Integrated Cardio Metabolic Centre

The rat, mouse and human estrogen receptor (ER) exists as two subtypes, ER alpha and ER beta, which differ in the C-terminal ligand-binding domain and in the N-terminal transactivation domain. In this study, we investigated the estrogenic activity of environmental chemicals and phytoestrogens in competition binding assays with ER alpha or ER beta protein, and in a transient gene expression assay using cells in which an acute estrogenic response is created by cotransfecting cultures with recombinant human ER alpha or ER beta complementary DNA (cDNA) in the presence of an estrogen-dependent reporter plasmid. Saturation ligand-binding analysis of human ER alpha and ER beta protein revealed a single binding component for [3H]-17beta-estradiol (E2) with high affinity [dissociation constant (Kd) = 0.05 - 0.1 nM]. All environmental estrogenic chemicals [polychlorinated hydroxybiphenyls, dichlorodiphenyltrichloroethane (DDT) and derivatives, alkylphenols, bisphenol A, methoxychlor and chlordecone] compete with E2 for binding to both ER subtypes with a similar preference and degree. In most instances the relative binding affinities (RBA) are at least 1000-fold lower than that of E2. Some phytoestrogens such as coumestrol, genistein, apigenin, naringenin, and kaempferol compete stronger with E2 for binding to ER beta than to ER alpha. Estrogenic chemicals, as for instance nonylphenol, bisphenol A, o, p'-DDT and 2',4',6'-trichloro-4-biphenylol stimulate the transcriptional activity of ER alpha and ER beta at concentrations of 100-1000 nM. Phytoestrogens, including genistein, coumestrol and zearalenone stimulate the transcriptional activity of both ER subtypes at concentrations of 1-10 nM. The ranking of the estrogenic potency of phytoestrogens for both ER subtypes in the transactivation assay is different; that is, E2 >> zearalenone = coumestrol > genistein > daidzein > apigenin = phloretin > biochanin A = kaempferol = naringenin > formononetin = ipriflavone = quercetin = chrysin for ER alpha and E2 >> genistein = coumestrol > zearalenone > daidzein > biochanin A = apigenin = kaempferol = naringenin > phloretin = quercetin = ipriflavone = formononetin = chrysin for ER beta. Antiestrogenic activity of the phytoestrogens could not be detected, except for zearalenone which is a full agonist for ER alpha and a mixed agonist-antagonist for ER beta. In summary, while the estrogenic potency of industrial-derived estrogenic chemicals is very limited, the estrogenic potency of phytoestrogens is significant, especially for ER beta, and they may trigger many of the biological responses that are evoked by the physiological estrogens.

BACKGROUND: Microalbuminuria and hypertension are risk factors for diabetic nephropathy. Blockade of the renin-angiotensin system slows the progression to diabetic nephropathy in patients with type 1 diabetes, but similar data are lacking for hypertensive patients with type 2 diabetes. We evaluated the renoprotective effect of the angiotensin-II-receptor antagonist irbesartan in hypertensive patients with type 2 diabetes and microalbuminuria. METHODS: A total of 590 hypertensive patients with type 2 diabetes and microalbuminuria were enrolled in this multinational, randomized, double-blind, placebo-controlled study of irbesartan, at a dose of either 150 mg daily or 300 mg daily, and were followed for two years. The primary outcome was the time to the onset of diabetic nephropathy, defined by persistent albuminuria in overnight specimens, with a urinary albumin excretion rate that was greater than 200 microg per minute and at least 30 percent higher than the base-line level. RESULTS: The base-line characteristics in the three groups were similar. Ten of the 194 patients in the 300-mg group (5.2 percent) and 19 of the 195 patients in the 150-mg group (9.7 percent) reached the primary end point, as compared with 30 of the 201 patients in the placebo group (14.9 percent) (hazard ratios, 0.30 [95 percent confidence interval, 0.14 to 0.61; P< 0.001] and 0.61 [95 percent confidence interval, 0.34 to 1.08; P=0.081 for the two irbesartan groups, respectively). The average blood pressure during the course of the study was 144/83 mm Hg in the placebo group, 143/83 mm Hg in the 150-mg group, and 141/83 mm Hg in the 300-mg group (P=0.004 for the comparison of systolic blood pressure between the placebo group and the combined irbesartan groups). Serious adverse events were less frequent among the patients treated with irbesartan (P=0.02). CONCLUSIONS: Irbesartan is renoprotective independently of its blood-pressure-lowering effect in patients with type 2 diabetes and microalbuminuria.

INTRODUCTION: Microalbuminuria and hypertension are risk factors for the development of diabetic nephropathy. Blockade of the renin-angiotensin system reduces progression to diabetic nephropathy in type 1 diabetic patients, whereas similar data are lacking for hypertensive type 2 diabetic subjects. We evaluated the renoprotective effect of an angiotensin II receptor antagonist, irbesartan, in hypertensive type 2 diabetic patients with microalbuminuria. MATERIAL AND METHODS: Five hundred and ninety hypertensive type 2 diabetic patients with microalbuminuria were enrolled in this multinational, randomised, double-blind, placebo-controlled study of irbesartan 150 mg/daily or 300 mg/daily or matching placebo for two years. The primary outcome was time to progression to diabetic nephropathy, defined as a persistent overnight albuminuria > 200 micrograms/min and at least a 30 per cent increase from baseline. RESULTS: Baseline characteristics in the three groups were similar. Ten patients (5.2 per cent) receiving irbesartan 300 mg and 19 patients (9.7 per cent) receiving irbesartan 150 mg daily reached the primary end point, as compared to 30 (14.9 per cent) patients on placebo (hazard ratio 0.30 [95 per cent confidence interval 0.14 to 0.61], p < 0.001 and 0.61 [95 per cent confidence interval 0.34 to 1.08] p = 0.08), respectively). The average blood pressure throughout the study was 144/83, 143/83, and 141/81 mmHg in the placebo, irbesartan 150 mg and 300 mg group, respectively (p = 0.004 for systolic blood pressure). Serious adverse events were less frequent in the patients treated with irbesartan (p = 0.02). DISCUSSIONS: Irbesartan is renoprotective independently of its blood pressure lowering effect in type 2 diabetic subjects with microalbuminuria. It is safe and well tolerated.

