherical HDL can interact functionally with SR-BI and these interactions lead to selective lipid uptake and net efflux of cholesterol and thus remodel HDL. Defective apoA-I/ABCA1 interactions prevent lipidation of apoA-I that is necessary for the formation of HDL particles. In the same way, specific mutations in apoA-I or LCAT prevent the conversion of discoidal to spherical HDL particles. The interactions of lipid-bound apoA-I with SR-BI are affected in vitro by specific mutations in apoA-I or SR-BI. Furthermore, deficiency of SR-BI affects the lipid and apolipoprotein composition of HDL and is associated with increased susceptibility to atherosclerosis. Here we review the current status of the pathway of HDL biogenesis and mutations in apoA-I, ABCA1, and SR-BI that disrupt different steps of the pathway and may lead to dyslipidemia and atherosclerosis in mouse models. The phenotypes generated in experimental mouse models for apoA-I, ABCA1, LCAT, SR-BI, and other proteins of the HDL pathway may facilitate early diagnosis of similar phenotypes in the human population and provide guidance for proper treatment.

Suicidal death of erythrocytes in recurrent hemolytic uremic syndrome.

Abstract: Hemolytic uremic syndrome (HUS) is characterized by hemolytic anemia with fragmented erythrocytes, thrombocytopenia, and acute renal failure. Lack of complement inactivating factor H predisposes to the development of atypical HUS. Little is known about mechanisms linking complement activation with loss of erythrocyte integrity during HUS. Recent studies disclosed that increased cytosolic Ca2+ activity and cellular ceramide trigger programmed erythrocyte death or eryptosis, characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte surface. In the present study, we investigated whether eryptosis occurs during the course of HUS. To this end, erythrocytes from healthy volunteers were exposed to plasma from a patient with severe idiopathic recurrent HUS secondary to factor H depletion. Phosphatidylserine exposure (Annexin binding), cell volume (forward scatter), cytosolic Ca2+ activity (Fluo3 fluorescence), and ceramide formation [anti-ceramide antibody and enzymatic (diacylgycerol kinase) analysis] were determined. Exposure of erythrocytes to plasma from the patient, but not to plasma from healthy individuals, triggered Annexin binding. The effect of plasma on erythrocyte Annexin binding was abolished by plasmapheresis or filtration at 30 kDa. It was paralleled by formation of ceramide and increase of cytosolic Ca2+ activity. Enhanced Annexin binding of erythrocytes from healthy individuals was observed after exposure to plasma from three other patients with HUS. The proeryptotic effect of patient plasma was mimicked by exposure to the Ca2+ ionophore ionomycin, and eryptosis was potentiated in the presence of cell membrane-permeable C6-ceramide. Furthermore, in vitro complement activation similarly triggered erythrocyte phosphatidylserine exposure, an effect which was blunted by the addition of factor H. In conclusion, our present observations disclose a novel, pathophysiological, factor-H dependent mechanism leading to injury of erythrocytes during the course of hemolytic uremic syndrome.

Enzymes involved in the biosynthesis of leukotriene B4 and prostaglandin E2 are active in sebaceous glands.

Abstract: The expression of enzymes involved in leukotriene and prostaglandin signalling pathways, of interleukins 6 and 8 and of peroxisome proliferator-activated receptors in sebaceous glands of acne-involved facial skin was compared with those of non-involved skin of acne patients and of healthy individuals. Moreover, 5-lipoxygenase and leukotriene A4 hydrolase were expressed at mRNA and protein levels in vivo and in SZ95 sebocytes in vitro (leukotriene A4 hydrolase > 5-lipoxygenase), while 15-lipoxygenase-1 was only detected in cultured sebocytes. Cyclooxygenase-1 and cyclooxygenase-2 were also present. Peroxisome proliferator-activated receptors were constitutively expressed. Enhanced 5-lipoxygenase, cyclooxygenase 2 and interleukin 6 expression was detected in acne-involved facial skin. Arachidonic acid stimulated leukotriene B4 and interleukin 6 release as well as prostaglandin E2 biosynthesis in SZ95 sebocytes, induced abundant increase in neutral lipids and down-regulated peroxisome proliferator-activated receptor-α, but not receptor-γ1 mRNA levels, which were the predominant peroxisome proliferator-activated receptor isotypes in SZ95 sebocytes. In conclusion, human sebocytes possess the enzyme machinery for functional leukotriene and prostaglandin pathways. A comprehensive link between inflammation and sebaceous lipid synthesis is provided.

The sarcomeric Z-disc: a nodal point in signalling and disease

Abstract: The perception of the Z-disc in striated muscle has undergone significant changes in the past decade. Traditionally, the Z-disc has been viewed as a passive constituent of the sarcomere, which is important only for the cross-linking of thin filaments and transmission of force generated by the myofilaments. The recent discovery of multiple novel molecular components, however, has shed light on an emerging role for the Z-disc in signal transduction in both cardiac and skeletal muscles. Strikingly, mutations in several Z-disc proteins have been shown to cause cardiomyopathies and/or muscular dystrophies. In addition, the elusive cardiac stretch receptor appears to localize to the Z-disc. Various signalling molecules have been shown to interact with Z-disc proteins, several of which shuttle between the Z-disc and other cellular compartments such as the nucleus, underlining the dynamic nature of Z-disc-dependent signalling. In this review, we provide a systematic view on the currently known Z-disc components and the functional significance of the Z-disc as an interface between biomechanical sensing and signalling in cardiac and skeletal muscle functions and diseases.

