Kathleen M. Gillespie

Diabetes and gender.

Abstract It is often assumed that there is little or no sex bias within either Type I (insulin-dependent) or Type II (non-insulin-dependent) diabetes mellitus. This review considers evidence that sex effects of interest and importance are present in both forms of the disease. Type I diabetes is the only major organ-specific autoimmune disorder not to show a strong female bias. The overall sex ratio is roughly equal in children diagnosed under the age of 15 but while populations with the highest incidence all show male excess, the lowest risk populations studied, mostly of non-European origin, characteristically show a female bias. In contrast, male excess is a consistent finding in populations of European origin aged 15–40 years, with an approximate 3:2 male:female ratio. This ratio has remained constant in young adults over two or three generations in some populations. Further, fathers with Type I diabetes are more likely than affected mothers to transmit the condition to their offspring. Women of childbearing age are therefore less likely to develop Type I diabetes, and – should this occur – are less likely to transmit it to their offspring. Type II diabetes showed a pronounced female excess in the first half of the last century but is now equally prevalent among men and women in most populations, with some evidence of male preponderance in early middle age. Men seem more susceptible than women to the consequences of indolence and obesity, possibly due to differences in insulin sensitivity and regional fat deposition. Women are, however, more likely to transmit Type II diabetes to their offspring. Understanding these experiments of nature might suggest ways of influencing the early course of both forms of the disease. [Diabetologia (2001) 44: 3–15]

The rising incidence of childhood type 1 diabetes and reduced contribution of high-risk HLA haplotypes

The incidence of childhood type 1 diabetes has risen over the past 50 years. We compared the frequency of HLA class II haplotypes in 194 patients diagnosed more than 50 years ago and 582 age-matched and sex-matched individuals diagnosed between 1985 and 2002. The proportion of high-risk susceptibility genotypes was increased in the earlier cohort (p=0.003), especially in those diagnosed at age 5 years or younger, which is consistent with the hypothesis that the rise of type 1 diabetes is due to a major environmental effect.

HLA Genotyping Supports a Nonautoimmune Etiology in Patients Diagnosed With Diabetes Under the Age of 6 Months

Children with permanent diabetes are usually assumed to have type 1 diabetes. It has recently been shown that there are genetic subgroups of diabetes that are often diagnosed during the neonatal period but may present later. A recent Italian study proposed that type 1 diabetes is rare before 6 months of age. We aimed to examine genetic susceptibility to type 1 diabetes in patients diagnosed with diabetes before the age of 2 years. We analyzed HLA class II genotypes, markers of autoimmune diabetes, in 187 children with permanent diabetes diagnosed at <2 years of age. Of the 79 subjects diagnosed at <6 months of age, 41% (95% CI 0.30–0.51) had type 1 diabetes–associated high-risk genotypes, a proportion similar to that in healthy population control subjects (44%, P = 0.56). This group included 32 patients with mutations in the KCNJ11 gene, which encodes Kir6.2 (44% high-risk HLA class II genotypes), and 47 in whom the etiology of diabetes was unknown (38% high-risk HLA class II genotypes). Of 108 patients diagnosed between 6 and 24 months of age, 93% (0.86–0.99) had high-risk HLA class II genotypes compared with 44% of the population control subjects (P < 0.0001). We conclude that infants diagnosed with diabetes before 6 months of age are unlikely to have autoimmune type 1 diabetes and are most likely to have a monogenic etiology.

THE INTERLEUKIN-10-1082 G/A POLYMORPHISM: ALLELE FREQUENCY IN DIFFERENT POPULATIONS AND FUNCTIONAL SIGNIFICANCE

Abstract. Genotypic variation in the human interleukin-10 (IL-10) promoter may account for marked inter-individual variation in IL-10 production and may influence susceptibility to autoimmune diseases. The G/A polymorphism at position -1082 has been linked to high/low IL-10 producer status. We directly tested the functional significance of this polymorphism using DNA-binding assays and reporter gene assays, examined allele frequencies in two geographically distinct populations and assessed intra- and inter-individual variation in IL-10 production in vitro according to genotype. Functional analyses showed that the -1082 region contains a putative ETS-like transcription factor-binding site, and nuclear factors from a monocyte cell line bind to this region. Transient transfection studies in an Epstein-Barr virus-transformed B cell line indicated that the -1082 A allele confers a two fold increase in transcriptional activity of the IL-10 promoter compared to the G allele. There was marked inter-individual variation in IL-10 production by peripheral blood mononuclear cells in vitro, with no consistent effect of genotype.

Heterogeneous vascular endothelial growth factor (VEGF) isoform mRNA and receptor mRNA expression in human glomeruli, and the identification of VEGF148 mRNA, a novel truncated splice variant.

