The role of transmembrane protein 27 (TMEM27) in islet physiology and its potential use as a beta cell mass biomarker.

AIMS/HYPOTHESIS: Transmembrane protein 27 (TMEM27) is a membrane protein cleaved and shed by pancreatic beta cells that has been proposed as a beta cell mass biomarker. Despite reports of its possible role in insulin exocytosis and cell proliferation, its function in beta cells remains controversial. We aimed to characterise the function of TMEM27 in islets and its potential use as a beta cell mass biomarker. METHODS: To determine TMEM27 function, we studied TMEM27 gene expression and localisation in human healthy and diabetic islets, the correlation of its expression with cell cycle and insulin secretion genes in human islets, its expression in tungstate-treated rats, and the effects of its overproduction on insulin secretion and proliferation in a beta cell line and islets. To elucidate its utility as a beta cell mass biomarker, we studied TMEM27 cleavage in a beta cell line, islets and primary proximal tubular cells. RESULTS: TMEM27 mRNA levels in islets are lower in diabetic donors than in controls. Its gene expression correlates with that of insulin and SNAPIN in human islets. TMEM27 expression is downregulated in islets of tungstate-treated rats, which exhibit decreased insulin secretion and increased proliferation. TMEM27 overproduction in a beta cell line and islets significantly enhanced glucose-induced insulin secretion, with modest or no effects on proliferation. Finally, TMEM27 is cleaved and shed by renal proximal tubular cells and pancreatic islets. CONCLUSIONS/INTERPRETATION: Our data support a role for TMEM27 in glucose-induced insulin secretion but not in cell proliferation. The finding that its cleavage is not specific to beta cells challenges the current support for its use as a potential beta cell mass biomarker.

Gene expression profiles for the human pancreas and purified islets in type 1 diabetes: new findings at clinical onset and in long-standing diabetes.

Type 1 diabetes (T1D) is caused by the selective destruction of the insulin-producing beta cells of the pancreas by an autoimmune response. Due to ethical and practical difficulties, the features of the destructive process are known from a small number of observations, and transcriptomic data are remarkably missing. Here we report whole genome transcript analysis validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and correlated with immunohistological observations for four T1D pancreases (collected 5 days, 9 months, 8 and 10 years after diagnosis) and for purified islets from two of them. Collectively, the expression profile of immune response and inflammatory genes confirmed the current views on the immunopathogenesis of diabetes and showed similarities with other autoimmune diseases; for example, an interferon signature was detected. The data also supported the concept that the autoimmune process is maintained and balanced partially by regeneration and regulatory pathway activation, e.g. non-classical class I human leucocyte antigen and leucocyte immunoglobulin-like receptor, subfamily B1 (LILRB1). Changes in gene expression in islets were confined mainly to endocrine and neural genes, some of which are T1D autoantigens. By contrast, these islets showed only a few overexpressed immune system genes, among which bioinformatic analysis pointed to chemokine (C-C motif) receptor 5 (CCR5) and chemokine (CXC motif) receptor 4) (CXCR4) chemokine pathway activation. Remarkably, the expression of genes of innate immunity, complement, chemokines, immunoglobulin and regeneration genes was maintained or even increased in the long-standing cases. Transcriptomic data favour the view that T1D is caused by a chronic inflammatory process with a strong participation of innate immunity that progresses in spite of the regulatory and regenerative mechanisms.

[Hepatitis type A with atypical course]. / Epatite A ad evoluzione atipica.

Hepatitis A virus infection is usually asymptomatic in children. Classic symptomatic forms and atypical clinical manifestations are known. We report a paediatric case of hepatitis A with marked cholestasis, treated with steroids, and with an unusual prolonged course.

[Acute acalculous cholecystitis as a complication of hepatitis A: report of 2 pediatric cases]. / Colecistite acuta acalcolotica in corso di epatite A: 2 casi pediatrici.

Acute acalculous cholecystitis (CAA) is very rare in children and it is usually related to infectious agents. We report 2 paediatric cases of CAA complicating hepatitis type A, with a favourable evolution with conservative treatment.

[Hexadactylism of the four limbs: a case report]. / Esadattilia dei quattro arti: un insolito caso.