Our appreciation of the physiological functions of estrogens and the mechanisms through which estrogens bring about these functions has changed during the past decade. Just as transgenic mice were produced in which estrogen receptors had been inactivated and we thought that we were about to understand the role of estrogen receptors in physiology and pathology, it was found that there was not one but two distinct and functional estrogen receptors, now called ER alpha and ER beta. Transgenic mice in which each of the receptors or both the receptors are inactive have revealed a much broader role for estrogens in the body than was previously thought. This decade also saw the description of a male patient who had no functional ER alpha and whose continued bone growth clearly revealed an important function of estrogen in men. The importance of estrogen in both males and females was also demonstrated in the laboratory in transgenic mice in which the aromatase gene was inactivated. Finally, crystal structures of the estrogen receptors with agonists and antagonists have revealed much about how ligand binding influences receptor conformation and how this conformation influences interaction of the receptor with coactivators or corepressors and hence determines cellular response to ligands.

In most PCR-based tissue typing techniques the PCR amplification is followed by a post-amplification specificity step. In typing by PCR amplification with sequence-specific primers (PCR-SSP), typing specificity is part of the amplification step, which makes the technique almost as fast as serological tissue typing. In the present study primers were designed for DR "low-resolution" typing by PCR-SSP, i.e. identifying polymorphism corresponding to the serologically defined series DR1-DRw18. This resolution was achieved by performing 19 PCR reactions per individual, 17 for assigning DR1-DRw18 and 2 for the DRw52 and DRw53 superspecificities. Thirty cell lines and 121 individuals were typed by the DR "low-resolution" PCR-SSP technique, TaqI DRB-DQA-DQB RFLP analysis and serology. The concordance between PCR-SSP typing and RFLP analysis was 100%. The reproducibility was 100% in 40 samples typed on two separate occasions. No false-positive or false-negative typing results were obtained. All homozygous and heterozygous combinations of DR1-DRw18 could be distinguished. Amplification patterns segregated according to dominant Mendelian inheritance. DNA preparation, PCR amplification and post-amplification processing, including gel detection, documentation and interpretation, were performed in 2 hours. In conclusion, PCR-SSP is an accurate typing technique with high sensitivity, specificity and reproducibility. The method is rapid and inexpensive. DR "low-resolution" typing by the PCR-SSP technique is ideally suited for analyzing small numbers of samples simultaneously and is an alternative to serological DR typing in routine clinical practice including donor-recipient matching in cadaveric transplantations.

Hormone-secreting cells within pancreatic islets of Langerhans play important roles in metabolic homeostasis and disease. However, their transcriptional characterization is still incomplete. Here, we sequenced the transcriptomes of thousands of human islet cells from healthy and type 2 diabetic donors. We could define specific genetic programs for each individual endocrine and exocrine cell type, even for rare δ, γ, ε, and stellate cells, and revealed subpopulations of α, β, and acinar cells. Intriguingly, δ cells expressed several important receptors, indicating an unrecognized importance of these cells in integrating paracrine and systemic metabolic signals. Genes previously associated with obesity or diabetes were found to correlate with BMI. Finally, comparing healthy and T2D transcriptomes in a cell-type resolved manner uncovered candidates for future functional studies. Altogether, our analyses demonstrate the utility of the generated single-cell gene expression resource.

Single-cell RNA sequencing is an increasingly used method to measure gene expression at the single cell level and build cell-type atlases of tissues. Hundreds of single-cell sequencing datasets have already been published. However, studies are frequently deposited as raw data, a format difficult to access for biological researchers due to the need for data processing using complex computational pipelines. We have implemented an online database, PanglaoDB, accessible through a user-friendly interface that can be used to explore published mouse and human single cell RNA sequencing studies. PanglaoDB contains pre-processed and pre-computed analyses from more than 1054 single-cell experiments covering most major single cell platforms and protocols, based on more than 4 million cells from a wide range of tissues and organs. The online interface allows users to query and explore cell types, genetic pathways and regulatory networks. In addition, we have established a community-curated cell-type marker compendium, containing more than 6000 gene-cell-type associations, as a resource for automatic annotation of cell types.

The estrogen receptor (ER) is a ligand-activated transcription factor that mediates the effects of the steroid hormone 17 beta-estradiol, in both males and females. Since the isolation and cloning of ER, the consensus has been that only one such receptor exists. The finding of a second subtype of ER (ER beta) has caused considerable excitement amongst endocrinologists. In this article, we present data regarding the genomic structure and chromosomal localization of the human ER beta gene, demonstrating that two independent ER genes do exist in the human. Furthermore, we present data regarding the tissue distribution of human ER beta, showing that this receptor is expressed in multiple tissues. For instance, ER beta is found in developing spermatids of the testis, a finding of potential relevance for the ongoing debate on the effects of environmental estrogens on sperm counts. In addition, we find ER beta in ovarian granulosa cells, indicating that estrogens also participate in the regulation of follicular growth in the human.