Antigen presentation in autoimmunity and CNS inflammation: how T lymphocytes recognize the brain.

Abstract: The central nervous system (CNS) is traditionally viewed as an immune privileged site in which overzealous immune cells are prevented from doing irreparable damage. It was believed that immune responses occurring within the CNS could potentially do more damage than the initial pathogenic insult itself. However, virtually every aspect of CNS tissue damage, including degeneration, tumors, infection, and of course autoimmunity, involves a significant cellular inflammatory component. While the blood-brain barrier (BBB) inhibits diffusion of hydrophilic (immune) molecules across brain capillaries, activated lymphocytes readily pass the endothelial layer of postcapillary venules without difficulty. In classic neuro-immune diseases such as multiple sclerosis or acute disseminated encephalomyelitis, it is thought that neuroantigen-reactive lymphocytes, which have escaped immune tolerance, now invade the CNS and are responsible for tissue damage, demyelination, and axonal degeneration. The developed animal model for these disorders, experimental autoimmune encephalomyelitis (EAE), reflects many aspects of the human conditions. Studies in EAE proved that auto-reactive encephalitogenic T helper (Th) cells are responsible for the onset of the disease. Th cells recognize their cognate antigen (Ag) only when presented by professional Ag-presenting cells in the context of major histocompatibility complex class II molecules. The apparent target structures of EAE immunity are myelinating oligodendrocytes, which are not capable of presenting Ag to invading encephalitogenic T cells. A compulsory third party is thus required to mediate between the attacking T cells and the myelin-expressing target. This review will discuss the recent advances in this field of research and we will discuss the journey of an auto-reactive T cell from its site of activation into perivascular spaces and further into the target tissue.

The marijuana component cannabidiol inhibits β-amyloid-induced tau protein hyperphosphorylation through Wnt/β-catenin pathway rescue in PC12 cells

Abstract: Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder A massive accumulation of β-amyloid (Aβ) peptide aggregates has been proposed as pivotal event in AD Aβ-induced toxicity is accompanied by a variegated combination of events including oxidative stress The Wnt pathway has multiple actions in the cascade of events triggered by Aβ, and drugs that rescue Wnt activity may be considered as novel therapeutics for AD treatment Cannabidiol, a non-psychoactive marijuana component, has been recently proposed as an antioxidant neuroprotective agent in neurodegenerative diseases Moreover, it has been shown to rescue PC12 cells from toxicity induced by Aβ peptide However, the molecular mechanism of cannabidiol-induced neuroprotective effect is still unknown Here, we report that cannabidiol inhibits hyperphosphorylation of tau protein in Aβ-stimulated PC12 neuronal cells, which is one of the most representative hallmarks in AD The effect of cannabidiol is mediated through the Wnt/β-catenin pathway rescue in Aβ-stimulated PC12 cells These results provide new molecular insight regarding the neuroprotective effect of cannabidiol and suggest its possible role in the pharmacological management of AD, especially in view of its low toxicity in humans

Modification of gene expression of the small airway epithelium in response to cigarette smoking

Abstract: The earliest morphologic evidence of changes in the airways associated with chronic cigarette smoking is in the small airways. To help understand how smoking modifies small airway structure and function, we developed a strategy using fiberoptic bronchoscopy and brushing to sample the human small airway (10th-12th order) bronchial epithelium to assess gene expression (Affymetrix HG-U133A and HG-133 Plus 2.0 array) in phenotypically normal smokers (n = 16, 25 +/- 7 pack-years) compared to matched nonsmokers (n = 17). Compared to samples from large (second to third order) bronchi, the small airway samples had a higher proportion of ciliated cells, but less basal, undifferentiated, and secretory cells, and contained Clara cells. Even though the smokers were phenotypically normal, microarray analysis of gene expression of the small airway epithelium of the smokers compared to the nonsmokers demonstrated up- and downregulation of genes in multiple categories relevant to the pathogenesis of chronic obstructive lung disease (COPD), including genes coding for cytokines/innate immunity, apoptosis, mucin, response to oxidants and xenobiotics, and general cellular processes. In the context that COPD starts in the small airways, these gene expression changes in the small airway epithelium in phenotypically normal smokers are candidates for the development of therapeutic strategies to prevent the onset of COPD.

Hereditary breast and ovarian cancer: review and future perspectives

Abstract: Breast cancer (BC) is the most frequent carcinoma in women. The cumulative risk for the disease is 10% up to the age of 80 years. A familial history of BC and ovarian cancer (OC) is a significant risk factor. Some 5–10% of all cases of BC and 25–40% of cases in patients under the age of 35 years have a hereditary origin. BRCA1/BRCA2 mutations are responsible for 3–8% of all cases of BC and 30–40% of familial cases. Ten percent of patients with OC have a genetic predisposition. About 80% of families with a history of OC have BRCA1 mutations, while 15% have BRCA2 mutations. Women at risk can receive counseling from interdisciplinary cancer genetics clinics, while those at high risk can receive genetic testing. Risk calculation programs can define the risks and assist in decision making for genetic testing and clinical options. Clinical options require information on the risks of the disease and its mutation status. Chemoprevention is currently a controversial topic, while the use of oral contraceptives can be regarded as reducing the risk for OC. Prophylactic mastectomy and bilateral ovariectomy are the only options that lead to a demonstrable reduction in risk, but they do, of course, affect the patient’s physical integrity. It is not currently known whether intensified early cancer detection is individually beneficial, but this is currently the option that is the least invasive and least burdensome to the patient. Although hereditary BC has different pathological characteristics and the BRCA mutation is an independent negative prognostic factor, there are currently no special treatment guidelines. Without adjuvant hormone therapy or chemotherapy, the overall survival in BRCA mutation carriers is reduced. Chemotherapy regimens involving platinum are particularly beneficial in the treatment of hereditary BC.

