Effects of shear stress on endothelial cells: go with the flow.

Haemodynamic forces influence the functional properties of vascular endothelium. Endothelial cells (ECs) have a variety of receptors, which sense flow and transmit mechanical signals through mechanosensitive signalling pathways to recipient molecules that lead to phenotypic and functional changes. Arterial architecture varies greatly exhibiting bifurcations, branch points and curved regions, which are exposed to various flow patterns. Clinical studies showed that atherosclerotic plaques develop preferentially at arterial branches and curvatures, that is in the regions exposed to disturbed flow and shear stress. In the atheroprone regions, the endothelium has a proinflammatory phenotype associated with low nitric oxide production, reduced barrier function and increased proadhesive, procoagulant and proproliferative properties. Atheroresistant regions are exposed to laminar flow and high shear stress that induce prosurvival antioxidant signals and maintain the quiescent phenotype in ECs. Indeed, various flow patterns contribute to phenotypic and functional heterogeneity of arterial endothelium whose response to proatherogenic stimuli is differentiated. This may explain the preferential development of endothelial dysfunction in arterial sites with disturbed flow.

Vascular smooth muscle cell in atherosclerosis.

Vascular smooth muscle cells (VSMCs) exhibit phenotypic and functional plasticity in order to respond to vascular injury. In case of the vessel damage, VSMCs are able to switch from the quiescent 'contractile' phenotype to the 'proinflammatory' phenotype. This change is accompanied by decrease in expression of smooth muscle (SM)-specific markers responsible for SM contraction and production of proinflammatory mediators that modulate induction of proliferation and chemotaxis. Indeed, activated VSMCs could efficiently proliferate and migrate contributing to the vascular wall repair. However, in chronic inflammation that occurs in atherosclerosis, arterial VSMCs become aberrantly regulated and this leads to increased VSMC dedifferentiation and extracellular matrix formation in plaque areas. Proatherosclerotic switch in VSMC phenotype is a complex and multistep mechanism that may be induced by a variety of proinflammatory stimuli and hemodynamic alterations. Disturbances in hemodynamic forces could initiate the proinflammatory switch in VSMC phenotype even in pre-clinical stages of atherosclerosis. Proinflammatory signals play a crucial role in further dedifferentiation of VSMCs in affected vessels and propagation of pathological vascular remodelling.

Contribution of neovascularization and intraplaque haemorrhage to atherosclerotic plaque progression and instability.

Atherosclerosis is a continuous pathological process that starts early in life and progresses frequently to unstable plaques. Plaque rupture leads to deleterious consequences such as acute coronary syndrome, stroke and atherothrombosis. The vulnerable lesion has several structural and functional hallmarks that distinguish it from the stable plaque. The unstable plaque has large necrotic core (over 40% plaque volume) composed of cholesterol crystals, cholesterol esters, oxidized lipids, fibrin, erythrocytes and their remnants (haeme, iron, haemoglobin), and dying macrophages. The fibrous cap is thin, depleted of smooth muscle cells and collagen, and is infiltrated with proinflammatory cells. In unstable lesion, formation of neomicrovessels is increased. These neovessels have weak integrity and leak thereby leading to recurrent haemorrhages. Haemorrhages deliver erythrocytes to the necrotic core where they degrade promoting inflammation and oxidative stress. Inflammatory cells mostly presented by monocytes/macrophages, neutrophils and mast cells extravagate from bleeding neovessels and infiltrate adventitia where they support chronic inflammation. Plaque destabilization is an evolutionary process that could start at early atherosclerotic stages and whose progression is influenced by many factors including neovascularization, intraplaque haemorrhages, formation of cholesterol crystals, inflammation, oxidative stress and intraplaque protease activity.

[Contribution of non-HLA genes to juvenile idiopathic arthritis susceptibility].

Juvenile idiopathic arthritis (JAL4) is the most common chronic rheumatologic disease in children. JIA is a group of disorders that share the clinical manifestation of chronic joint inflammation. The Human Leukocyte Antigen region (HLA) seems to be a major susceptibility locus for JIA that is estimated to account for 17% of familial segregation of the disease. Genome-wide association studies (GWAS), case-control studies and meta-analyses of the post-GWAS era revealed over 20 non-HLA loci conferring susceptibility to JIA. At least a half of those are shared between JIA and rheumatoid arthritis, an adult rheumatic disease, thereby suggesting for similarity of pathogenic mechanisms of both diseases. New findings also suggest for a likely role of epigenetic alterations in the pathogenesis of JIA that should be investigated in the future.