The rat, mouse and human estrogen receptor (ER) exists as two subtypes, ERα and ERβ, which differ in the C-terminal ligand-binding domain and in the N-terminal transactivation domain. In this study, we investigated the estrogenic activity of environmental chemicals and phytoestrogens in competition binding assays with ERα or ERβ protein, and in a transient gene expression assay using cells in which an acute estrogenic response is created by cotransfecting cultures with recombinant human ERα or ERβ complementary DNA (cDNA) in the presence of an estrogen-dependent reporter plasmid. Saturation ligand-binding analysis of human ERα and ERβ protein revealed a single binding component for[ 3H]-17β-estradiol (E2) with high affinity[ dissociation constant (Kd) = 0.05 - 0.1 nm]. All environmental estrogenic chemicals [polychlorinated hydroxybiphenyls, dichlorodiphenyltrichloroethane (DDT) and derivatives, alkylphenols, bisphenol A, methoxychlor and chlordecone] compete with E2 for binding to both ER subtypes with a similar preference and degree. In most instances the relative binding affinities (RBA) are at least 1000-fold lower than that of E2. Some phytoestrogens such as coumestrol, genistein, apigenin, naringenin, and kaempferol compete stronger with E2 for binding to ERβ than to ERα. Estrogenic chemicals, as for instance nonylphenol, bisphenol A, o, p′-DDT and 2′,4′,6′-trichloro-4-biphenylol stimulate the transcriptional activity of ERα and ERβ at concentrations of 100-1000 nm. Phytoestrogens, including genistein, coumestrol and zearalenone stimulate the transcriptional activity of both ER subtypes at concentrations of 1–10 nm. The ranking of the estrogenic potency of phytoestrogens for both ER subtypes in the transactivation assay is different; that is, E2 ≫ zearalenone = coumestrol > genistein > daidzein > apigenin = phloretin > biochanin A = kaempferol = naringenin> formononetin = ipriflavone = quercetin = chrysin for ERα and E2 ≫ genistein = coumestrol > zearalenone > daidzein > biochanin A = apigenin = kaempferol = naringenin > phloretin = quercetin = ipriflavone = formononetin = chrysin for ERβ. Antiestrogenic activity of the phytoestrogens could not be detected, except for zearalenone which is a full agonist for ERα and a mixed agonist-antagonist for ERβ. In summary, while the estrogenic potency of industrial-derived estrogenic chemicals is very limited, the estrogenic potency of phytoestrogens is significant, especially for ERβ, and they may trigger many of the biological responses that are evoked by the physiological estrogens.

BACKGROUND: Sirolimus (rapamycin) is a potent immunosuppressant with a mechanism of action different from cyclosporine (CsA) or tacrolimus. METHODS: In 11 European centers, first cadaveric renal allograft recipients were randomized to CsA (n=42) or sirolimus (n=41). Dosing of these agents was concentration-controlled and open-labeled. All patients received corticosteroids and azathioprine. RESULTS: At 12 months, graft survival (98% sirolimus vs. 90% CsA), patient survival (100% vs. 98%), and incidence of biopsy-confirmed acute rejection (41% vs. 38%) were similar. Serum creatinine was lower with sirolimus, significantly (P< or =0.05) so at 3 and 4 months, and serum uric acid and magnesium were normal. Laboratory abnormalities reported significantly more often with sirolimus included hypertriglyceridemia (51% vs. 12%), hypercholesterolemia (44% vs. 14%), thrombocytopenia (37% vs. 0%), leukopenia (39% vs. 14%), and, of lesser importance, increased liver enzymes and hypokalemia. These abnormalities improved 2 months after transplantation when the sirolimus target trough level was lowered from 30 to 15 ng/ml. Occurrence of cytomegalovirus was comparable (14% vs. 12%); incidences of herpes simplex (24% vs. 10%, P=0.08) and pneumonia (17% vs. 2%, P=0.03) were higher with sirolimus. No gingival hyperplasia was seen with sirolimus, tremor was rare, and hypertension was less frequent (17% vs. 33%). Two malignancies were observed with CsA and none with sirolimus. CONCLUSIONS: Results at 12 months suggest that sirolimus can be used as base therapy in the prophylaxis of acute renal transplant rejection, and has a safety profile that differs from CsA.

BACKGROUND: Cognitive impairment in MS impacts negatively on many patients at all disease stages and in all subtypes. Full clinical cognitive assessment is expensive, requiring expert staff and special equipment. Test versions and normative data are not available for all languages and cultures. OBJECTIVE: To recommend a brief cognitive assessment for multiple sclerosis (MS) that is optimized for small centers, with one or few staff members, who may not have neuropsychological training and constructed to maximize international use. METHODS: An expert committee of twelve members representing the main cultural groups that have so far contributed considerable data about MS cognitive dysfunction was convened. Following exhaustive literature review, peer-reviewed articles were selected to cover a broad spectrum of cultures and scales that targeted cognitive domains vulnerable to MS. Each was rated by two committee members and candidates scales were rated on psychometric qualities (reliability, validity, and sensitivity), international application, ease of administration, feasibility in the specified context, and acceptability to patients. RESULTS: The committee recommended the Symbol Digit Modalities Test, if only 5 minutes was available, with the addition of the California Verbal Learning Test - Second Edition and the Brief Visuospatial Memory Test - Revised learning trials if a further 10 minutes could be allocated for testing. CONCLUSIONS: A brief cognitive assessment for MS has been recommended. A validation protocol has been prepared for language groups and validation studies have commenced.