Role of L-type Ca2+ channels in iron transport and iron-overload cardiomyopathy

Abstract: Excessive body iron or iron overload occurs under conditions such as primary (hereditary) hemochromatosis and secondary iron overload (hemosiderosis), which are reaching epidemic levels worldwide. Primary hemochromatosis is the most common genetic disorder with an allele frequency greater than 10% in individuals of European ancestry, while hemosiderosis is less common but associated with a much higher morbidity and mortality. Iron overload leads to iron deposition in many tissues especially the liver, brain, heart and endocrine tissues. Elevated cardiac iron leads to diastolic dysfunction, arrhythmias and dilated cardiomyopathy, and is the primary determinant of survival in patients with secondary iron overload as well as a leading cause of morbidity and mortality in primary hemochromatosis patients. In addition, iron-induced cardiac injury plays a role in acute iron toxicosis (iron poisoning), myocardial ischemia–reperfusion injury, Friedreich ataxia and neurodegenerative diseases. Patients with iron overload also routinely suffer from a range of endocrinopathies, including diabetes mellitus and anterior pituitary dysfunction. Despite clear connections between elevated iron and clinical disease, iron transport remains poorly understood. While low-capacity divalent metal and transferrin-bound transporters are critical under normal physiological conditions, L-type Ca2+ channels (LTCC) are high-capacity pathways of ferrous iron (Fe2+) uptake into cardiomyocytes especially under iron overload conditions. Fe2+ uptake through L-type Ca2+ channels may also be crucial in other excitable cells such as pancreatic beta cells, anterior pituitary cells and neurons. Consequently, LTCC blockers represent a potential new therapy to reduce the toxic effects of excess iron.

Inhibition of Notch/RBP-J signaling induces hair cell formation in neonate mouse cochleas

Abstract: Mammalian inner ear hair cells in cochleas are believed to be incapable of regeneration after birth, which hampers treatment of sensorineural hearing impairment mainly caused by hair cell loss. Sensory epithelia of cochleas are composed of hair cells and supporting cells, both of which originate from common progenitors. Notch/RBP-J signaling is an evolutionally conserved pathway involved in specification of various cell types in developmental stage and even in some of postnatal mammalian organs. The specification of hair cell fate from the progenitors is inhibited by Notch/RBP-J signaling in embryonic inner ears. However, its function in postnatal inner ears is unknown. We showed that inhibition of Notch/RBP-J signaling, by either conditional disruption of the Rbpsuh gene or treatment with a γ-secretase inhibitor, could give rise to ectopic hair cells in the supporting cell region in organs of Corti from neonatal mouse cochleas where hair cells have not been considered to regenerate after birth. We also showed that down-regulation of Hes5 and up-regulation of Math1 were associated with ectopic hair cell induction. These results suggest that Notch/RBP-J signaling inhibits supporting cells from differentiation into hair cells even in postnatal days, implying that inhibitors of Notch/RBP-J signaling can be used to help regenerating hair cells after birth and thus serve for potential treatment of intractable sensorineural hearing impairment caused by hair cell loss without genetical manipulation.

Common variants in the TCF7L2 gene and predisposition to type 2 diabetes in UK European Whites, Indian Asians and Afro-Caribbean men and women

Abstract: Common variants of TCF7L2, encoding a β-cell-expressed transcription factor, are strongly associated with increased risk of type 2 diabetes (T2D). We examined this association using both prospective and case-control designs. A total of 2,676 healthy European white middle-aged men from the prospective NPHSII (158 developed T2D over 15 years surveillance) were genotyped for two intronic SNPs [rs 7903146 (IVS3C > T) and rs12255372 (IVS4G > T)] which showed strong linkage disequilibrium (D′ = 0.88, p T in all studies. Linkage disequilibrium between the two SNPs was high in Indian Asians (D′ = 0.94), but much weaker in Afro-Caribbeans (D′ = 0.17) and haplotype frequencies differed markedly in this group. These results extend previous observations to other ethnic groups, and strongly confirm that TCF7L2 genotype is a major risk factor for development of T2D.

Aberrant intestinal expression and allelic variants of mucin genes associated with inflammatory bowel disease.