Role of microRNAs in mechanisms of glioblastoma resistance to radio- and chemotherapy.

Low-grade gliomas and multiform glioblastoma are characterized by highly pronounced anaplasia, malignization, proliferation, and invasiveness; moreover, they are highly resistant to chemo- and radiotherapy. The very low efficiency of traditional approaches in the treatment of patients with glioblastomas is due to the intensive invasive growth of the tumor resulting in deep infiltration of adjacent normal perivascular and nervous tissue and formation of areas of perineural infiltration differently remote from the tumor epicenter. MicroRNAs are key posttranscriptional regulators of gene activities, and their expression is markedly increased in tumors, in particular in gliomas. MicroRNAs have been shown to promote the growth, proliferation, migration, and survival of tumor stem and non-stem cells. However, a population of microRNA possessing antitumor effects is also detected in gliomas. As a rule, the expression of antitumor microRNAs is suppressed in tumors. In this review, we consider microRNAs, their influence on radio- and chemoresistance of gliomas, and prospects for their use as specific agents in targeted therapy of gliomas. The pool of these microRNAs has distinct therapeutic value, because on use in combined therapy it can decrease the resistance of glioma tumor stem cells to existing pharmaceuticals and improve the efficiency of radio- and chemotherapy.

A variant of the Il2ra / Cd25 gene predisposing to graves' disease is associated with increased levels of soluble interleukin-2 receptor.

Alpha-subunit of the IL-2 receptor (IL-2Rα) encoded by the IL2RA/CD25 gene binds IL-2 that plays a pivotal role in the regulation of T cell function. Levels of a soluble form of IL-2Rα (sIL-2Rα) lacking the transmembrane and cytoplasmic domains were shown to be increased in several autoimmune diseases including Graves' disease (GD). Recent studies showed association between the IL2RA/CD25 gene variants and several autoimmune diseases including GD. In this study, we analyzed whether polymorphic markers rs2104286, rs41295061, and rs11594656 located at the IL2RA/CD25 locus confer susceptibility to GD and are related to increased concentrations of sIL-2Rα. A total of 1474 Russian GD patients and 1609 control subjects were genotyped for rs2104286, rs41295061, and rs11594656 using a Taqman assay. Concentrations of sIL-2Rα in sera of affected and non-affected individuals were measured using an ELISA test. A minor allele A of rs41295061 showed significant association with increased risk of GD [odds ratio (OR) = 1.43, P(c) = 0.00102]. The allele A of rs41295061 and allele A of rs11594656 constitute a higher risk haplotype AA (OR = 1.47, P(c) = 0.0477). Compared to carriers of the protective haplogenotype GT/GT, the carriage of two copies of the haplogenotype AA/AA was associated with elevated levels of sIL-2Rα in both GD patients (AA/AA versus GT/GT: 1.35 ± 0.47 ng/ml versus 1.12 ± 0.45 ng/ml, P = 0.0065) and healthy controls (AA/AA versus GT/GT: 0.67 ± 0.28 ng/ml versus 0.51 ± 0.33 ng/ml, P = 0.0098). This is the first report presenting correlation between the carriage of disease-associated variants of IL2RA/CD25 with increased levels of sIL-2Rα in GD.

A variant of the interleukin-1beta gene in European sea bass, Dicentrarchus labrax L., is associated with increased resistance against Vibrio anguillarum.

Vibriosis caused by the pathogenic bacterium Vibrio (Listonella) anguillarum leads to serious losses in European sea bass, Dicentrarchus labrax. Because of its pleiotropic activity in controlling immune and inflammatory responses against various pathogens, interleukin-1beta (IL-1beta) is an attractive candidate for resistance to bacterial vibriosis. Four polymorphisms c.76 + 52C>T, c.76 + 157A>G, c.76 + 215A>and c76 + 310A>G of IL1B were genotyped in progeny of four families of wild sea bass captured in geographically distinct regions of the Black Sea and Sea of Azov and challenged with V. anguillarum. In the transmission disequilibrium test, the TGGG haplotype of IL1B showed significant overtransmission from parents to surviving progeny, thereby suggesting an association with higher resistance to V. anguillarum infection (Odds Ratio 0.38, P < 10(-7)). Using a luciferase reporter assay, we found a 1.4-fold increase in transcription activity of the protective IL1B TGGG variant compared to the susceptible CAAA variant of IL1B. The higher transcriptional activity of IL1B TGGG may arise from the functional effects of c.76 + 157A>G and c.76 + 215A>G polymorphisms disrupting potential binding sites for glucocorticoid receptor and YY1, both are negative transcription regulators.