It is believed that malnutrition is common in patients with chronic renal failure (CRF). They have reduced body weight, depleted energy (fat tissue) stores, loss of somatic protein (low muscle mass) and low levels of serum albumin, transferrin, pre‐albumin and other visceral proteins. Various studies show signs of malnutrition in 23–76% of haemodialysis (HD) and 18–50% of peritoneal dialysis (PD) patients [1–4]. Such variations in the prevalence of malnutrition may be related to factors such as age, case mix, co‐morbid conditions and quality of dialysis therapy. The aetiology of malnutrition in CRF is complex and may include many factors, e.g. poor food intake because of anorexia, nausea and vomiting due to uraemic toxicity, hormonal derangements, acidosis and increased resting energy expenditure. While malnutrition by definition is caused by poor nutritional intake, laboratory or anthropometric measurements are generally used to define it clinically. Other factors can cause the same changes in body and plasma protein composition, especially inflammatory and infectious complications [5,6] and chronic heart failure (CHF). In addition, factors directly associated with the dialytic procedure, such as bio‐incompatibility, nutrient losses in the dialysate and, during PD, poor appetite due to abdominal discomfort and uptake of glucose may also contribute to what we define as malnourishment in CRF. These may exert their action either by direct nutrient loss or by triggering the inflammatory response. However, since malnutrition also occurs in pre‐dialysis patients [7], it is evident that dialysis‐unrelated factors, e.g. infectious and inflammatory complications as well as co‐morbidity, may also be important contributors to malnutrition in CRF. Many methods have been used to assess the presence of malnutrition in patients with CRF. A history of weight loss and symptoms such as anorexia, nausea and vomiting may indicate impending or established malnutrition. Anthropometric measurements, such as mid‐arm muscle circumference, skinfold thickness and hand‐grip strength may all be useful tools for estimating malnutrition. Hand‐grip strength, in particular, has been shown to be an inexpensive, reliable and easily performed parameter of nutrition [4,8] that also predicts mortality in CRF patients (unpublished observation). Creatinine kinetics have also been advocated as a method to assess nutritional status. However, recent evidence suggests that it is unreliable in individual CRF patients [8,9]. More sophisticated methods used to evaluate nutritional status include bio‐electrical impedance, dual‐emission X‐ray absorptiometry (DXA), nuclear magnetic resonance, computerized tomography, total body potassium and total body nitrogen, but mostly as research tools. Finally, several biochemical markers [e.g. serum albumin, pre‐albumin, insulin‐like growth factor‐1 (IGF‐1) and transferrin] have been used to evaluate nutritional status. Of these biochemical markers, serum albumin so far has been the most common to assess malnutrition, and hypoalbuminaemia has sometimes, perhaps erroneously (see below), been used to diagnose malnutrition [10]. The protein requirements in maintenance dialysis patients are not well defined. It can be assumed that the variation in protein requirements is much greater among dialysis patients than in healthy subjects, due to additional causes of variation, such as endocrine and biochemical abnormalities, anaemia, drugs, physical inactivity and co‐morbid conditions, e.g. cardiovascular disease, diabetes and infections. In addition, specific effects of the dialytic process may increase the protein requirements, especially in patients treated with HD. The daily protein intake recommended is ∼0.6 g/kg body weight/day in non‐dialysed patients and at least 1.2 g/kg body weight/day in dialysis patients [11]. The energy requirements are dependent on the level of physical activity. In healthy subjects, an energy intake of 35–40 kcal/kg body weight/day is recommended for those not performing heavy physical exercise. There is no strong evidence that the energy requirements of chronic dialysis patients always differ from those of normal subjects [12,13] although increased energy expenditure has been reported in the former group [14]. A sufficient energy intake is needed to prevent protein from being utilized as an energy source via gluconeogenesis, and variations in energy intake can probably explain, at least partly, the inter‐individual variations in nitrogen balance in CRF patients on similar protein intake [15]. However, the inter‐patient variation in nitrogen balance with similar protein intake may also be related in part to the presence and degree of inflammation and/or co‐morbid conditions. Thus, more studies are needed to evaluate dietary protein intake and energy requirements in clinically stable dialysis patients and in dialysis patients with co‐morbid conditions and/or an inflammatory response [11]. It is well established that a low serum albumin level is a strong independent predictor of total and cardiovascular mortality in HD [16] and PD [17,18] patients. On the other hand, in non‐renal patients, no association was found between serum albumin and cardiovascular disease [19], suggesting that a low serum albumin level per se does not necessarily contribute to cardiovascular mortality. Moreover, Koch et al. [20] have reported that the nutritional status alone does not predict overall or cardiovascular mortality, and Struijk et al. [21] have shown that serum albumin merely reflects the presence of systemic disease in dialysis patients. It has been proposed that the primary cause of hypoalbuminaemia in CRF is malnutrition [22]. However, poor food intake does not often result in hypoalbuminaemia if CRF is not present [23] and, although the food intake is markedly lower in patients with anorexia nervosa, serum albumin levels and the catabolic rate of albumin have been shown to be similar to those of control subjects [24]. Furthermore, in a prospective 24 week study in which healthy volunteers were subjected to semi‐starvation (1500 kcal/24 h), serum albumin decreased only moderately (from 42.8 to 38.6 g/l) despite a 23% reduction in body weight (from 69.3 to 53.6 kg) and muscle mass [25]. Serum albumin levels may be low even in apparently well nourished HD patients, and they decrease in relation to the degree of malnutrition [4]. Although they differ markedly in patients with or without inflammation, they do not differ significantly between well‐nourished and malnourished pre‐dialysis patients [8], suggesting that serum albumin is a poor nutritional marker also in dialysis patients [26,27]. Despite these findings and the fact that today several alternative methods are available for assessing nutritional status, serum albumin still seems to be, by far, the most commonly used nutritional marker in CRF patients. Serum albumin concentration is best seen as being regulated by several factors, especially protein malnutrition, inflammation and external losses. The latter is important in PD, where transperitoneal losses may be as great as 20 g/24 h [28], and in HD, especially following re‐use with bleach [29], although other processes may also increase dialyser permeability to albumin. While dietary protein insufficiency may also cause a modest reduction in serum albumin concentration, inflammation by itself can lead to a very marked reduction [30]. Inflammation can cause hypoalbuminaemia by suppressing albumin synthesis [30] and by causing transfer of albumin from the vascular to the extravascular space. The combination of inflammation and reduced protein intake will lead to a significant reduction in serum albumin concentration [31], as shown in Figure 1. Relationship between subjective global assessment (SGA), C‐reactive protein (CRP) and serum albumin in 95 pre‐dialysis patients in Stockholm (top) and between normalized protein catabolic rate (nPCR), CRP and serum albumin in 79 haemodialysis patients in Sacramento (bottom). Both CRP and markers of nutrition (SGA and nPCR, respectively) independently controlled serum albumin concentrations in both models. In recent years, several reports have suggested that inflammation, alone or in combination with a low protein intake, plays a significant role in causing hypoalbuminaemia in CRF patients [31–34] (Figure 1). This is not unexpected since both serum albumin and C‐reactive protein (CRP) participate reciprocally in the same acute‐phase process. It has been established recently that moderately elevated plasma concentrations of CRP are associated with an increased risk of cardiovascular disease in otherwise healthy subjects. In non‐renal patient populations, elevated levels of CRP are associated with cardiovascular morbidity [35], ischaemic stroke [36] and mortality in the elderly [37]. Likewise, several groups recently have reported that an increased CRP is also a strong risk factor for death [33,38,39], cardiovascular mortality [38] and hospitalization [40] in dialysis patients. Furthermore, recently we have found a strong relationship between malnutrition, elevated CRP levels and atherosclerosis in pre‐dialysis patients [7]. The prevalence of an increased CRP (>8–10 mg/l) has been reported to be high in dialysis [4,38,41] and pre‐dialysis [7] patients. Serum levels of CRP appear to reflect generation of pro‐inflammatory cytokines [interleukin‐1 (IL‐1), IL‐6 and tumour necrosis factor‐α (TNF‐α)] which have also been reported to be increased in CRF patients [42,43]. It is well documented that high levels of pro‐inflammatory cytokines may cause muscle wasting by stimulating protein catabolism via the ubiquitin–proteosome pathway [44] by reducing albumin synthesis and by inhibiting appetite [45]. It has also been observed that IL‐1, TNF‐α and endotoxins may induce net catabolism of muscle protein by stimulating branched‐chain ketoacid dehydrogenase, which leads to greater oxidation of branched‐chain amino acids [46]. In consequence, increased plasma levels of pro‐inflammatory cytokines predict hypoalbuminaemia [34] and mortality [34,43] in dialysis patients. Furthermore, whereas serum albumin and CRP predict mortality in univariate analysis, only CRP is a significant predictor in multivariate analysis [33,38]. Taken together, available evidence suggests that the increased mortality rate observed in CRF may be associated with an acute‐phase response rather than low serum albumin levels caused by other mechanisms. Energy deficiency due to low energy intake and increased resting energy expenditure (REE) may be another important cause of wasting in dialysis patients. In general, failure to down‐regulate REE as an adaptation to anorexia is often said to be a major cause of weight loss and wasting. Although REE may be increased in patients with CHF [47], cancer [48,49], rheumatoid arthritis [50] and AIDS [51], either normal [12,13,52] or increased [14] REE have been observed in CRF patients. These discrepant REE findings in dialysis patients are not understood. However, inflammation repeatedly has been shown to be associated with increased REE in other patients with wasting disorders [48–50,53]. On the basis of these observations, some of the reported variations in REE in dialysis patients may reflect differences in the prevalence of inflammation. Thus, the presence of the acute‐phase response permits identification of hypermetabolic patients [48]. Indeed, Schneeweiss et al. [13] have reported increased REE in dialysis patients with infections. Therefore, since inflammation increases REE, we suggest that this may be a factor contributing to protein calorie malnutrition in CRF patients. Oxidative stress, which occurs in the presence of excessive free‐radical production or low anti‐oxidant levels, is an important co‐factor for development of endothelial dysfunction and atherogenesis. Recent