Abstract: Loss of intestinal mucosa integrity is an important factor in the pathogenesis of inflammatory bowel disease (IBD). The aim of this study was to characterize expression changes and allelic variants of genes related to intestinal epithelial barrier function in this disease. Therefore, ileal and colonic mucosal biopsies from nonaffected regions of patients with ulcerative colitis (UC) and Crohn’s disease (CD), as well as non-IBD probands, were subjected to Affymetrix DNA-microarray analysis. Real-time reverse transcription polymerase chain reaction was used for verification in larger IBD sample numbers. Disturbed mRNA expression was identified for several mucin genes in both disease groups and tissues. A significant downregulation in the colon was obtained for MUC2 in CD and MUC12 in CD and UC. Expression analysis of all dysregulated mucins in a broad human tissue panel revealed dominant epithelial tissue-specific transcription. In silico analysis of the regulatory regions of these mucins indicated nuclear factor κB (NFκB) binding sites in each promoter. Furthermore, NFκB was overrepresented in mucin promoters and a component of a specific combination of transcription factors (composite module). In vivo stimulation experiments in the adenocarcinoma cell line LS174T showed inducible mucin expression by the cytokines tumor necrosis factor-α and transforming growth factor-β, which could be blocked by NFκB signaling inhibitors. Allelic discrimination screening obtained statistically significant associations for the MUC2–V116M (P = 0.003) polymorphism with CD and for MUC4–A585S (P = 0.025), as well as MUC13–R502S (P = 0.0003) with UC. These data suggest that the disturbed expression of mucin genes and the connection to the NFκB pathway may influence the integrity of the intestine and therefore contribute to the pathophysiology of IBD.

Proliferation and cell–cell fusion of endometrial carcinoma are induced by the human endogenous retroviral Syncytin-1 and regulated by TGF-β

Abstract: Endometrial carcinomas (EnCa) predominantly represent a steroid hormone-driven tumor initiated from prestages. The human endogenous retrovirus HERV-W envelope gene Syncytin-1 was significantly increased at the mRNA and protein levels in EnCa and prestages compared to controls. Steroid hormone treatment of primary EnCa cells and cell lines induced Syncytin-1 due to a new HERV-W estrogen response element and resulted in increased proliferation. Activation of the cAMP-pathway also resulted in Syncytin-1 upregulation, but in contrast to proliferation, classic cell-cell fusions similar to placental syncytiotrophoblasts occurred. Cell-cell fusions were also histologically identified in endometrioid EnCa tumors in vivo. Clonogenic soft agar experiments showed that Syncytin-1 is also involved in anchorage-independent colony growth as well as in colony fusions depending on steroid hormones or cAMP-activation. The posttranscriptional silencing of Syncytin-1 gene expression and a concomitant functional block of induced cell proliferation and cell-cell fusion with siRNAs proved the essential role of Syncytin-1 in these cellular processes. TGF-beta1 and TGF-beta3 were identified as main regulative factors, due to the finding that steroid hormone inducible TGF-beta1 and TGF-beta3 inhibited cell-cell fusion, whereas antibody-mediated TGF-beta neutralization induced cell-cell fusions. These results showed that induced TGF-beta could override Syncytin-1-mediated cell-cell fusions. Interactions between Syncytin-1 and TGF-beta may contribute to the etiology of EnCa progression and also help to clarify the regulation of cell-cell fusions occurring in development and in other syncytial cell tumors.

Gene amplification, mutation, and protein expression of EGFR and mutations of ERBB2 in serous ovarian carcinoma

Abstract: EGFR and erbB-2 are targets for specific cancer therapy. The purpose of this study was to examine the frequency and clinicopathological correlations of gene amplification, protein expression, and mutations of EGFR and ERBB2 in serous carcinoma, the most common and aggressive type of ovarian cancer. Tissue microarray constructed of 398 carcinomas was examined by chromogenic in situ hybridization (CISH) and by immunohistochemistry. Cases with amplification of EGFR by CISH were further analyzed by fluorescence in situ hybridization. One hundred ninety-eight samples were analyzed for mutations in exons 18, 19, or 21 of EGFR and in exon 20 of ERBB2 using denaturating high-performance liquid chromatography and direct sequencing. Amplification of EGFR was present in 12% (41/333), low-level gain in 43% (144/333), and protein overexpression in 17% (66/379) of the tumors. Both increased copy number and overexpression of EGFR were associated with high tumor grade, greater patient age, large residual tumor size, high proliferation index, aberrant p53, and poor patient outcome. Furthermore, increased copy number of EGFR was associated with increased copy number of ERBB2. No mutations were identified in EGFR, whereas one tumor had an insertion mutation in exon 20 of ERBB2. Both amplification and protein overexpression of EGFR occur in serous ovarian carcinoma, but EGFR copy number has a stronger prognostic value. This makes EGFR amplification a potentially useful criterion for selecting patients in clinical trials testing the effect of EGFR inhibitors in serous ovarian carcinoma.