An unusual case of postaxial hexadactylism of the hands and feet in one female Caucasian neonate is described. The clinical picture was characterized by symmetrical duplication of the 5th finger in both hands and of the 4th finger in both feet. Malformations of the extremities both in the paternal and in the maternal family were reported. No other associated malformations have been found in the baby and her karyotype was normal. The performed analysis of the literature confirmed the peculiarity of the associated features of this case.

Soluble sugars in soft drinks.

The qualitative quantitative composition of soluble carbohydrates have been determined in 16 frequently consumed soft drinks. The qualitative analysis was carried out by thin layer chromatography. The quantitative determination was done by column chromatography and spectrophotometric technique. Most of the soft drinks analyzed contain the monosaccharides glucose and fructose and the disaccharide sucrose. For some of the drinks, the amounts of these sugars vary from bottle to bottle of the same soft drink. This is probably due to a hydrolytic process of sucrose taking place during storage as a result of the acidic pH of the media. The content of total soluble carbohydrates of most of the drinks analyzed is rather high and may represent an important caloric supplement in the diet, considering the high consumption of these drinks by the Spanish population.

Chromatographic measurement of the carbohydrate content of some commonly used soft drinks.

The composition of soluble carbohydrates has been determined in some frequently consumed soft drinks. Qualitative analysis was carried out by thin layer and gas liquid chromatography. Quantitative determination was done by column chromatography and spectrophotometry, as well as by gas liquid chromatography. The results obtained by both methods were similar. In most of the soft drinks analyzed, the fructose and glucose contents ranged between 0.5 and 1.5 g/100 ml, and that of sucrose between 7 and 10 g/100 ml which, in total sugar content, is equal to 10 to 12 g/100 ml. This value may represent an important energy supplement in the diet, considering the high consumption of these drinks by the Spanish population.

Effects of an androgenic derivative on pre-established mammary tumours chemically induced in the rat.

The effects were studied of an androgenic derivative--danazol--administered at doses of 10-12 mg kg-1 day-1 during 97 days to rats with dimethylbenz[a] anthracene-induced mammary tumours. Our main observations were as follows. (a) Danazol did not influence ovarian function at the end of the assay. (b) The treatment with danazol reduced the incidence (P less than 0.05), number of tumours (P less than 0.05) and volume of malignant mammary tumours; on the other hand, the values of these parameters for benign tumours and those of doubtful expression were similar in both experimental groups. (c) Such differential action of Danazol seems to be due to the different incidence and/or content of receptors of both types of tumours. (d) The latter results lead to a hypothesis for the mechanism of action of danazol based on its behaviour at different levels.

Effects of an androgenic derivative on the development of chemically-induced mammary carcinogenesis in the rat.

The effects of an androgenic derivative--danazol--on the development of dimethylbenz(a)anthracene (DMBA)-rat mammary carcinogenesis were studied. Animals in the treated group received danazol (10-12 mg/kg/day) during 169 days, starting 5 days after DMBA induction. As compared with tumours in control animals, those treated with danazol appear later and are significantly smaller. Moreover, this treatment reduced the incidence of animals affected by mammary cancer and the average number of malignant tumours in each animal. In the same way, the incidence of animals with missing nodules was significantly higher in the group treated with danazol. It is concluded that danazol has an inhibitory effect on experimental mammary tumours. This effect seems to be greater the earlier the treatment is started and the longer time it is applied.

Experimental diets for the study of lipid influence on the induced mammary carcinoma in rats: II--Suitability of the diets.

We have previously reported one method for obtention of experimental diets for the study of the effects of dietary lipids on the rat breast carcinoma. The purpose of this second part of the study was to develop a quality control system for demonstrating the suitability of these diets. This system is essentially based on the animals' growth control, their period clinical examination as well as the anatomopathological postmortem study of the animals submitted to such diets. Two groups of weaning rats, control (C) and hyperlipidic (HL), were submitted to a low-fat diet (N3) or a high-fat polyunsaturated--corn oil--diet (HL20) respectively. At 53 days of age all animals were induced with 5mg of dimethylbenz (a) anthracene. Experiments were ended when animals reached a mean age of 214 days. The results show: 1) a normal ponderal evolution of the animals in the two experimental groups with respect to two series of the same strain fed with a standard diet, and 2) the homogeneity of growth determined by the coefficient of variance study. On the other hand, neither the weekly clinical examination nor the anatomopathological post-mortem studies revealed any pathology that could be specifically attributed to nutritional imbalance. These results confirm the suitability of both diets for rat growth. Their use in the study of the effects of dietary lipids on the mammary carcinoma would satisfy the initial aim of guaranteeing the specificity of the results.