data indicate that increased oxidative stress occurs in dialysis patients [54]. Malnourished pre‐dialysis patients have biochemical evidence of more oxidative stress than well‐nourished ones [55]. Moreover, malnourished pre‐dialysis patients have lower plasma levels of vitamin E [7], suggesting a low intake of anti‐oxidants when nutritional intake is low. In addition, recent data show that inflammation is associated with an increase in oxidative stress [56] and that advanced oxidation protein products are associated with monocyte activation in CRF patients [57]. On the basis of these findings, one can speculate that an increase in oxidative stress may contribute to the high prevalence of cardiovascular disease in malnourished CRF patients. Human plasma contains a variety of anti‐oxidant biomolecules and it is of interest that serum albumin may act as the principal anti‐oxidant in plasma [58]. Thus, low serum albumin levels associated with malnutrition and/or inflammation may increase atherogenesis by raising oxidative stress. Indeed, Soejima et al. [59] have shown that hypoalbuminaemia accelerates erythrocyte membrane lipid peroxidation in HD patients. One mechanism by which greater oxidative stress may accelerate atherogenesis is by affecting endothelial function. In fact, increased oxidant stress appears to play a pathophysiological role in the deleterious endothelial effects of homocysteine [60]. Atherosclerosis is related to endothelial dysfunction and reduced bio‐availability of nitric oxide (NO). It is not yet known whether CRF patients have reduced or increased NO production because, although whole‐body NO production is reduced [61], excretion of NO is increased in exhaled air of patients with CRF [62]. However, both PD [63] and HD [64] patients have impaired endothelium‐dependent vasodilation. It should be noted that NO forms an adduct with serum albumin that has endothelium‐derived relaxing properties [65]. Thus, low albumin levels could be associated with impaired endothelial‐dependent vasodilation. Although Kim et al. [66] showed correlations between serum albumin, CRP and serum markers of endothelial function, an infusion of albumin did not normalize endothelial function. Consequently, their findings strongly suggest that the relationship between low serum albumin levels and endothelial dysfunction may be secondary to other factors, such as inflammation. Indeed, Kessler et al. [67] have reported in rabbits that pro‐inflammatory mediators inhibit the formation of endothelium‐dependent hyperpolarizing factor which may contribute to endothelial dysfunction. Moreover, an increased CRP is associated with endothelial dysfunction in patients with diabetes [68]. Altogether, available evidence suggests that inflammation can be associated with endothelial dysfunction, which may accelerate atherosclerosis. It has been well documented that CHF is associated with malnutrition (cardiac cachexia) and increased levels of pro‐inflammatory cytokines. More than 50% of patients with CHF have signs of cardiac cachexia [69]. Evidence of muscle wasting is present even in mild CHF [70], and muscle wasting appears to be related to reduced exercise capacity [71]. The pathogenesis of cardiac cachexia is not understood, and no relationship between nutritional intake and cardiac cachexia has been established. However, elevated serum levels of TNF‐α are related to cardiac cachexia [72]. The serum levels of IL‐6 are also significantly higher even in mild or moderate CHF [73]. It has been suggested that pro‐inflammatory cytokines, generated in response to factors such as reduced tissue perfusion, altered gut permeability and congestion, may play an important role in the loss of lean body mass in these patients. Indeed, cardiac cachexia is a strong independent risk factor for mortality in patients with CHF [74]. Since the prevalence of CHF is high among dialysis patients [75], CHF may contribute to wasting, elevated levels of pro‐inflammatory cytokines and increased mortality even in dialysis patients. It may seem puzzling that whereas hypoalbuminaemia and inflammation have been shown to be important predictors of mortality in dialysis patients, complications from malnutrition as such are not common causes of mortality in dialysis patients [76]. In fact, malnutrition accounts for <5% of deaths in renal patients [77] while atherosclerotic cardiovascular disease is by far the most common cause of mortality in the dialysis population [78]. How can this finding be explained? A recent study suggests that strong interactions exist between cardiovascular disease and inflammatory as well as nutritional parameters in CRF patients [7]. We have therefore suggested the existence of a syndrome consisting of malnutrition, inflammation and atherosclerosis (MIA syndrome) in some patients with CRF [79]. Indeed, inflammation is more common in malnourished HD patients [4], and Ikizler et al. [40] have shown that the nutritional status and inflammatory response are independent predictors of hospitalization in HD patients. Moreover, malnutrition [80] and inflammation [38] are associated with a higher cardiovascular mortality rate in HD patients. Taken together, available evidence suggests that nutritional and inflammatory markers are closely linked to cardiovascular disease in CRF. It therefore seems likely that elevated levels of pro‐inflammatory cytokines could be the link between the high prevalence of inflammation, malnutrition and cardiovascular disease in patients with CRF (Figure 2). The vicious circle of malnutrition, inflammation and atherosclerotic cardiovascular disease (MIA syndrome) in patients with chronic renal failure. A central role in this scenario is played by the pro‐inflammatory cytokines generated in response to factors such as chronic heart failure and infectious/inflammatory co‐morbid diseases. On the basis of findings discussed above, it seems reasonable to conclude that the acute‐phase response and malnutrition are closely linked and that both these conditions may contribute to the excessive atherosclerotic cardiovascular mortality observed in dialysis patients. Therefore, like Baltzan and Shoker [81], we believe that at least two types of malnutrition may occur in dialysis patients (Table 1). The first type (type 1) is associated with the uraemic syndrome per se or factors associated with uraemia (such as physical inactivity, underdialysis, dietary restrictions and psychosocial factors). It is characterized by a modest reduction in serum albumin levels, because of lower protein and energy intake due to uraemic toxicity. The first signs of protein and energy malnutrition begin early in the course of progressive renal failure [82] and the development of renal failure is associated with a spontaneous decrease in dietary protein intake [83]. Significant co‐morbidity, such as CHF, and elevated levels of pro‐inflammatory cytokines are usually not present in this type of malnutrition. The main feature is a low protein energy intake due to uraemic anorexia, with a corresponding decrease in protein catabolism. REE may be normal in this type of malnutrition. More marked hypoalbuminaemia, higher REE, markedly increased oxidative stress and increased protein catabolism, on the other hand, would characterize the other type of malnutrition (type 2). Significant co‐morbid conditions, such as CHF, frequently are found in this type of malnutrition, and there is usually an inflammatory response, as evidenced by higher levels of CRP and pro‐inflammatory cytokines. However, the spectrum of malnutrition in CRF may involve a continuous overlap between both types of malnutrition (Figure 3), and most dialysis patients probably have a mixed type of malnutrition. It is of interest that if one uses either normalized protein catabolic rate in HD patients, or subjective global assessment in pre‐dialysis patients as markers of nutrition, reduction in serum albumin concentration of is only when CRP levels are increased (Figure 1). Thus, hypoalbuminaemia in these patient may well reflect this combination between malnutrition and inflammation. It seems likely that type malnutrition occurs not only in CRF patients but also in those with wasting e.g. CHF, chronic inflammatory rheumatoid arthritis and advanced cancer and chronic of (such as low intake of protein and energy due to uraemic anorexia, underdialysis, physical inactivity, and the inflammatory of malnutrition in patients with type and type malnutrition, of type and type malnutrition of type and type malnutrition studies have the effects of nutritional in patients with CRF the findings have been to usually because of the of control A recent that the data the of nutrition are One for this may be that the patient were both patients with type and type malnutrition were In whereas nutritional and dialysis is probably sufficient to type malnutrition, this will the nutritional status in type malnutrition. Indeed, pro‐inflammatory effects of the dialytic per se (such as of the dialyser the to endotoxins in the dialysis and may this type of malnutrition. Therefore, it is that type malnutrition can be treated co‐morbid and/or of chronic inflammation are Indeed, some recent findings suggest that the of nutritional may be if co‐morbid conditions and/or inflammation are recent data show that co‐morbid conditions seem to the on the nutritional in PD patients of dialysis Moreover, et al. have found recently that inflammatory the of growth in HD patients. Finally, nutritional to increase serum albumin have been reported to especially in patients with low Despite the in dialysis the mortality rate from cardiovascular disease is still high in dialysis patients and are needed for the Indeed, several may be of interest and should be in prospective in patients malnutrition associated with In of the strong between CHF, muscle wasting and elevated levels of pro‐inflammatory cytokines, we believe that it is very important to cardiac in dialysis patients. In this with may be of This of has been shown not only to cardiac and the mortality rate in patients with cardiovascular disease but also to be associated with a nutritional status and lower levels of TNF‐α in CRF patients et al. recently found that with reduced the risk of weight loss in patients with It is that in to their may also the response to and in CRF. In of the documented association between increased oxidative stress and endothelial dysfunction we speculate that anti‐oxidant may endothelial in malnourished dialysis patients. Indeed, vitamin endothelial dysfunction of the in patients Since there is evidence suggesting that and may play a role in the pathogenesis of cardiovascular disease prospective studies are needed to whether or may the cardiovascular and nutritional status of dialysis patients. Finally, TNF‐α and in other patient groups with wasting disorders has been found to be associated with a not only in findings but also in inflammatory It should also be noted that the production of by the of TNF‐α the wasting syndrome associated with and studies are therefore needed to whether are and may have a on cardiovascular and nutritional status and mortality rate in CRF patients with signs of wasting and inflammation. inflammation, as evidenced by increased levels of pro‐inflammatory cytokines and is common in CRF patients and may cause malnutrition and progressive atherosclerotic cardiovascular disease by several mechanisms. We therefore that at least two types of malnutrition exist in one without (type 1) and one with (type a inflammatory response and significant It is likely that the for these types of malnutrition should be very We suggest that in prospective in which the effects of on nutritional and/or cardiovascular status are in CRF patients, an assessment of inflammatory status and between the two types of malnutrition should be and of This study was by the and the

