Genetic susceptibility factors in type 1 diabetes: linkage, disequilibrium and functional analyses

Rehabilitation after soft-tissue autograft reconstructions is controversial because there is indirect evidence that some grafts fail by creeping over time. The vulnerability of soft-tissue grafts to creep over healing time and the effects of the load environment during healing on this vulnerability have never been studied specifically. We hypothesized that immobilization would decrease the magnitude of the vulnerability of ligament grafts to creep. Thirty-nine skeletally mature New Zealand White rabbits underwent a standardized medial collateral ligament autograft procedure to the right hindlimb, and 19 of the rabbits also had the limb rigidly pinned into flexion. Subgroups were killed at 3 or 8 weeks, and all isolated tibia/medial collateral ligament/femur complexes were tested for creep at 4.1 MPa under a standardized protocol. Eight normal medial collateral ligament controls were tested similarly. Results showed that all grafts were quantitatively more susceptible to cyclic and static creep than were normal medial collateral ligament controls (p < 0.05). By 3 weeks of healing, immobilization significantly increased the magnitude of the vulnerability of the grafts to cyclic, static, and total creep (all: p < 0.05). Furthermore, the grafts had more unrecovered creep strain than did the controls following a 20-minute recovery period. Contrary to our hypothesis, immobilization resulted in increased vulnerability of these ligament autografts to creep even with this relatively nonprovocative test of short duration and low stress. We postulate that following immobilization, this increase in the magnitude of susceptibility of the grafts to creep will result in functionally significant elongation of the graft if it is exposed to higher loads and over longer periods of time in vivo.     

Enhanced susceptibility of low-density lipoprotein to in vitro oxidation in type 1 and type 2 diabetic patients

Macrovascular disease represents a major cause of morbidity and mortality in patients with diabetes mellitus. Low-density lipoprotein (LDL) is involved in the pathogenesis of atherosclerotic lesions, through modifying processes such as oxidation. We examined the in vitro susceptibility to oxidation and the oxidizability of LDL isolated from the plasma of Type 1 and Type 2 diabetic patients. Two groups of diabetic patients (20 Type 1, 20 Type 2) were compared with sex- and age-matched non-diabetic control groups. In vitro oxidation of the purified LDL preparations was assessed by determination of the kinetics for the formation of conjugated dienes (lag phase duration, maximal rate and maximal dienes concentration) and by measurement of thiobarbituric acid-reacting substances (TBARS) in the presence of copper ions. LDL from both Type 1 and Type 2 diabetic patients exhibited a shorter lag phase duration for conjugated dienes formation (94 +/- 14 vs. 108 +/- 20 and 97 +/- 26 vs. 112 +/- 18 min for Type 1 and Type 2 diabetic groups vs. respective control groups, P < 0.05). We also observed an increase in maximal rate of conjugated dienes formation (2.21 +/- 0.55 vs. 1.52 +/- 0.31 and 2.02 +/- 0.55 vs. 1.52 +/- 0.31 nmol/mg LDL/min, P < 0.01) and of maximal production of TBARS (77.9 +/- 11.8 vs. 65.5 +/- 10.4 and 76.7 +/- 9.9 vs. 65.3 +/- 9.4 nmol/mg LDL protein, P < 0.05) in diabetic groups. Our results demonstrate both a higher susceptibility to oxidation and a higher oxidizability of LDL from diabetic patients, as much for Type 1 as Type 2 diabetic subjects with or without pre-existent vascular complications. This enhanced propensity of LDL oxidation in patients with diabetes mellitus could at least partly be attributable to quantitative and qualitative alterations in the chemical composition of LDL and to the glycoxidation process occurring on these lipoproteins.     

