Overexpression of PAI-1 prevents the development of abdominal aortic aneurysm in mice.

Vessel wall inflammation and matrix destruction are critical to abdominal aortic aneurysm (AAA) formation and rupture. We have previously shown that urokinase plasminogen activator (uPA) is highly expressed in experimental AAA and is essential for AAA formation and expansion. In this study, we examined the effects of overexpression of a natural inhibitor of uPA, plasminogen activator inhibitor-1 (PAI-1), on the development of angiotensin (Ang) II-induced AAA in ApoE-deficient (ApoE(-/-)) mice. Mice were treated with recombinant adenovirus containing either the human PAI-1 gene (Ad5.CMV.PAI-1) or the luciferase gene (Ad5.CMV.Luc) delivered either locally by intra-adventitial injection or systemically by tail vein injection. Our results show that local delivery of the PAI-1 gene completely prevented AAA formation (0 vs 55.6% in Ad5.CMV.Luc controls, P<0.05). In contrast, systemic delivery of the PAI-1 gene did not affect AAA incidence (78 vs 90% in Ad5.CMV.Luc controls, P=0.125). Local delivery of the PAI-1 gene 2 weeks after Ang II infusion prevented further expansion of small aneurysms, but had no significant effect on the progression of larger aneurysms. These data suggest that local PAI-1 gene transfer could be used to stabilize small AAA and reduce the rate of expansion and risk of rupture.

Effective treatment of vascular endothelial growth factor refractory hindlimb ischemia by a mutant endothelial nitric oxide synthase gene.

Gene delivery of angiogenic growth factors is a promising approach for the treatment of ischemic cardiovascular diseases. However, success of this new therapeutic principle is hindered by the lack of critical understanding as to how disease pathology affects the efficiency of gene delivery and/or the downstream signaling pathways of angiogenesis. Critical limb ischemia occurs in patients with advanced atherosclerosis often exhibiting deficiency in endothelial nitric oxide production. Similar to these patients, segmental femoral artery resection progresses into severe ischemic necrosis in mice deficient in endothelial nitric oxide synthase (ecNOS-KO) as well as in balb/c mice. We used these models to evaluate the influence of severe ischemia on transfection efficiency and duration of transgene expression in the skeletal muscle following plasmid injection in combination with electroporation. Subsequently, we also explored the potential therapeutic effect of the phosphomimetic mutant of ecNOS gene (NOS1177D) using optimized delivery parameters, and found significant benefit both in ecNOS-KO and balb/c mice. Our results indicate that NOS1177D gene delivery to the ischemic skeletal muscle can be efficient to reverse critical limb ischemia in pathological settings, which are refractory to treatments with a single growth factor, such as vascular endothelial growth factor.

Maternal and fetal amino acid concentrations and fetal outcomes during pre-eclampsia.

The pathophysiology of pre-eclampsia is contested, but one hypothesis indicates that it is a heterogeneous condition in which only a subset of affected women bear small-for-gestational age (SGA) babies. In intrauterine growth-restricted (IUGR) infants, placental transport of amino acids is diminished and the resulting decrease in cord-blood amino acid concentrations is thought to contribute to their stunted growth. In contrast, the metabolic syndrome (dyslipidaemia, hyperinsulinaemia, hyperglycaemia, hypertension and obesity) which is associated with high amino acid concentrations is more prevalent in women with pre-eclampsia. The focus of this study was to compare maternal and fetal serum amino acid concentrations during normal pregnancy and pre-eclampsia and to evaluate the associations between the amino acid concentrations and fetal growth. The results indicate that maternal and cord-blood amino acid concentrations were significantly higher in women with pre-eclampsia compared with normal pregnant women and the concentrations were inversely associated with measures of infant growth. Maternal and cord-blood amino acid concentrations were also significantly higher in pre-eclamptic mothers with SGA infants compared with pre-eclamptic mothers whose babies were not SGA. These data indicate that, in contrast to IUGR, pre-eclampsia is associated with enhanced placental amino acid transport or reduced fetal amino acid utilization. Furthermore, the data are consistent with the hypothesis that pre-eclampsia is a heterogeneous disease associated with the metabolic syndrome.

Increased nitric oxide accounts for decreased basal vascular tone and responsiveness in the resistance vessels of high-cholesterol-fed rabbits.