Genetic variations in the pancreatic ATP-sensitive potassium channel, beta-cell dysfunction, and susceptibility to type 2 diabetes.

The KCNJ11 and ABCC8 genes encode the components of the pancreatic ATP-sensitive potassium (KATP) channel, which regulates insulin secretion by beta-cells and hence could be involved in the pathogenesis of type 2 diabetes (T2D). The KCNJ11 E23K and ABCC8 exon 31 variants have been studied in 127 Russian T2D patients and 117 controls using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) approach. The KCNJ11 E23 variant and the ABCC8 exon 31 allele A were associated with higher risk of T2D [Odds ratio (OR) of 1.53 (P=0.023) and 2.41 (P=1.95 x 10(-5))], respectively. Diabetic carriers of the ABCC8 G/G variant had reduced 2 h glucose compared to A/A+A/G (P=0.031). The G/G genotype of ABCC8 was also significantly associated with increased both fasting and 2 h serum insulin in diabetic and non-diabetic patients. A HOMA-beta value characterizing the beta-cell homeostasis was higher in the non-diabetic carriers homozygous for G/G (98.0+/-46.9) then for other genotypes (HOMA-beta = 85.6+/-45.5 for A/A+A/G, P=0.0015). The KCNJ11 E23K and ABCC8 exon 31 variants contribute to susceptibility to T2D diabetes, glucose intolerance and altered insulin secretion in a Russian population.

A combined AFLP and microsatellite linkage map and pilot comparative genomic analysis of European sea bass Dicentrarchus labrax L.

European sea bass (Dicentrarchus labrax L., Moronidae, Teleostei) sustains a regional fishery and is commonly farmed in the Mediterranean basin, but has not undergone much long-term genetic improvement. An updated genetic linkage map of the European sea bass was constructed using 190 microsatellites, 176 amplified fragment length polymorphisms and two single nucleotide polymorphisms. From the 45 new microsatellite markers (including 31 type I markers) reported in this study, 28 were mapped. A total of 368 markers were assembled into 35 linkage groups. Among these markers, 28 represented type I (coding) markers, including those located within the peptide Y, SOX10, PXN1, ERA and TCRB genes (linkage groups 1, 7, 16, 17 and 27 respectively). The sex-averaged map spanned 1373.1 centimorgans (cM) of the genome. The female map measured 1380.0 cM, whereas the male map measured 1046.9 cM, leading to a female-to-male (F:M) recombination rate ratio of 1.32:1. The intermarker spacing of the second-generation linkage map of the European sea bass was 3.67 cM, which is smaller than that of the first-generation linkage map (5.03 cM). Comparative mapping of microsatellite flanking regions was performed with five model teleosts and this revealed a high percentage (33.6%) of evolutionarily conserved regions with the three-spined stickleback.

The 262T>C promoter polymorphism of the catalase gene is associated with diabetic neuropathy in type 1 diabetic Russian patients.

OBJECTIVE: Oxidative stress plays an important role in the development of diabetic neuropathy (DN). Antioxidant enzymes reduce enhanced oxidative stress in the peripheral nerve. Genetic variations within the antioxidant genes therefore could be implicated in the pathogenesis of DN. METHODS: Using a PCR-RFLP assay, a total of 216 Russian type 1 diabetic (T1D) patients with DN and 250 T1D individuals without DN have been tested to verify whether the -262T > C and 1167C > T polymorphisms of the catalase (CAT), 197Pro > Leu amino acid substitution of the glutathione peroxidase 1 (GPX1) and +/null polymorphism of the glutathione S-transferase M1 (GSTM1) and T1 (GSTT1) genes contribute to susceptibility to DN. RESULTS: Association between the -262T > C polymorphism of the CAT gene and DN was shown. The -262TT genotype of the CAT gene was significantly associated with higher erythrocyte catalase activity in blood of DN patients compared to the -262CC genotype (17.8 +/- 2.7 x 104 IU/g Hb vs. 13.5 +/- 3.2 x 104 IU/g Hb, P = 0.0022). CONCLUSIONS: These data suggest a protective role of the -262T allele of the CAT gene against the rapid development of DN in T1D (Odds Ratio = 0.7 [95% confidence interval 0.54-0.9], P = 0.002).