A defective Vkappa A2 allele in Navajos which may play a role in increased susceptibility to haemophilus influenzae type b disease

The antibody response to H. influenzae type b (Hib) is pauciclonal, and is dominated by antibodies using the VkappaA2 gene. Navajos have a 5-10-fold increased incidence of Hib disease compared with control populations. We hypothesized that a polymorphism in one of the genes in this oligoclonal response may lead to increased disease susceptibility. Since the predominant A2+ anti-Hib antibodies have high avidity for Hib and can be unmutated, the A2 Vkappa gene was analyzed. Over half of the Navajos studied, but only one control individual, had a new allele of A2, termed A2b, with three changes from the published A2 germline sequence. One of the changes was in the recombination signal sequence, suggesting that the A2b allele might not undergo V-J rearrangement very frequently. This possibility was confirmed by analyzing the relative frequency of non-productive A2 rearrangements in A2a/b heterozygous Navajos. Many fewer A2b rearrangements were observed, showing that the A2b allele is defective in its ability to undergo rearrangement. The prevalence of this allele in Navajos may play a role in their increased susceptibility to invasive Hib disease. If so, it would underscore the importance of the germline Ig repertoire for protective antibody responses to pathogenic bacteria in unimmunized children.     

A role for T-helper type 1 and type 2 cytokines in the pathogenesis of various human diseases

Mosmann first proposed the existence of subsets of CD4+ T cells that produce distinct types of cytokines. Native T lymphocytes (Thp cells) differentiates into either CD4+ Th1 cells that produce IL-2, IFN gamma, and lymphotoxin which promote cell-mediated immunity, or into Th2 cells that produce IL-4, IL-5, IL-6, IL-10 and IL-13, which promote antibody production and humoral immunity. These T cell subsets reciprocally regulate one another since one of the Th1 products, IFN gamma, inhibits the proliferation and functions of Th2 cells, whereas the Th2 products, IL-4 and IL-10, suppress cytokine production by Th1 cells. A distinct Th1/Th2 divergence determine resistance versus susceptibility to diseases such as leishmaniasis and toxoplasmosis in mice. In allergic diseases such as atopic dermatitis and allergic asthma, allergen-specific T cells acquired the Th2 phenotype. These Th2 cells produce IL-4, IL-5, IL-6, IL-10 and IL-13. These cytokines induce eosinophilia and an Ig class switch to IgG4 and IgE. These Th2 cells are responsible for the enhanced production of IgE antibodies. These findings indicate that Th2 cytokines play an important role in the development of allergic diseases. The importance of cell-mediated immunity, particularly donor-anti-host CTL, in mediating acute GVHD suggests that Th1 cytokines may be important in the induction of acute GVHD. To further characterize the roles of Th1 and Th2 cytokines in the development of acute GVHD, analysis of IL-2, IFN gamma, IL-4 and IL-10 cytokine genes was performed by RT-PCR on biopsied skin specimen. An increase in mRNA expression for IL-2 and IFN gamma was observed, whereas there was no significant increase in IL-4 and IL-10 mRNA. These data suggest that Th1 cytokines may be essential for the development of acute GVHD. It is apparent that Th1 cytokines are generally harmful to the maintenance of pregnancy. We have shown that Th2 cytokines are produced by maternal T lymphocytes at the maternal-fetal surface (retroplacental blood lymphocytes). This finding strengthens the hypothesis of a significant contribution of Th2 cytokines to a successful pregnancy.     

Comparison of granulocyte function in diabetes mellitus type 1 and type 2

The phagocytosis rate of polymorphonuclear leucocytes was measured by flow-cytometry. Vital bacteria were incubated in whole blood. 111 blood samples were measured, 54 in diabetic patients (14 type 1 and 40 type 2), the rest of 57 samples in healthy controls. Results showed firstly, that a decompensation in glucose metabolism in diabetic patients correlated with a decrease in phagocytosis. The HbA1 level was more closely correlated than the glucose level. The second result was, that despite a similar grade of decompensation in type 1 and type 2 diabetic patients, the phagocytosis was significantly lower in type 1 diabetes. No correlation was found concerning age and sex. These findings show, that the impact on granulocytic function in diabetes is of multifactorial origin, not only a shorter or longer elevation of the serum glucose level can explain it solely.     