Vascular endothelial growth factor (VEGF) mediates increased vascular permeability and endothelial mitogenesis, and may orchestrate normal glomerular permselectivity and proteinuria. Distinct isoforms result from differential gene splicing. VEGF binds to two cell surface tyrosine-kinase receptors, KDR (kinase domain region) and Flt-1 (fms-like tyrosine kinase-1). The latter also exists in a soluble form (sFlt), which is inhibitory. We have studied patterns of VEGF-isoform and VEGF-receptor expression in isolated single normal human glomeruli. mRNA from 190 glomeruli (from 20 individuals) was harvested on to magnetic beads, and nested reverse transcription-PCR was performed using primers for the VEGF isoforms and VEGF receptors. Simultaneous nested reverse transcription-PCR for CD45 was conducted in order to exclude leucocyte contamination. Unexpected products were isolated, cloned and sequenced. Multiple patterns of glomerular VEGF mRNA isoform expression were identified. Most frequently (58%), all three common forms were expressed. VEGF(189) (i.e. 189-amino-acid form of VEGF) was expressed in 63%, VEGF(165) in 85% and VEGF(121) in 84% of glomeruli. Two unexpected PCR products were also identified: 18% of glomeruli expressed VEGF(145), and 27% of glomeruli expressed a new truncated VEGF splice variant, VEGF(148), lacking exon 6, the terminal part of exon 7 and exon 8. Multiple patterns of VEGF-receptor expression were also identified, the most common being expression of all three isoforms (28%). Overall, KDR was seen in 59% of glomeruli, Flt-1 in 45% and sFlt in 57%. Thus the expression of VEGF within normal glomeruli is complex and variable, with inter- and intra-individual variation. Furthermore, sFlt appears to be the co-dominant form of VEGF receptor expressed within glomeruli, suggesting that, in healthy individuals, a degree of VEGF autoregulation is the norm. The physiological importance of VEGF(148) remains to be confirmed.

Levamisole induces interleukin-18 and shifts type 1/type 2 cytokine balance

Immune responses can be classified, according to the predominant cytokines involved, into type 1 (featuring interferon-gamma, IFN-gamma) and type 2 (featuring interleukin-4, IL-4); imbalance between type 1 and type 2 cytokine compartments has been implicated in many human diseases. Levamisole is a drug with an unknown mode of action that has been used to boost immunity in infectious diseases including leprosy, and in some cancers. To test the hypothesis that levamisole acts by inducing a shift to a type 1 immune response, we used Brown Norway (BN) rats, which are markedly biased to type 2 responses. BN rats treated with levamisole showed a dose-dependent rise in serum IFN-gamma and fall in serum immunoglobulin E (IgE) level. Detailed analysis of cytokine gene expression showed upregulation of IFN-gamma and downregulation of IL-4 messenger RNA. This coincided with marked upregulation of IL-18, a recently characterized cytokine with potent activity in stimulating IFN-gamma production. IL-12 was not induced. Further, the type 2 response induced in BN rats by mercuric chloride was markedly attenuated when rats were pretreated with levamisole: there was a 2-log reduction in maximum serum IgE level and marked attenuation of IL-4 gene upregulation. These data indicate that levamisole acts by resetting the immune balance towards a type 1 response via induction of IL-18. Our findings provide a direction for development of more specific immunomodulating therapy.

The genetic basis for type 1 diabetes

BACKGROUND Type 1 diabetes (T1D) is characterized by autoimmune destruction of insulin-producing beta-cells in the pancreas resulting from the action of environmental factors on genetically predisposed individuals. The increasing incidence over recent decades remains unexplained, but the capacity of identifying infants at highest genetic risk has become an increasing requirement for potential therapeutic intervention trials. SOURCES OF DATA Literature searches on T1D and genes were carried out, and key papers since the 1970s were highlighted for inclusion in this review. AREAS OF AGREEMENT Early genetic studies identified the most important region for genetic susceptibility to T1D-the human leukocyte antigen genes on chromosome 6; later shown to contribute approximately half of the genetic determination of T1D. The other half is made up of multiple genes, each having a limited individual impact on genetic susceptibility. AREAS OF CONTROVERSY Historically, there have been many controversial genetic associations with T1D, mostly caused by underpowered case-control studies but these are now decreasing in frequency. AREAS OF GROWTH: The functional effect of each gene associated with T1D must be investigated to determine its usefulness both in risk assessment and as a potential therapeutic target. AREAS TIMELY FOR DEVELOPING RESEARCH Recently identified copy number variants in DNA and epigenetic modifications (heritable changes not associated with changes in the DNA sequence) are also likely to play a role in genetic susceptibility to T1D.