SGK1-dependent cardiac CTGF formation and fibrosis following DOCA treatment

Abstract: The mineralocorticoids aldosterone and deoxycorticosterone acetate (DOCA) stimulate renal tubular salt reabsorption, increase salt appetite, induce extracellular volume expansion, and elevate blood pressure. Cardiac effects of mineralocorticoids include stimulation of matrix protein deposition leading to cardiac fibrosis, which is at least partially due to the direct action of the hormones on cardiac cells. The signaling mechanisms mediating mineralocorticoid-induced cardiac fibrosis have so far remained elusive. Mineralocorticoids have been shown to upregulate the serum- and glucocorticoid-inducible kinase 1 (SGK1), which participates in the effects of mineralocorticoids on renal tubular Na+ reabsorption and salt appetite. To explore the involvement of SGK1 in the pathogenesis of mineralocorticoid-induced cardiac fibrosis, SGK1 knockout mice (sgk1-/-) and wild-type littermates (sgk1+/+) were implanted a 21-day-release 50-mg DOCA pellet and supplied with 1% NaCl in drinking water for 18 days. This DOCA/high-salt treatment increased blood pressure in both genotypes but led to significant cardiac fibrosis only in sgk1+/+ but not in sgk1-/- mice. According to real-time polymerase chain reaction and Western blotting, DOCA/high-salt treatment enhanced transcript levels and protein expression of cardiac connective tissue growth factor (CTGF) only in sgk1+/+ but not in sgk1-/- mice. Furthermore, DOCA (10 microM) upregulated CTGF expression and enhanced CTGF promoter activity in lung fibroblasts isolated from sgk1+/+ but not from sgk1-/- mice, an effect involving spironolactone-sensitive mineralocorticoid receptors and activation of nuclear factor-kappaB (NFkappaB). Our results suggest that SGK1 plays a decisive role in mineralocorticoid-induced CTGF expression and cardiac fibrosis.

Human genetics of adiponectin in the metabolic syndrome

Abstract: Adiponectin, an adipose-derived plasma protein, has been well established to be an important biomarker for metabolic syndrome and its complications after exhausted studies in humans. Animal and cell culture experiments also support most claims from human observations of its roles in the metabolic syndrome. Reproducible results of human genetic studies of diverse ethnic origin and by different investigators may provide the evidence for its causative roles in the pathogenesis of the metabolic syndrome and further insight into the genetic constitutions of the metabolic syndrome. Some of the common polymorphisms in the promoter region, exon and intron 2, and the rare nonsynonymous mutations in exon 3 of the human adiponectin gene were repeatedly shown in many studies from many different ethnic populations to associate with the phenotypes related to body weight, glucose metabolism, insulin sensitivity, and risk of type 2 diabetes mellitus and coronary artery disease. The association of adiponectin genetic variations with dyslipidemia and blood pressure was less explored. The common polymorphisms and rare mutations of the human adiponectin gene itself were demonstrated to associate with differential expression of adiponectin at the plasma protein level and mRNA level in adipose tissue. The PPARγ2 Pro12Ala variants were also shown to influence insulin sensitivity in interaction with adiponectin genotype or to influence plasma adiponectin levels. However, the results were not consistent. Three genome-wide scans for the loci that regulate plasma adiponectin concentration suggest further exploration on chromosomes 5, 9, 14, 15, and 18 is required. These human genetic studies on adiponectin and the metabolic syndrome strongly suggest that adiponectin is one of the causative factors in its pathogenesis and provide significant insights into the genetic makeup of the metabolic syndrome. Extension from these studies may accelerate the discovery of new molecular targets for future therapeutic interventions.

Lessons from SARS: control of acute lung failure by the SARS receptor ACE2.

Abstract: Angiotensin-converting enzyme 2 (ACE2), a second angiotensin-converting enzyme (ACE), regulates the renin-angiotensin system by counterbalancing ACE activity. Accumulating evidence in recent years has demonstrated a physiological and pathological role of ACE2 in the cardiovascular systems. Recently, it has been shown that severe acute respiratory syndrome (SARS) coronavirus, the cause of SARS, utilizes ACE2 as an essential receptor for cell fusion and in vivo infections in mice. Intriguingly, ACE2 acts as a protective factor in various experimental models of acute lung failure and, therefore, acts not only as a key determinant for SARS virus entry into cells but also contributes to SARS pathogenesis. Here we review the role of ACE2 in disease pathogenesis, including lung diseases and cardiovascular diseases.

Autophagy in innate and adaptive immunity against intracellular pathogens.

Abstract: Autophagy delivers cytoplasmic constituents for lysosomal degradation. Recent studies have demonstrated that this pathway mediates resistance to pathogens and is targeted for immune evasion by viruses and bacteria. Lysosomal degradation products, including pathogenic determinants, are then surveyed by the adaptive immune system to elicit antigen-specific T cell responses. CD4+ T helper cells have been shown to recognize nuclear and cytosolic antigens via presentation by major histocompatibility complex (MHC) class II molecules after autophagy. Furthermore, some sources of natural MHC class II ligands display characteristics of autophagy substrates, and autophagosomes fuse with late endosomes, in which MHC class II loading is thought to occur. Although MHC class II antigen processing via autophagy has so far mainly been described for professional antigen-presenting cells like B cells, macrophages, and dendritic cells, it might be even more important for cells with less endocytic potential, like epithelial cells, when these express MHC class II at sites of inflammation. Therefore, autophagy might contribute to immune surveillance of intracellular pathogens via MHC class II presentation of intracellular pathogen-derived peptides.