HLA-C genes and susceptibility to type 1 autoimmune hepatitis

Susceptibility to autoimmune hepatitis (AIH) is associated with the HLA A1-B8-DR3 haplotype, DR4 antigen, and, more specifically, the HLA DRB3*0101, DRB1*0301, and DRB1*0401 alleles. Few investigators, however, have examined the HLA C locus in AIH, which warrants detailed study in view of its recently described roles in immunoregulation. Eighty-seven adult, white patients with well-characterized type 1 AIH and 100 controls were studied. HLA C and HLA DRB1 alleles were assigned by polymerase chain reaction (PCR)-based genotyping. HLA A and B antigens were determined by standard microlymphocytotoxicity assay. Extended haplotypes were constructed according to known patterns of linkage disequilibrium. Only one HLA C locus allele, Cw*0701, which was present in 54% of patients versus 34% of controls (P = .006; relative risk [RR] = 1.54) was associated with AIH. The overall increase in the frequency of the Cw*07 gene (70.1% of patients vs. 54% of controls; P = .024; RR = 1.3) was due entirely to inheritance of the Cw*0701 allele rather than the other Cw*07 alleles, Cw*0702, *0703, and *0704. The RR for Cw*0701 (RR = 1.54) is greater than that for HLA A1 (RR = 1.33) and DRB1*0301 (RR = 1.49), but less than that for HLA-B8 (RR = 1.75). The present findings suggest that the gene or genes conferring susceptibility to AIH lie in the region centromeric to the HLA A locus between HLA C and DRB1. Although linkage disequilibrium with both B8 and DRB1*0301 may account for our finding of an increased frequency of Cw*0701, it is also possible that this allele contributes to disease susceptibility, perhaps by interaction with natural killer cells or cytotoxic T lymphocytes.     

Molecular analysis of the functional role of beta-adrenergic receptor kinase 1 amino-terminal

Receptor phosphorylation is a key step in the process of rapid desensitization. beta-adrenergic receptor kinase (betaARK) is a specific receptor kinase that is known to phosphorylate and induce desensitization of several G-coupled receptors only when they are occupied by their agonists. In the present study we have done several modifications to the amino-terminal of betaARK1, in order to clarify its functional role. The recombinant mutants were tested for their ability to phosphorylate rhodopsin present in purified bovine ROS membranes which serves as a substrate for betaARK1. Their expression levels were detected by Western blot analysis. We found that when the amino-terminal of betaARK1 is modified its expression level is very low, hence it is not able to phosphorylate over the basal. These findings suggest that this region is crucial for the normal processing of the protein.     

Genetic susceptibility in diabetes mellitus: analysis of the HLA association

Two hundred and eighty-eight patients with insulin-dependent diabetes w,o were aged 30 or under at onset and 150 patients with late-onset diabetes, 50 of them dependent on insulin and 100 not dependent on insulin, were HLA-typed. There was a significant positive association between the young-onset insulin-dependent patients and HLA-B8, BW15, and B18 and a significant negative association with B7. These data were combined with those from two other centres. There was a significant concordance for the distribution of all the HLA antigens among these three series, producing evidence in favour of an HLA-linked diabetogenic gene (or genes) having a major role in all cases of juvenile-onset insulin-dependent diabetes. There was a positive association between late-onset insulin-dependent diabetes and B8, but no association between non-insulin-dependent diabetes and the HLA system. This provides further evidence for the existence of different pathogenetic mechanisms in the two major clinical forms of diabetes mellitus.     

Evidence by allelic association-dependent methods for a type 1 diabetes polygene (IDDM6) on chromosome 18q21