BACKGROUND: An association between polymyositis and cancer was first proposed in 1916, but the existence of the association has been disputed. An association between dermatomyositis and cancer is better accepted, but its magnitude is not known. METHODS: We undertook a study to provide accurate estimates of the risk of cancer in patients with dermatomyositis or polymyositis. We studied the incidence of cancer and the rate of mortality from cancer in a population-based cohort of 788 patients with dermatomyositis or polymyositis in Sweden from 1963 through 1983. The results were compared with those for the general population. RESULTS: Among the 396 patients with polymyositis, 42 cancers were diagnosed at the same time or after polymyositis was diagnosed in 37 patients (9 percent). The relative risk of cancer was 1.8 (95 percent confidence interval, 1.1 to 2.7) in the male patients and 1.7 (95 percent confidence interval, 1.0 to 2.5) in the female patients. Eighty-four males and 85 females died, and in 24 of these cases (14 percent) cancer was the principal cause of death. The mortality ratio (the rate of mortality from cancer in these patients as compared with that in the general population) was 0.90 (95 percent confidence interval, 0.6 to 1.4). Among the 392 patients with dermatomyositis, 61 cancers were diagnosed at the same time or after dermatomyositis was diagnosed in 59 patients (15 percent). The relative risk of cancer was 2.4 (95 percent confidence interval, 1.6 to 3.6) in the male patients and 3.4 (95 percent confidence interval, 2.4 to 4.7) in the female patients. Fifty-seven males and 110 females died, and in 67 of these cases (40 percent) cancer was the principal cause of death (mortality ratio, 3.8; 95 percent confidence interval, 2.9 to 4.8). CONCLUSIONS: The risk of cancer is increased in patients with polymyositis or dermatomyositis. In patients with dermatomyositis there is also a higher rate of mortality from cancer.