B-vitamin mixture improves the analgesic effect of diclofenac in patients with osteoarthritis: a double blind study.

According to the high consumption of the mixture of B vitamins and diclofenac in several countries, this combination has constituted a frequently used option in pain therapy from inflammatory origin. Although the evidence obtained from inflammatory pain animal models has shown the existence of analgesic synergy between diclofenac and the B vitamins mixture, the corresponding clinical evidence is scarce. A double-blind, randomized clinical trial study was designed to characterize the analgesic effect and safety of diclofenac and B vitamins against diclofenac alone in patients with severe osteoarthritis. Forty eight patients programmed to total knee arthroplasty with a pain level ≥7 in a 1-10 cm visual analogue scale were allocated to receive a single intramuscular injection of sodium diclofenac (75 mg) alone or combined with thiamine (100 mg), pyridoxine (100 mg) and cyanocobalamin (5 mg), and the pain level was evaluated during 12 h post-injection. Diclofenac+B vitamins mixture showed a superior analgesic effect during the assessed period and also a better assessment of the pain relief perception by patients than diclofenac alone. This study constitutes a clinical support on the improvement of the analgesic effect of diclofenac by B vitamins in patients with osteoarthritis programmed to total knee arthroplasty, as a clinical model of inflammatory pain.

Hexose transport by chicken cecum during development.

Hexose accumulation during development has been studied in tissue slices from chicken cecum. The age of birds ranged from 0 to 7 weeks after hatch. Ceca were divided into six portions according to their situation either proximal (PC), medial (MC) or distal (DC) to the ileocecal junction. In 0-day-old chicks all segments can accumulate 3-O-methyl-D-glucose (0.5 mmol/l) against a concentration gradient through a phloridzin-sensitive mechanism. Cumulative capacity is lower in DC than in PC and declines with development. Distal segments lose sugar transport ability 1-2 days after hatch whereas the medial region retains some concentrative ability in older birds. In 7-week chickens, PC slices have a similar cumulative ability to that of jejunum (yolk sac region). Kinetic studies showed that in PC the apparent Km for phloridzin-sensitive transport was half that in 1-day- than in 7-week-old birds; apparent Vm increased by 50% in this time range. The ability to transport sugars by the cecum was further confirmed in isolated enterocytes from 5- to 7-week-old chickens using alpha-methyl-D-glucoside (0.1 mmol/l) as substrate. Cell sugar concentration was greater in PC than in jejunal cells and jejunal greater than MC enterocytes. Sugar present in cells from DC was the same as in phloridzin-treated cells. It is concluded that cecal epithelium may play a significant role in the absorption of sugars during development.

[Entamoeba histolytica stained with Warthin-Starry stain]. / Tinción de la Entamoeba histolítica con la coloración de Warthin-Starry.

The Warthin-Starry stain have been used for coloring a different microorganism like spirochetas, Donovan bodies and Campylobacter and also melanin granules demonstration in soft tissues tumors. We started using the stain, as we know it, in order to stain the Histolytica amebas. We observed parasite stained black and brown as well as a good differentiation between endoplasm and ectoplasm where granular appearance, bacterial remnants and red cells were apparent. The stain was also usefull in differentiating amebas from histiocytes. We advice its use in amebiasis since is inexpensive and easy to do.

Autoantibodies against pancreatic beta-cells: characterization by western blot analysis in the non-obese diabetic (NOD) mouse.