Type 1 diabetes is a common polygenic disease. Fine mapping of polygenes by affected sibpair linkage analysis is not practical and allelic association or linkage disequilibrium mapping will have to be employed to attempt to detect founder chromosomes. Given prior evidence of linkage of the Jk-D18S64 region of chromosome 18q12-q21 to type 1 diabetes, we evaluated the 12 informative microsatellite markers in the region for linkage with disease by the transmission disequilibrium test (TDT) in a UK data set of type 1 diabetic families (n = 195). Increased transmission of allele 4 of marker D18S487 to affected children was detected (P = 0.02). Support for this was extended in a total of 1067 families from four different countries by isolating, and evaluating by the TDT, two novel microsatellites within 70 kb of D18S487. Evidence for linkage and association was P = 5 x 10(-5) and 3 x 10(-4), respectively. There was no evidence for increased transmission of associated alleles to nonaffected siblings. Analysis of an additional 390 families by the TDT did not extend the evidence further, and reduced support in the total 1457 families to P = 0.001 for linkage and P = 0.003 for association. However, evidence for linkage by affected sibpair allele sharing was strong (P = 3.2 x 10(-5)) in the second data set. Heterogeneity in TDT results between data sets was, in part, accounted for by the presence of more than one common disease-associated haplotype (allelic heterogeneity) which confounds the analysis of individual alleles by the TDT. Guidelines for strategies for the mapping of polygenes are suggested with the emphasis on collections of large numbers of families from multiple populations that should be as genetically homogeneous as possible.     

Affected-sib-pair mapping of a novel susceptibility gene to insulin-dependent diabetes mellitus (IDDM8) on chromosome 6q25-q27

Affected-sib-pair analyses were performed using 104 Caucasian families to map genes that predispose to insulin-dependent diabetes mellitus (IDDM). We have obtained linkage evidence for D6S446 (maximum lod score [MLS] = 2.8) and for D6S264 (MLS = 2.0) on 6q25-q27. Together with a previously reported data set, linkage can be firmly established (MLS = 3.4 for D6S264), and the disease locus has been designated IDDM8. With analysis of independent families, we confirmed linkage evidence for the previously identified IDDM3 (15q) and DDM7 (2q). We also typed additional markers in the regions containing IDDM3, IDDM4, IDDM5, and IDDM8. Preliminary linkage evidence for a novel region on chromosome 4q (D4S1566) has been found in 47 Florida families (P < .03). We also found evidence of linkage for two regions previously identified as potential linkages in the Florida subset: D3S1303 on 3q (P < .04) and D7S486 on 7q (P < .03). We could not confirm linkage with eight other regions (D1S191, D1S412, D4S1604, D8S264, D8S556, D10S193, D13S158, and D18S64) previously identified as potential linkages.     

Dermatoglyphics in type 1 diabetes mellitus

Although fingerprints and handprints are widely used in criminology, it is only recently that this approach has been applied to the field of medical and genetic diagnoses. In order to investigate dermatoglyphics in Type 1 diabetes mellitus, quantitative characteristics of fingers and palms (ridge count and main line indices) as well as qualitative parameters such as digital and interdigital patterns, the position of the palmar axial triradii and main line courses were analysed in 88 male and 108 female Type 1 diabetic patients and compared with data from 100 male and 99 female normal controls. Type 1 diabetic patients show a lower third finger ridge count (p < 0.05) and a-b ridge count (p < 0.001) and higher transversality of the main lines as indicated by the main line index value (p < 0.001) or the ending of the main line A in a specific sector 5, 5', and 5" (p < 0.001) compared with controls. In addition, diabetic patients show higher frequency of palmar axial t' and t" triradii (p < 0.001) and a lower frequency of 'true' patterns in the fourth interdigital and thenar area (p < 0.001) than controls. By multivariate analysis of quantitative and qualitative variables a predictive value of 78.6% and 77.3%, respectively, for male, and 81.4% and 82.2%, respectively, for female Type 1 diabetic patients was found. In conclusion, dermatoglyphics seem to be an interesting tool for genetic studies related to Type 1 diabetes.     

The role of ethnicity in cancer susceptibility gene polymorphisms: the example of CYP1A1

Individual susceptibility to cancer from environmental agents may be influenced by polymorphic metabolic genes such as CYP1A1. The CYP1A1 gene contains four major polymorphisms identified to date. A modern nomenclature system, used with other genes, is presented to clarify the identity of these polymorphisms. The various CYP1A1 alleles exhibit population frequencies that depend on ethnicity. The association of these alleles with cancer at several sites has also been found to depend on racial or ethnic origin of the study population. Statistical considerations, such as the need for large studies when the power to detect a rare polymorphism is low, and ethnic differences in genetic linkage disequilibrium are among possible reasons for ethnic-specific effects on cancer susceptibility related to metabolic gene polymorphisms. New efforts to determine population frequencies of such polymorphisms are essential for future research in this area.     