The discovery of genetic loci associated with complex diseases has outpaced the elucidation of mechanisms of disease pathogenesis. Here we conducted a genome-wide association study (GWAS) for coronary artery disease (CAD) comprising 181,522 cases among 1,165,690 participants of predominantly European ancestry. We detected 241 associations, including 30 new loci. Cross-ancestry meta-analysis with a Japanese GWAS yielded 38 additional new loci. We prioritized likely causal variants using functionally informed fine-mapping, yielding 42 associations with less than five variants in the 95% credible set. Similarity-based clustering suggested roles for early developmental processes, cell cycle signaling and vascular cell migration and proliferation in the pathogenesis of CAD. We prioritized 220 candidate causal genes, combining eight complementary approaches, including 123 supported by three or more approaches. Using CRISPR-Cas9, we experimentally validated the effect of an enhancer in MYO9B, which appears to mediate CAD risk by regulating vascular cell motility. Our analysis identifies and systematically characterizes >250 risk loci for CAD to inform experimental interrogation of putative causal mechanisms for CAD.

Many important cell types in adult vertebrates have a mesenchymal origin, including fibroblasts and vascular mural cells. Although their biological importance is undisputed, the level of mesenchymal cell heterogeneity within and between organs, while appreciated, has not been analyzed in detail. Here, we compare single-cell transcriptional profiles of fibroblasts and vascular mural cells across four murine muscular organs: heart, skeletal muscle, intestine and bladder. We reveal gene expression signatures that demarcate fibroblasts from mural cells and provide molecular signatures for cell subtype identification. We observe striking inter- and intra-organ heterogeneity amongst the fibroblasts, primarily reflecting differences in the expression of extracellular matrix components. Fibroblast subtypes localize to discrete anatomical positions offering novel predictions about physiological function(s) and regulatory signaling circuits. Our data shed new light on the diversity of poorly defined classes of cells and provide a foundation for improved understanding of their roles in physiological and pathological processes.

BACKGROUND: Mesenchymal stem cells (MSCs) can reduce the incidence of graft-versus-host disease because of their ability to inhibit T-lymphocyte proliferation. There are no publications on the effect that MSCs have on cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, effector cells vital for the graft-versus-leukemia effect. METHODS: Cytotoxic T cells were primed in mixed lymphocyte culture (MLC) against irradiated stimulator lymphocytes, and irradiated third-party MSCs were added at different time points. The CTLs were collected, and their cytotoxic potential was analyzed in a chromium-release assay against the same stimulator cells as in the MLC. Purified NK cells were mixed with irradiated MSCs, and the lysis was measured in chromium-release assay against K562 target cells. RESULTS: We found that MSCs inhibited CTL-mediated lysis by 70% if added at the beginning of the 6-day MLC. The lysis was not affected on day 3 or in the cytotoxic phase. Furthermore, MSCs inhibited the formation of cytotoxic lymphocytes when the cells were separated in a transwell system, which indicates that the effect is mediated by a soluble factor. NK cell-mediated lysis of K562 cells was not inhibited by MSCs. MSCs did not induce proliferation of allogeneic lymphocytes, and they were not lysed by allogeneic CTLs or NK cells. CONCLUSION: Our findings indicate that MSCs escape recognition by CTLs and alloreactive NK cells, and inhibit the formation of cytotoxic T cells by secreting a soluble factor, but that they do not interfere with CTLs and NK cell lysis.