The objective of this study was to determine the effects of hypercholesterolemia on basal vascular tone and vascular responses to pharmacologic agents in hindquarter resistance vessels. Blood pressure and hindquarter blood flow were measured in conscious rabbits fed a high cholesterol diet (1%) for 17 weeks (HC) compared to age-matched rabbits fed a normal diet (control). Basal hindquarter blood flow and vascular conductance were significantly higher in HC than in control rabbits. Administration of a non-selective nitric oxide synthase (NOS) inhibitor, L-NAME (100 mg/kg) decreased basal hindquarter blood flow and vascular conductance in a greater magnitude in HC than in control rabbits, thus, abolished the differences in both the flow and conductance between 2 groups, indicating that increased NO was responsible for reduced basal vascular tone in the HC rabbits. L-NIL (30 mg/kg), a selective inducible NOS (iNOS) inhibitor had no effects on either flow or conductance. This result does not support the involvement of iNOS. In separate experiments, animals were anesthetized and instrumented with an extracorporeal circuit to measure perfusion pressure under constant blood flow to the hindquarter vascular bed. In the HC group, vascular responses to acetylcholine, S-nitroso-N-acetyl-penicillamine and phenylephrine were all attenuated when compared to the responses in the control rabbits. These results indicate that local overproduction of NO due to hypercholesteremia could desensitize smooth muscle reactivity, thus causing general vascular hyporesponsiveness to vasoactive agents.

Angiotensin II increases urokinase-type plasminogen activator expression and induces aneurysm in the abdominal aorta of apolipoprotein E-deficient mice.

Urokinase-type plasminogen activator (uPA) is increased in human abdominal aortic aneurysm (AAA). Chronic infusion of angiotensin II (Ang II) results in AAA in apolipoprotein E-deficient mice. We tested the hypothesis that Ang II infusion results in an elevation of uPA expression contributing to aneurysm formation. Ang II or vehicle was infused by osmotic pumps into apoE-KO mice. All mice treated with Ang II developed a localized expansion of the suprarenal aorta (75% increase in outer diameter), accompanied by an elevation of blood pressure (22 mmHg), compared to the vehicle-treated group. Histological examination of the dilated aortic segment revealed similarities to human AAA including focal elastin fragmentation, macrophage infiltration, and intravascular hemorrhage. Ang II treatment resulted in a 13-fold increase in the expression of uPA mRNA in the AAA segment in contrast to a twofold increase in the atherosclerotic aortic arch. Increased uPA protein was detected in the abdominal aorta as early as 10 days after Ang II infusion before significant aorta expansion. Thus, Ang II infusion results in macrophage infiltration, increased uPA activity, and aneurysm formation in the abdominal aorta of apoE-KO mice. These data are consistent with a causal role for uPA in the pathogenesis of AAA.

Nitric oxide synthase inhibition increases aortic stiffness measured by pulse wave velocity in rats.

OBJECTIVE: The present study was to examine whether endogenous nitric oxide (NO) plays a role in the regulation of vascular stiffness. METHODS: Pulse wave velocity (PWV) was determined as the time delay between the foot of pressure waves recorded simultaneously at the aortic arch and abdominal aorta (just above the bifurcation) in anesthetized Sprague-Dawley rats. A decrease in vascular compliance results in an increase in PWV. RESULTS: A bolus injection of a NO synthase inhibitor, L-NAME (30 mg/kg), significantly increased PWV, accompanied by an increase in blood pressure. Since changes in blood pressure are known to affect PWV, phenylephrine (PE) was administered to mimic the blood pressure changes induced by L-NAME, thus compensating for the pressure-dependent component of the PWV changes. At each given level of mean arterial pressure (MAP), PWV was significantly higher with L-NAME than with PE treatment, suggesting that acute withdrawal of endogenous NO reduces aortic compliance independent of changes in MAP. In rats chronically treated with L-NAME (0.5 g/l in drinking water) for 3 weeks, PWV was even higher than those acutely treated with L-NAME (at MAP=150 mmHg). This additional increase in vascular stiffness may be due to the remodeling of the vascular wall as a result of chronic NOS inhibition and hypertension. CONCLUSION: These data demonstrate that NO modulates vascular compliance independent of blood pressure changes and that an intact endogenous NO system is required to maintain normal vascular compliance.

Oral contraceptives, other methods of contraception, and risk reduction for ovarian cancer.

Oral contraceptives reduce the risk of ovarian cancer, but the impact of other methods of contraception has not been fully explored. This population-based, case-control study involved women 20-69 years of age who had ever had intercourse. We compared cases with a recent diagnosis of ovarian cancer (N = 727) with community controls (N = 1,360). All methods of contraception evaluated were associated with a reduced risk for ovarian cancer. After adjustment for age, race, pregnancies, and family history of ovarian cancer, the odds ratios for ever-use of each method as compared with never-use were: oral contraceptives for contraception, 0.6 (95% confidence interval = 0.5-0.8); intrauterine device, 0.8 (95% confidence interval = 0.6-1.0); barrier methods, 0.8 (95% confidence interval = 0.6-0.9); tubal ligation, 0.5 (95% confidence interval 0.4-0.7); and vasectomy, 0.8 (95% confidence interval = 0.6-1.1). Nulligravid women were not protected by any of these contraceptive methods. Multigravid women, however, were protected by all methods. We conclude that various methods of contraception reduce ovarian cancer risk. This effect does not appear to result from contraceptive use being a nonspecific marker of fertility. The results imply mechanisms other than hormonal or ovulatory by which ovarian cancer risk is reduced.

Hemodynamic changes in apolipoprotein E-knockout mice.