Polymorphic structural features of modelled HLA-DQ molecules segregate according to susceptibility or resistance to IDDM

The structural features of HLA-DQ alleles which are susceptible and resistant to insulin-dependent diabetes mellitus (IDDM) have been examined using a model of their three-dimensional structure obtained by energy minimisation, based on the published structure of HLA-DR1. The model shows DQ molecules to have an overall shape nearly identical to that of DR molecules, but with significant differences in the fine structure: 1) the antigen-binding groove of DQ molecules has a polymorphic first pocket; this pocket can be either amphiphilic or hydrophilic, 2) The beta 49-56 dimerisation domain of DQ is polymorphic: hydrophobic, or amphiphilic, or hydrophilic and positively charged, leading to spontaneous or T-cell receptor-induced homodimer formation, or T-cell receptor-induced homodimer formation, or difficulty of the formation of such dimers, respectively; 3) a prominent Arg-Gly-Asp loop is formed by some DQ alleles (beta 167-169) and probably functions in cell adhesion. There are also small differences in the residues and sequences implicated in CD4 binding (mostly in DQ beta 134-148) but the significance of these differences cannot be evaluated at present. All seven DQ alleles which confer susceptibility to IDDM possess a hydrophilic first pocket in the antigen-binding groove, a hydrophobic or amphiphilic beta 49-56 dimerisation patch that allows for spontaneous or T-cell receptor-induced dimerisation, and the Arg-Gly-Asp loop. By contrast, in the protective alleles at least one of these three features is absent. This segregation of phenotypes according to susceptibility or resistance can well explain the model of tighter autoantigen binding by the protective alleles compared to the susceptible alleles, previously proposed for the pathogenesis of IDDM.     

Differential susceptibility of type 1 and type 4 gonococci to chlorhexidine (Hibitane)

Virulent Type 1 gonococci have been shown to be much more suceptible to the killing action of Hibitane gluconate than the avirulent Type 4. Those exploring the possibility of genital prophylaxis as a means of controlling gonorrhoea might find Hibitane worthy of consideration.     

GLP-1 tablet in type 2 diabetes in fasting and postprandial conditions

OBJECTIVE: To examine the absorption of glucagon-like peptide (GLP)-1(7-36) amide from the buccal mucosa of type 2 diabetic patients. Previously, the effects of the peptide have been studied following intravenous and subcutaneous injection. Now, a mucoadhesive, biodegradable buccal GLP-1 tablet (9 mm) containing 119 nmol has been developed as a possible alternative to injection. RESEARCH DESIGN AND METHODS: A total of 10 type 2 diabetic patients received a single tablet under fasting conditions and before a standard meal in this randomized placebo-controlled study. RESULTS: The mean peak GLP-1 concentration was 125.1 pmol/l and occurred 30 min after application. The mean placebo-adjusted area under the curve was 5,334 min pmol/l, consistent with a relative bioavailability of 6% vs. intravenous injection and 42% vs. subcutaneous injection. The half-life of total peptide activity after buccal administration was 17 min. The placebo-adjusted glucose concentrations decreased by 1.4 mmol/l in fasting experiments and by 4.2 mmol/l after a standard mixed meal. In the fasting state at 30 min, plasma insulin increased by 185% and glucagon decreased by 20%, consistent with the increase in plasma GLP-1 concentrations. The peptide exerted a significant insulinotropic effect during meals (calculated as an insulinogenic index, 0-120 min; 84.1 vs. 45.7 in placebo experiments). CONCLUSIONS: Potentially therapeutic plasma levels of GLP-1 were achieved after administration of a single buccal tablet in type 2 diabetic patients. The peptide had a marked glucose-lowering effect during the first 2 h. This new GLP-1 tablet may become a feasible alternative treatment for type 2 diabetic patients, although a more prolonged pharmacokinetic profile is required.