Screening of SNPs at 18 positional candidate genes, located within the GD-1 locus on chromosome 14q23-q32, for susceptibility to Graves' disease: a TDT study.

Graves' disease (GD) is a complex autoimmune thyroid disorder with a strong genetic component. Genome-wide screens resolved several susceptibility loci that contribute to the development of GD. One of the susceptibility loci (GD-1 locus) was mapped on chromosome 14q31. However, a susceptibility gene located within the GD-1 locus remains undefined. Here we screen eighteen single nucleotide polymorphisms (SNPs), each is situated at a corresponding positional candidate gene, located within the GD-1 susceptibility locus on chromosome 14q23-q32, for predisposition to GD using the transmission disequilibrium test in 126 simplex Russian families affected with GD. Among SNPs tested, a significant preferential transmission of the Ala allele (41 transmissions vs. 17 nontransmissions, corrected P=0.031) of the Thr92Ala SNP within the DIO2 gene, encoding type II iodothyronine deiodinase, from parents to affected children was found in a Russian family data set. The Thr92Ala SNP of the DIO2 gene and the D727E substitution of the thyrotropin receptor (TSHR) gene have been found to be in pair-wise linkage disequilibrium. The A92/E727 haplotype showed significant preferential transmission from parents to affected sibling (17 transmissions vs. 8 nontransmissions, P=0.039) in simplex families. This suggests that the Thr92Ala variant of the DIO2 gene is associated or may be in linkage disequilibrium with a functional DIO2 polymorphism which involves in the development of GD in a Russian population.

Evidence for a type 1 diabetes susceptibility locus (IDDM10) on chromosome 10p11-q11 in a Russian population.

Around 20 susceptibility loci for type 1 diabetes mellitus (T1DM) have been mapped. One of these loci, IDDM10, was found on chromosome 10p11-q11. Here, we investigated whether the IDDM10 locus contributes in the susceptibility to T1DM in a Russian family dataset. One hundred and fourteen simplex Russian families, each containing two siblings (one affected with T1DM diagnosed and one nondiabetic sibling), and 97 multiplex families, containing 106 affected full sibling pairs, were studied. Genomic DNA from the venous blood of the patients was genotyped by PCR using 12 microsatellites (D10S193, D10S548, D10S565, D10S586, D10S588, D10S675, D10S1243, D10S1426, D10S1733, D10S1772, D10S1780 and D10S1783) located on chromosome 10p11-q11. Using the multipoint linkage analysis, the region of suggestive linkage, with a multipoint logarithm of odds (LOD) ratio (MLS) value of more than 2.2, was found between markers D10S1733 and D10S1780, an area of 9.0 cM on the genetic map. The maximum linkage peak (MLS = 2.85 and nonparametric logarithm = 2.68) was observed between markers D11S565 and D11S1243. Using the transmission disequilibrium test, an association of these markers, D10S565 (P overall = 0.0082) and D10S1243 (P overall = 0.017), with T1DM was shown. These results suggest the evidence for the IDDM10 susceptibility locus on chromosome 10p11-q11.

A new type 1 diabetes susceptibility locus containing the catalase gene (chromosome 11p13) in a Russian population.