Quantitative assessment of DNA methylation: Potential applications for disease diagnosis, classification, and prognosis in clinical settings

Abstract: Deregulation of the epigenome is now recognized as a major mechanism involved in the development and progression of human diseases such as cancer. As opposed to the irreversible nature of genetic events, which introduce changes in the primary DNA sequence, epigenetic modifications are reversible and leave the original DNA sequence intact. There is now evidence that the epigenetic landscape in humans undergoes modifications as the result of normal aging, with older individuals exhibiting higher levels of promoter hypermethylation compared to younger ones. Thus, it has been proposed that the higher incidence of certain disease in older individuals might be, in part, a consequence of an inherent change in the control and regulation of the epigenome. These observations are of remarkable clinical significance since the aberrant epigenetic changes characteristic of disease provide a unique platform for the development of new therapeutic approaches. In this review, we address the significance of DNA methylation changes that result or lead to disease, occur with aging, or may be the result of environmental exposure. We provide a detailed description of quantitative techniques currently available for the detection and analysis of DNA methylation and provide a comprehensive framework that may allow for the incorporation of protocols which include DNA methylation as a tool for disease diagnosis and classification, which could lead to the tailoring of therapeutic approaches designed to individual patient needs.

Defective glycosylation of decorin and biglycan, altered collagen structure, and abnormal phenotype of the skin fibroblasts of an Ehlers–Danlos syndrome patient carrying the novel Arg270Cys substitution in galactosyltransferase I (β4GalT-7)

Abstract: The Ehlers–Danlos syndrome (EDS) is a heterogeneous group of connective tissue disorders affecting skin and joint function. Molecular defects in extracellular matrix proteins, including collagen (type I, III, and V) and tenascin X are associated with different forms of EDS. Compound heterozygous mutations in the B4GALT7 gene, resulting in aberrant glycosylation of the dermatan sulfate proteoglycan decorin, had been described in a single patient affected with the progeroid form of EDS. We have studied the molecular phenotype of decorin, biglycan, and collagen type I containing fibrils in skin fibroblasts of a patient carrying the novel homozygous C808T point mutation in the B4GALT7 gene, which causes an Arg270Cys substitution in β4GalT-7. Compared to control fibroblasts, galactosyltransferase activity in β4GalT-7Arg270Cys cells was approximately three times reduced over a temperature range of 25–41°C. Pulse-chase experiments and confocal microscopy demonstrated that synthesis and secretion of decorin were normal in β4GalT-7Arg270Cys cells. However, about 50% of decorin were synthesized as a protein core in addition to its proteoglycan form. Biglycan was found in a monoglycanated form in addition to its mature form. Glycosaminoglycan chains were of the dermatan/chondroitin sulfate type both in β4GalT-7Arg270Cys and control cells, and epimerization was reduced for decorin and biglycan. Compared to control cells, β4GalT-7Arg270Cys cells showed altered, highly spread or stretched phenotypes and decreased proliferation rates. At the ultrastructural level, an intracellular accumulation of multiple secondary lysosomes and degenerative vacuoles was seen in β4GalT-7Arg270Cys cells. Furthermore, the collagen suprastructures were altered in the β4GalT-7Arg270Cys cells. The reduced β4GalT-7 activity resulting in defective glycosylation of decorin and biglycan may be responsible for the complex molecular pathology in β4GalT-7 deficient EDS patients, given the role of these proteoglycans in bone formation, collagen fibrillogenesis, and skeletal muscle development.

Modulation of Hsp90 function in neurodegenerative disorders: a molecular-targeted therapy against disease-causing protein

Abstract: Abnormal accumulation of disease-causing protein is a commonly observed characteristic in chronic neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and polyglutamine (polyQ) diseases. A therapeutic approach that could selectively eliminate would be a promising remedy for neurodegenerative disorders. Spinal and bulbar muscular atrophy (SBMA), one of the polyQ diseases, is a late-onset motor neuron disease characterized by proximal muscle atrophy, weakness, contraction fasciculations, and bulbar involvement. The pathogenic gene product is polyQ-expanded androgen receptor (AR), which belongs to the heat shock protein (Hsp) 90 client protein family. 17-Allylamino-17-demethoxygeldanamycin (17-AAG), a novel Hsp90 inhibitor, is a new derivative of geldanamycin that shares its important biological activities but shows less toxicity. 17-AAG is now in phase II clinical trials as a potential anti-cancer agent because of its ability to selectively degrade several oncoproteins. We have recently demonstrated the efficacy and safety of 17-AAG in a mouse model of SBMA. The administration of 17-AAG significantly ameliorated polyQ-mediated motor neuron degeneration by reducing the total amount of mutant AR. 17-AAG accomplished the preferential reduction of mutant AR mainly through Hsp90 chaperone complex formation and subsequent proteasome-dependent degradation. 17-AAG induced Hsp70 and Hsp40 in vivo as previously reported; however, its ability to induce HSPs was limited, suggesting that the HSP induction might support the degradation of mutant protein. The ability of 17-AAG to preferentially degrade mutant protein would be directly applicable to SBMA and other neurodegenerative diseases in which the disease-causing proteins also belong to the Hsp90 client protein family. Our proposed therapeutic approach, modulation of Hsp90 function by 17-AAG treatment, has emerged as a candidate for molecular-targeted therapies for neurodegenerative diseases. This review will consider our research findings and discuss the possibility of a clinical application of 17-AAG to SBMA and other neurodegenerative diseases.

Gene expression profiling of human alveolar macrophages of phenotypically normal smokers and nonsmokers reveals a previously unrecognized subset of genes modulated by cigarette smoking.