OBJECTIVE: To assess the impact of possible risk factors on intestinal resection and postoperative recurrence in Crohn's disease (CD) and to evaluate the disease course. SUMMARY BACKGROUND DATA: The results of previous studies on possible risk factors for surgery and recurrence in Crohn's disease have been inconsistent. Varying findings may be explained by referral biases and small numbers of patients in some studies. METHODS: Data on initial intestinal resection and postoperative recurrence were evaluated retrospectively in a population-based cohort of 1,936 patients. The influence of concomitant risk factors was assessed using uni- and multivariate analyses. RESULTS: The cumulative rate of intestinal resection was 44%, 61%, and 71% at 1, 5, and 10 years after diagnosis. Postoperative recurrences occurred in 33% and 44% at 5 and 10 years after resection. The relative risk of surgery was increased in patients with CD involving any part of the small bowel, in those having perianal fistulas, and in those who were 45 to 59 years of age at diagnosis. Female gender and perianal fistulas, as well as small bowel and continuous ileocolonic disease, increase the relative risk of recurrence. CONCLUSIONS: Three of four patients with CD will undergo an intestinal resection; half of them will ultimately relapse. The extent of disease at diagnosis and the presence of perianal fistulas have an impact on the risk of surgery and the risk of postoperative recurrence. Women run a higher risk of postoperative recurrence than men. The frequency of surgery has decreased over time, but the postoperative relapse rate remains unchanged.

The theory that cancer may arise under conditions of reduced immune capacity is supported by observations of humans with immune deficiencies such as occur following organ transplants. However, no study on humans has been done in which the reference population was the same as that in which the cancer cases arose and in which there was a sufficiently long period of follow-up. Information on 5,692 Nordic recipients of renal transplants in 1964-1982 was linked with the national cancer registries (1964-1986) and population registries. Person-years at risk were calculated from the date of first transplantation until death or the end of the study period and were multiplied by the appropriate age- and calender-specific incidence rates to obtain the expected numbers of cancers. Standardized incidence ratios (SIR) were calculated after stratification by a number of recorded variables. Altogether, 32,392 person-years were accrued, and 471 cancers occurred, yielding overall SIR of 4.6 (95% CI, 4.0 to 5.2) for males and 4.5 (95% CI, 4.0 to 5.2) for females. Significant overall 2- to 5-fold excess risks in both sexes were seen for cancers of the colon, larynx, lung and bladder, and in men also for cancers of the prostate and testis. Notably high risks, 10-fold to 30-fold above expectation, were associated with cancers of the lip, skin (non-melanoma), kidney and endocrine glands, also with non-Hodgkin's lymphoma, and in women also with cancers of the cervix and vulva-vagina. Among a number of donor and recipient variables studied, including tissue types and compatibility (ABO, HLA, DR), age below 45 years at the time of transplantation was the most important determinant for increased risk at most sites. Kidney transplantation increases the risk of cancer in the short and in the long term, consistent with the theory that an impaired immune system allows carcinogenic factors to act. The tumor risk is small in comparison with the benefits of transplants, but patients should be followed up for signs of cancer.

BACKGROUND: A consensus has not been reached about the optimal duration of oral anticoagulant therapy after a second episode of venous thromboembolism. METHODS: In a multicenter trial, we compared six months of oral anticoagulant therapy with anticoagulant therapy continued indefinitely in patients who had had a second episode of venous thromboembolism. Of 227 patients enrolled, 111 were randomly assigned to six months of anticoagulation and 116 were assigned to receive anticoagulant therapy indefinitely; for both groups, the target international normalized ratio was 2.0 to 2.85. The initial episodes of deep-vein thrombosis (n = 193) and pulmonary embolism (n = 34), as well as recurrent episodes, were all objectively confirmed. RESULTS: After four years of follow-up, there were 26 recurrences of venous thromboembolism that fulfilled the diagnostic criteria, 23 in the group assigned to six months of therapy (20.7 percent) and 3 in the group assigned to continuing therapy (2.6 percent). The relative risk of recurrence in the group assigned to six months of therapy, as compared with the group assigned to therapy of indefinite duration, was 8.0 (95 percent confidence interval, 2.5 to 25.9). There were 13 major hemorrhages, 3 in the six-month group, (2.7 percent) and 10 in the infinite-treatment group (8.6 percent). The relative risk of major hemorrhage in the six-month group, as compared with the infinite-treatment group was 0.3 (95 percent confidence interval, 0.1 to 1.1). There was no difference in mortality between the two groups. CONCLUSIONS: Prophylactic oral anticoagulation that was continued for an indefinite period after a second episode of venous thromboembolism was associated with a much lower rate of recurrence during four years of follow-up than treatment for six months. However, there was a trend toward a higher risk of major hemorrhage when anticoagulation was continued indefinitely.

The three-dimensional structure of the DNA-binding domain (DBD) of the glucocorticoid receptor has been determined by nuclear magnetic resonance spectroscopy and distance geometry. The structure of a 71-residue protein fragment containing two "zinc finger" domains is based on a large set of proton-proton distances derived from nuclear Overhauser enhancement spectra, hydrogen bonds in previously identified secondary structure elements, and coordination of two zinc atoms by conserved cysteine residues. The DBD is found to consist of a globular body from which the finger regions extend. A model of the dimeric complex between the DBD and the glucocorticoid response element is proposed. The model is consistent with previous results indicating that specific amino acid residues of the DBD are involved in protein-DNA and protein-protein interactions.