An association analysis of the HLA gene region in latent autoimmune diabetes in adults

Aims/hypothesisPathophysiological similarities between latent autoimmune diabetes in adults (LADA) and type 1 diabetes indicate an overlap in genetic susceptibility. HLA-DRB1 and HLA-DQB1 are major susceptibility genes for type 1 diabetes but studies of these genes in LADA have been limited. Our aim was to define patterns of HLA-encoded susceptibility/protection in a large, well characterised LADA cohort, and to establish association with disease and age at diagnosis.Materials and methodsPatients with LADA (n = 387, including 211 patients from the UK Prospective Diabetes Study) and non-diabetic control subjects (n = 327) were of British/Irish European origin. The HLA-DRB1 and -DQB1 genes were genotyped by sequence-specific PCR.ResultsAs in type 1 diabetes mellitus, DRB1*0301_DQB1*0201 (odds ratio [OR] = 3.08, 95% CI 2.32–4.12, p = 1.2 × 10−16) and DRB1*0401_DQB1*0302 (OR = 2.57, 95% CI 1.80–3.73, p = 4.5 × 10−8) were the main susceptibility haplotypes in LADA, and DRB1*1501_DQB1*0602 was protective (OR = 0.21, 95% CI 0.13–0.34, p = 4.2 × 10−13). Differential susceptibility was conferred by DR4 subtypes: DRB1*0401 was predisposing (OR = 1.79, 95% CI 1.35–2.38, p = 2.7 × 10−5) whereas DRB1*0403 was protective (OR = 0.37, 95% CI 0.13–0.97, p = 0.033). The highest-risk genotypes were DRB1*0301/DRB1*0401 and DQB1*0201/DQB1*0302 (OR = 5.14, 95% CI 2.68–10.69, p = 1.3 × 10−8; and OR = 6.88, 95% CI 3.54–14.68, p = 1.2 × 10−11, respectively). These genotypes and those containing DRB1*0401 and DQB1*0302 associated with a younger age at diagnosis in LADA, whereas genotypes containing DRB1*1501 and DQB1*0602 associated with an older age at diagnosis.Conclusions/interpretationPatterns of susceptibility at the HLA-DRB1 and HLA-DQB1 loci in LADA are similar to those reported for type 1 diabetes, supporting the hypothesis that autoimmune diabetes occurring in adults is an age-related extension of the pathophysiological process presenting as childhood-onset type 1 diabetes.

Association of intercellular adhesion molecule-1 gene with type 1 diabetes

Intercellular adhesion molecule-1 (ICAM-1) functions via its ligands, the leucocyte integrins, in adhesion of immune cells to endothelial cells and in T cell activation. The third immunoglobulin-like extracellular domain binds integrin Mac-1 and contains a common non-conservative aminoacid polymorphism, G241R. Phenotypically, ICAM-1 has been associated with type 1 diabetes, a T-cell-mediated autoimmune disease. We assessed two independent datasets, and noted that R241 was associated with lower risk of type 1 diabetes than is G241 (3695 families, relative risk 0.91, p=0.03; 446 families, 0.60, p=0.006). Our data indicate an aetiological role for ICAM-1 in type 1 diabetes, which needs to be confirmed in future genetic and functional experiments.

Vascular Endothelial Growth Factor (VEGF) isoform expression and activity in human and murine lung injury

BackgroundThe properties of vascular endothelial growth factor (VEGF) as a potent vascular permogen and mitogen have led to investigation of its potential role in lung injury. Alternate spliced VEGF transcript generates several isoforms with potentially differing functions. The purpose of this study was to determine VEGF isoform expression and source in normal and ARDS subjects and investigate the expression and regulation of VEGF isoforms by human alveolar type 2 (ATII) cells.MethodsVEGF protein expression was assessed immunohistochemically in archival normal and ARDS human lung tissue. VEGF isoform mRNA expression was assessed in human and murine lung tissue. Purified ATII cells were cultured with proinflammatory cytokines prior to RNA extraction/cell supernatant sampling/proliferation assay.Measurements and Main ResultsVEGF was expressed on alveolar epithelium, vascular endothelium and alveolar macrophages in normal and ARDS human lung tissue. Increases in VEGF expression were detected in later ARDS in comparison to both normal subjects and early ARDS (p < 0.001). VEGF121, VEGF165 and VEGF189 isoform mRNA expression increased in later ARDS (p < 0.05). The ratio of soluble to cell-associated isoforms was lower in early ARDS than normal subjects and later ARDS and also in murine lung injury. ATII cells constitutionally produced VEGF165 and VEGF121 protein which was increased by LPS (p < 0.05). VEGF165 upregulated ATII cell proliferation (p < 0.001) that was inhibited by soluble VEGF receptor 1 (sflt) (p < 0.05).ConclusionThese data demonstrate that changes in VEGF isoform expression occur in ARDS which may be related to their production by and mitogenic effect on ATII cells; with potentially significant clinical consequences.