Abstract: Cigarette smoking is the leading cause of the respiratory diseases collectively known as chronic obstructive pulmonary disease (COPD). While the pathogenesis of COPD is complex, there is abundant evidence that alveolar macrophages (AM) play an important role. Based on the concept that COPD is a slow-progressing disorder likely involving multiple mediators released by AM activated by cigarette smoke, the present study focuses on the identification of previously unrecognized genes that may be linked to early events in the molecular pathogenesis of COPD, as opposed to factors associated with the presence of disease. To accomplish this, microarray analysis using Affymetrix microarrays was used to carry out an unbiased survey of the differences in gene expression profiles in the AM of phenotypically normal, ∼20 pack-year smokers compared to healthy nonsmokers. Although smoking did not alter the global gene expression pattern of AM, 75 genes were modulated by smoking, with 40 genes up-regulated and 35 down-regulated in the AM of smokers compared to nonsmokers. Most of these genes belong to the functional categories of immune/inflammatory response, cell adhesion and extracellular matrix, proteolysis and antiproteolysis, lysosomal function, antioxidant-related function, signal transduction, and regulation of transcription. Of these 75 genes, 69 have not been previously recognized to be up- or down-regulated in AM in association with smoking or COPD, including genes coding for proteins belonging to all of the above categories, and others belonging to various functional categories or of unknown function. These observations suggest that gene expression responses of AM associated with the stress of cigarette smoking are more complex than previously thought, and offer a variety of new insights into the complex pathogenesis of smoking-induced lung diseases.

Thioredoxin in the cardiovascular system

Abstract: The thioredoxin (TRX) system (TRX, TRX reductase, and NADPH) is a ubiquitous thiol oxidoreductase system that regulates cellular reduction/oxidation (redox) status. The impairment of cell redox state alters multiple cell pathways, which may contribute to the pathogenesis of cardiovascular disorders including hypertension, atherosclerosis, and heart failure. In this manuscript, we review the essential roles that TRX plays by limiting oxidative stress directly via antioxidant effects and indirectly by protein-protein interactions with key signaling molecules such as thioredoxin interacting protein (TXNIP). TRX and its endogenous regulators may represent important future targets to develop clinical therapies for diseases associated with oxidative stress.

Association of angiotensin-converting enzyme 2 (ACE2) gene polymorphisms with parameters of left ventricular hypertrophy in men. Results of the MONICA Augsburg echocardiographic substudy.

Abstract: Angiotensin-converting enzyme (ACE) activity is considered to be of major importance for the conversion of angiotensin (Ang) I to Ang II. Recently, a second ACE, named ACE2, has been identified. Experimental data provide evidence that ACE2 might be involved in modulating cardiac structure and function. In the present explorative study, we assessed whether polymorphisms in the ACE2 gene are related to echocardiographically determined parameters of left ventricular mass, structure or function in the general population. Five intronic single nucleotide polymorphisms (SNPs) were genotyped using the 5′-exonuclease activity (TaqMan) assay in the echocardiographic substudy of the third MONICA Augsburg survey. As ACE2 is located on the X chromosome, women and men were analysed separately. Four SNPs showed high pairwise linkage disequilibrium (rs4646156, rs879922, rs4240157 and rs233575). The minor alleles of these four SNPs were associated with higher left ventricular mass index (LVMI) and higher septal wall thickness (SWT) in men. Likewise, male carriers of a common haplotype (frequency 29.9%) consisting of the minor alleles of these four SNPs displayed higher values for LVMI and SWT than non-carriers (LVMI: TGGC 98.8±1.52 vs non-TGGC 94.8±0.99 g/m2, p=0.027; SWT: TGGC 11.5±0.14 vs non-TGGC 11.1±0.09 mm, p=0.019). Furthermore, this haplotype was associated with an increased odds ratio (OR) for left ventricular hypertrophy (OR 3.10, p=0.006). In women, similar but less pronounced and consistent trends were observed. No association was observed between any of these SNPs and parameters of left ventricular systolic or diastolic function nor with blood pressure levels. This study provides evidence that genetic variants in the ACE2 gene may be associated with left ventricular mass, SWT and left ventricular hypertrophy in hemizygous men.

SHP-1 promoter 2 methylation in normal epithelial tissues and demethylation in psoriasis.

Abstract: SHP-1 promoter hypermethylation has been studied in hematopoietic cells and observed only in various types of lymphoma and leukemia. This study reports a contrasting situation in normal epithelial tissues and an association with skin pathogenesis, particularly in psoriasis. We investigated several cell lines, five of them were epithelial and six were hematopoietic, white blood cells from normal, healthy donors, and normal microdissected epithelium of kidney, liver, breast, cervix, lung, prostate, bladder, and skin. Interestingly, promoter 2 hypermethylation was apparent in all epithelial cell lines and tissues. However, distinctive degrees of demethylation were noted in some skin samples. The methylation patterns of each cell line corresponded to their mRNA isoforms, in that isoforms I and II could not be detected with either promoter 1 or 2 hypermethylation, respectively. We further explored whether an enhanced degree of demethylation could be observed in various dermatopathology lesions. While the promoter 2 methylation levels of squamous cell cancers, eczemas, and normal skins were not different, a significant degree of demethylation can be observed in psoriasis (p<0.005). In addition, psoriasis displays a higher level of SHP-1 isoform II than normal skin (p<0.05). In conclusion, this study discovered an unprecedented role of SHP-1 methylation in tissue-specific expression and its alteration in a nonmalignant human disease besides the transcription inhibition in leukemia and lymphoma. Furthermore, the promoter demethylation may play an important role in skin pathogenesis by enhancing SHP-1 isoform II transcription in psoriatic skin lesions.