The NOD mouse is a relevant model for studying autoimmune diabetes. As in human insulin-dependent diabetes mellitus, the nature of the autoantigen towards which the immune system is directed remains to be clarified. It has been shown that T cells are central to the disease process. However, autoantibodies may be used as a probe to identify islet autoantigens to which self tolerance is defective. Using Western blot analysis, we characterized autoantibodies which are specific for a 58 kDa islet antigen and a 29 kDa antigen. The 58 kDa autoantigen was present in cellular extracts prepared from rat tumoral insulin-secreting cells (Rin5F) and NOD islets but not from most other non-insulin-secreting cell lines. By contrast the 29 kDa antigen was a ubiquitous antigen expressed in all cell lines tested and was not further characterized since it is very likely to be responsible for secondary immunization rather than play any role in the NOD disease process. Anti-58 kDa autoantibodies were detected in all diabetic male and female NOD animals as well as in sera from old non-diabetic NOD animals. Anti-58 kDa antibodies were not detected in sera from young NOD mice (less than 6 weeks of age) or in sera from other conventional laboratory strains of mice including autoimmune prone animals such as MRL/lpr and (NZB x NZW)F1 mice. A monoclonal antibody (72.2) specific for the 58 kDa structure was obtained, which allowed further characterization of the corresponding islet cell antigen. The expression of the 58 kDa antigen was evidenced by Western blot analysis in normal islets and in a mouse neuroblastoma cell line.

Anti-alpha/beta T cell receptor monoclonal antibody provides an efficient therapy for autoimmune diabetes in nonobese diabetic (NOD) mice.

The nonobese diabetic (NOD) mouse is a relevant model for studying human insulin-dependent diabetes mellitus (IDDM). The selective destruction of insulin-secreting cells in this model is subsequent to an autoimmune reaction directed towards the beta cells inside the islets of Langerhans of the pancreas. Given the key role played by T cells in the development of IDDM, we investigated a model of IDDM prevention in NOD mice by administration of a monoclonal antibody to the alpha/beta dimer of the T cell receptor for antigen. Our data provide evidence that aiming at the T cell receptor protects against both spontaneous and cyclophosphamide-induced diabetes in the NOD mouse. Interestingly, potential clinical application is suggested by the efficient and durable reversal of recent onset diabetes in mice treated with anti-alpha/beta monoclonal antibody within 1 week following the clinical discovery of IDDM.

Dietary polyunsaturated n-6 lipids effects on the growth and fatty acid composition of rat mammary tumors.

The aim of this study was to analyze the effects of a polyunsaturated n-6 high-fat diet on rat DMBA-induced breast cancer at different stages of the carcinogenesis and to investigate if changes in the tumor fatty acid composition are one of the mechanisms by which dietary lipids could exert their effects. 14 fatty acids were evaluated in 6 lipid fractions. The results firstly showed that this high-fat diet stimulated the malignant mammary tumor growth, mainly all in the promotion group. The tumor lipid analysis indicated: 1) that each lipid fraction presented distinct major fatty acids (>5%) which were not the most abundant in the diet, except in the case of the triacylglicerides, suggesting the different resistance to dietary fatty acid modification of the tumor lipid fractions; 2) a higher arachidonic acid content in the fractions with less linoleic acid, above all in phospholipids, particularly in the phosphatidylethanolamine, indicating a different efficiency of conversion; 3) the three most abundant fatty acids in the dietary lipid (18:2n-6, 18:1n-9 and 16:0) were those which essentially displayed the differences between groups; thus, the high-fat diet changed the tumor lipid profile, increasing the 18:2n-6 relative content and decreasing that of the 18:1n-9; differences were significant in phosphatidylcholine, free fatty acids and triacylglycerides. Any change was obtained in the phosphatidylinositol. The greatest number of differences was found in the promotion group. Taken as a whole, our results suggest the different roles of lipid fractions in breast cancer cells and an association between cancer malignancy and the content of linoleic and oleic acids.

Peripherin: an islet antigen that is cross-reactive with nonobese diabetic mouse class II gene products.

The nonobese diabetic (NOD) mouse, in which major histocompatibility complex genes may be involved in the susceptibility to diabetes, has been developed as a model of autoimmune diabetes. The NOD mouse expresses I-A-encoded class II major histocompatibility complex antigens, which differ from those of other mouse haplotypes by the presence of a serine at position 57 of the A beta chain. Identifying islet autoantigens may help elucidate the role of class II antigens in the activation of autoreactive T cells and, thus, in the development of diabetes. We have detected autoantibodies directed against a 58-kDa islet cell antigen in NOD mice but not in other strains, including lupus-prone mice. Apart from insulin-secreting cells, the 58-kDa antigen was only found to be expressed by neuroblastoma cells and was identified as peripherin, an intermediate filament protein previously characterized in well-defined neuronal populations. This autoantigen cross-reacted with I-Anod class II antigens, suggesting that it may contribute to defective self-tolerance of islet beta cells in the NOD mouse.