BACKGROUND: Oxidative stress is involved in the origin of type 1 diabetes. Low efficiency of the scavenging antioxidant system has been shown to be related to the pathogenesis of the disease. This, therefore suggests that genes encoding catalase and other antioxidant enzymes may implicate in the development of type 1 diabetes. METHODS: Nine microsatellite markers that cover about 10 megabases around the catalase (CAT) gene on chromosome 11p13 were analyzed using polymerase chain reaction (PCR) and fluorescence-based genotyping on an automatic DNA sequencer. We also evaluated three single-nucleotide polymorphisms (SNP) within genes encoding catalase (T1667T and C(-262)T dimorphism) and ETS homologous factor (EHF) (C255T SNP) using a PCR-restriction fragment-length polymorphism approach. Multipont linkage analysis in 37 affected sibling pairs was performed using GENEHUNTER 2.1. We examined the markers for association with the disease by transmission disequilibrium tests in 57 discordant sibling pairs and by a case-control study in 258 unrelated healthy donors and 247 affected patients. RESULTS: We obtained close-to-suggestive evidence of linkage to type I diabetes, with the maximum linkage peak between markers D11S907 and D11S2008. Analysis of three SNPs at the CAT and EHF gene located within the region of maximum linkage showed that T1667T and C(-262)T markers of the CAT gene are strongly associated with the disease. CONCLUSION: Our findings support evidence of a new putative type 1 diabetes susceptibility locus on chromosome 11p13 and suggest that the CAT gene may play a role in conferring susceptibility to the disorder in Russian patients.

[Search for the association of polymorphic markers for genes coding for antioxidant defense enzymes, with development of diabetic polyneuropathies in patients with type 1 diabetes mellitus]. / Poisk assotsiatsii polimorfnykh markerov genov, kodiruiushchikh fermenty antiokislitel'noi zashchity, s razvitiem diabeticheskoi polineiropatii u bol'nykh sakharnym diabitom tipa 1.

The allele and genotype frequency distributions of polymorphic markers of genes coding for antioxidant enzymes were compared for type 1 diabetes mellitus patients with or without diabetic polyneuropathy (DPN). The groups (total 180 patients) had nonoverlapping (polar) phenotypes. Group DPN+ included 86 patients with DPN and diabetic record no more than 5 years. Control group DPN- included patients without DPN and diabetic record of at least 10 years. Comparative analysis with Fisher's exact test revealed a significant difference in allele and genotype frequency distributions of the T(-262)C polymorphic marker of the CAT gene. Polymorphic markers C1167T of the CAT gene, Pro/Leu of the GPX1 gene, 0/+ of the GSTT1 gene, and 0/+ of the GSTM1 gene showed no significant difference in allele or genotype frequency distribution. On this evidence, these markers were not associated with DPN in the sample examined.

Development and linkage relationships for new microsatellite markers of the sea bass (Dicentrarchus labrax L.).

Twenty-eight polymorphic microsatellites were isolated from the sea bass, Dicentrarchus labrax, using a microsatellite enrichment protocol and selective hybridization with oligonucleotide probes. Analysis for these markers and 11 recently described microsatellites of D. labrax found linkage between 26 loci and revealed eight linkage groups.

Thyroid-stimulating hormone receptor and its role in Graves' disease.

The thyroid-stimulating hormone (TSH, or thyrotropin) receptor (TSHR) mediates the activating action of TSH to the thyroid gland, resulting in the growth and proliferation of thyrocytes and thyroid hormone production. In Graves' disease, thyroid-stimulating autoantibodies can mimic TSH action and stimulate thyroid cells. This leads to hyperthyroidism and abnormal overproduction of thyroid hormone. TSHR-antibodies-binding epitopes on the receptor molecule are well studied. Mechanism of TSHR-autoantibodies production is more or less clear but a susceptibility gene, which is linked to their production, is still unknown. Genetic studies show no linkage between the TSHR gene and Graves' disease. Among three common polymorphisms in the TSHR gene, only the D727E germline polymorphism in the cytoplasmic tail of the receptor showed an association with the disease, and this association is weak. The absence of a strong genetic effect of the TSHR polymorphisms in such a common and complex disorder as Graves' disease may be explained by a high degree of evolutionary conservation in TSHR. This can be shown by naturally existing germline and somatic mutations in the TSHR gene that cause various types of nonautoimmune and hereditary thyroid disease.

CTLA-4 and its role in autoimmune thyroid disease.

Autoimmune thyroid disease (AITD) occurs in two common forms: Graves' disease and Hashimoto thyroiditis. On the basis of functional and experimental data, it has been suggested that the gene encoding cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) is a candidate gene for conferring susceptibility to thyroid autoimmunity. In this review, we critically evaluate the evidence for pathogenetic involvement of CTLA-4 in the various forms of AITD and focus on the possible role of genetic variation of the CTLA4 locus. Population genetics data strongly suggest a role for the CTLA4 region in susceptibility to AITD. However, further functional studies are required to understand the significance of CTLA4 polymorphisms in the pathogenic mechanism of AITD.