Identification of a novel frameshift mutation in the giant muscle filament titin in a large Australian family with dilated cardiomyopathy

Abstract: Dilated cardiomyopathy (DCM) is an etiologically heterogeneous cardiac disease characterized by left ventricular dilation and systolic dysfunction. Approximately 25-30% of DCM patients show a family history of mainly autosomal dominant inheritance. We and others have previously demonstrated that mutations in the giant muscle filament titin (TTN) can cause DCM. However, the prevalence of titin mutations in familial DCM is unknown. In this paper, we report a novel heterozygous 1-bp deletion mutation (c.62890delG) in TTN that cosegregates with DCM in a large Australian pedigree (A3). The TTN deletion mutation c.62890delG causes a frameshift, thereby generating a truncated A-band titin due to a premature stop codon (p.E20963KfsX10) and the addition of ten novel amino acid residues. The clinical phenotype of DCM in kindred A3 demonstrates incomplete penetrance and variable expressivity. Finally, protein analysis of a skeletal muscle biopsy sample from an affected member did not reveal the predicted truncated titin isoform although the aberrant mRNA was present, suggesting posttranslational modification and degradation of the truncated protein. The identification of a novel disease-causing mutation in the giant titin gene in a third large family with DCM indicates that mutations in titin may account for a significant portion of the genetic etiology in familial DCM.

The methionine synthase reductase 66A>G polymorphism is a maternal risk factor for spina bifida.

Abstract: The methionine synthase reductase (MTRR) enzyme restores methionine synthase (MTR) enzyme activity and therefore plays an essential role in homocysteine remethylation. In some studies, the 66A>G polymorphism in the MTRR gene was associated with increased neural tube defect (NTD) risk. Using a case-control design, we studied the association between the MTRR 66A>G polymorphism and spina bifida risk in 121 mothers, 109 spina bifida patients, 292 control women, and 234 pediatric controls. Possible interactions between the MTRR 66A>G variant and the MTR 2756A>G polymorphism, the MTHFR 677C>T variant, plasma vitamin B12, and plasma methylmalonic acid (MMA) levels were examined in the 121 mothers and 292 control women. Meta-analyses were conducted to set the results of the case-control study in the context of eligible literature on the relation between the MTRR 66A>G variant and NTD risk. Finally, a transmission disequilibrium test was performed for 82 complete mother–father–child triads to test for preferential transmission of the MTRR risk allele. In our case-control study, the MTRR 66A>G polymorphism had no influence on spina bifida risk in children [odds ratio (OR) 0.6, 95% confidence interval (CI) 0.4–1.1]. The MTRR 66GG genotype increased maternal spina bifida risk by 2.1-fold (OR 2.1, 95% CI 1.3–3.3). This risk became more pronounced in combination with the MTHFR 677TT genotype (OR 4.0, 95% CI 1.3–12.5). Moreover, we demonstrate a possible interaction between the MTRR 66GG genotype and high plasma MMA levels (OR 5.5, 95% CI 2.2–13.5). The meta-analyses demonstrated that the maternal MTRR 66GG genotype was associated with an overall 55% (95% CI 1.04–2.30) increase in NTD risk and that the MTRR 66GG genotype did not increase NTD risk in children (OR 0.96, 95% CI 0.46–2.01). These data show that the MTRR 66GG genotype is a maternal risk factor for spina bifida especially when intracellular vitamin B12 status is low.

Enhanced induction of dendritic cell maturation and HLA-A*0201-restricted CEA-specific CD8 + CTL response by exosomes derived from IL-18 gene-modified CEA-positive tumor cells

Abstract: Dendritic cells (DC)-derived or tumor-derived exosomes are a population of nanometer sized membrane vesicles that can induce specific anti-tumor immunity. However, the immunogenic potential and efficiency of exosomes-based tumor vaccine are not satisfactory enough to achieve a curative effect in clinical trials. In this article we investigated whether IL-18 genetic modification of tumor cells can increase the efficacy of exosomes derived from IL-18 gene-modified tumor cells. We transfected carcinoembryonic antigen (CEA)-expressing tumor cells with a recombinant adenovirus encoding human IL-18 (AdhIL-18) and prepared the exosomes, Exo/IL-18, from IL-18 gene-modified tumor cells. We found that Exo/IL-18 naturally contain CEA and bioactive IL-18. Moreover, Exo-IL-18 are potent in chemoattracting DC and T cells, enhancing the proliferation and Th1 cytokine release of PBMC, and promoting the phenotypic and functional maturation of DC. Furthermore, Exo/IL-18-pulsed DC are quite potent to induce HLA-A*0201-restricted, CEA-specific CD8+ CTL from the PBMC of HLA-A*0201 CEA+ cancer patients in vitro. In almost all of these experiments, Exo/IL-18 show more potent functions than the conventionally prepared exosomes derived from parent tumor cells without IL-18 gene modification. Our findings suggest that Exo/IL-18 has more potent capability to induce specific anti-tumor immunity, and our strategy of IL-18 modification of exosomes is a feasible approach to develop exosomes-based tumor vaccines.