[Association of the SOD2 Ala(-9)Val and SOD3 Arg213Gly polymorphisms with diabetic polyneuropathy in patients with diabetes mellitus type 1]. / Assotsiatsiia polimorfnykh markerov Ala(-9)Val gena SOD2 i Arg213Gly gena SOD3 s diabeticheskoi polineiropatiei u bol'nykh sakharnym diabetom tipa 1.

Single-nucleotide polymorphisms of the genes for mitochondrial (SOD2) and extracellular (SOD3) superoxide dismutases were tested for association with diabetic polyneuropathy (DPN) in diabetes mellitus (DM) type 1. Patients (n = 180) were divided into two groups with nonoverlapping (polar) phenotypes. Group DPN+ included 86 individuals with DPN and DM type 1 record of no more than 5 years. Group DPN-included 94 patients with DM type 1 record of more than 10 years but without clinical signs of DPN. Fisher's exact test revealed significant differences in allele and genotype frequencies for the two groups. Higher frequencies of SOD2 allele Val and genotype Val/Val and of SOD3 allele Arg and genotype Arg/Arg were established for group DPN+. On this evidence, SOD2 and SOD3 were associated with DPN in DM type 1.

[Polymorphic locus D6S392 near the Mn-dependent superoxide dismutase gene is associated with diabetes mellitus in the Moscow population]. / Polimofnyi lokus D6S392 riadom s genom Mn-zavisimoi superoksiddismutazy sviazan s sakharnym diabetom v Moskovskoi populiatsii.

AIM: Distribution of alleles of tetranucleotide microsatellite D6S392 located nearby the gene of Mn-dependent superoxide dismutase (SOD2) was studied in healthy donors (n = 143), patients with insulin-dependent (n = 166) and insulin-independent (n = 101) diabetes mellitus (IDDM and IIDM). MATERIALS AND METHODS: Alleles of the polymorphic locus D6S392 were amplified using polymerase chain reaction (PCR) on the basis of genome DNA isolated from the venous blood of the examinees. PCR products were analysed with gel-electrophoresis in polyacrylamide gel. Significance of differences of allele and genotypes distribution in the population control and patients were assessed with Fisher's criterion and Bonferroni's corrections. RESULTS: Locus D6S392 contains 31 allele from 210 to 330 pn in length and 37 to 67 tandem repeats. Compared to controls, IDDM patients had less frequent incidence of low molecular allele 41 and 42 as well as allele 62. CONCLUSION: Polymorphic locus D6S392 is closely associated with development of DM in Moscow population. This may indicate possible participation of the gene SOD2 in development of this pathology.

[Associative analysis of the connection between mutation C1167T in the catalase gene and polymorphic marker D6S392 nearby the Mn-dependent superoxide dismutase gene with diabetic microangiopathies]. / Assotsiativnyi analiz sviazi mutatsii C1167T v gene katalazy i polimorfnogo markera D6S392 riadom s genom Mn-zavisimoi superoksiddismutazy s diabeticheskimi mikroangiopatiiami.

AIM: To study allele polymorphism of two variable regions [C1167T substitution in the catalase (CAT) gene D6S392 microsatellite near the Mn-dependent superoxide dismutase (SOD2) gene] was studied in insulin-dependent diabetic (IDDM) patients with (n = 36) or without (n = 56) diabetic nephropathy, and with (n = 30) or without (n = 44) diabetic retinopathy. MATERIAL AND METHODS: Both polymorphic regions were amplified using polymerase chain reaction (PCR). PCR products were separated using polyacrylamide (D6S366) or agarose (C1167T) gel electrophoresis. In a case of C1167, PCR-amplified products were digested with BstXI restriction endonuclease before electrophoresis. A significance of the difference between allele distributions in complicated and uncomplicated IDDM patients was estimated using the exact Fisher's test. RESULTS: No significant difference was observed in allele and genotype frequencies in complicated and uncomplicated IDDM subjects. CONCLUSION: C1167 polymorphism in the CAT gene and D6S366 near the SOD2 gene are not associated with the development of diabetic nephropathy and diabetic retinopathy in IDDM.