Apolipoprotein E-knockout (ApoE-KO) mice develop advanced atherosclerotic lesions by 1 yr of age and have been well characterized pathologically and morphologically, but little is known regarding their cardiovascular physiology and hemodynamics. We used noninvasive Doppler ultrasound to measure aortic and mitral blood velocity and aortic pulse-wave velocity in 13-mo-old ApoE-KO and wild-type (WT) mice anesthetized with isoflurane. In other mice from the same colony, we measured systolic blood pressure, body weight, heart weight, cholesterol, and hematocrit. Heart rate and blood pressure were comparable (P = not significant) between ApoE-KO and WT mice, but significant decreases (P < 0.001) were found in body weight (-22%) and hematocrit (-11%), and significant increases were found in heart weight (+23%), aortic velocity (+60%), mitral velocity (+81%) (all P < 0.001), and pulse-wave velocity (+13%, P < 0.05). We also found inflections in the aortic arch velocity signal consistent with enhanced peripheral wave reflection. Thus ApoE-KO mice have phenotypic alterations in indexes of peripheral vascular resistance and compliance and significantly elevated cardiac outflow velocities and heart weight-to-body weight ratios.

Factors related to inflammation of the ovarian epithelium and risk of ovarian cancer.

Previous epidemiologic observations consistently suggest that suppression of ovulation, tubal ligation, and hysterectomy reduce the risk of ovarian cancer and that perineal talc use increases the risk. We examined these and other risk factors in the context of a new hypothesis: that inflammation may play a role in ovarian cancer risk. Ovulation entails ovarian epithelial inflammation; talc, endometriosis, cysts, and hyperthyroidism may be associated with inflammatory responses of the ovarian epithelium; gynecologic surgery may preclude irritants from reaching the ovaries via ascension from the lower genital tract. We evaluated these risk factors in a population-based case-control study. Cases 20-69 years of age with a recent diagnosis of epithelial ovarian cancer (767) were compared with community controls (1,367). We found that a number of reproductive and contraceptive factors that suppress ovulation, including gravidity, breast feeding, and oral contraception, reduced the risk of ovarian cancer. Environmental factors and medical conditions that increased risk included talc use, endometriosis, ovarian cysts, and hyperthyroidism. Gynecologic surgery including hysterectomy and tubal ligation were protective. Tubal ligation afforded a risk reduction even 20 or more years after the surgery. The spectrum of associations provides support for the hypothesis that inflammation may mediate ovarian cancer risk.

Racial differences in ovarian cancer risk.

We examined whether the previously observed lower risk of ovarian cancer among African-American women might be the result of differences in known risk factors. In a population-based, case-control study, sociodemographic, reproductive, and physical risk factors among white (669) and African-American (84) women aged 20 through 69 years with a recent diagnosis of epithelial ovarian cancer (study subjects) were compared with white (1110) and African-American (204) community control subjects. African-American women were more likely to have five or more pregnancies and to have a hysterectomy, whereas white women were more likely to have a family history of ovarian cancer. Yet, the risk and protective factors for ovarian cancer were similar among white and African-American women. As compared with white women, the odds of ovarian cancer among African-American women was significantly lower (odds ratio 0.7, 95% confidence interval [CI] 0.5 to 0.9) and remained somewhat lower after adjusting for known, important risk factors (odds ratio 0.8, 95% CI 0.6 to 1.0). Differences in the obstetric and gynecologic experiences of African-American and white women may explain some of the observed racial variability in ovarian cancer risk, but ovarian cancer risk remained lower among African-American women even after adjustment for these factors.

Risk of ovarian cancer in relation to estrogen and progestin dose and use characteristics of oral contraceptives. SHARE Study Group. Steroid Hormones and Reproductions.

Although past studies have shown that oral contraceptives with 50 microg or more of estrogen reduce the risk of ovarian cancer, it is not clear whether newer, lower-dose formulations do as well. We conducted a population-based, case-control study in the Delaware Valley to assess the impact of dose of oral contraception on risk of ovarian cancer. Cases aged 20-69 years with a diagnosis of epithelial ovarian cancer ascertained between May 1994 and July 1999 (n = 767) were compared with community controls (n = 1,367). Compared with never users, the adjusted risk of ovarian cancer was reduced by 40% for oral contraceptive users overall, with longer duration of use affording greater protection. The ovarian cancer risk reduction was similar for women who initiated oral contraception before 1972, when high-dose pills dominated the market; between 1972 and 1980; and after 1980, when newer, lower-dose pills dominated. Oral contraceptive estrogen and progestin content were compared for cases and controls after adjustment for current age, number of pregnancies, race, and family history of ovarian cancer. Use of low-estrogen/low-progestin pills afforded an estimated risk reduction (odds ratio = 0.5, 95% confidence interval: 0.3, 0.6) that was identical to that for high-estrogen/high-progestin pills (odds ratio = 0.5, 95% confidence interval: 0.3, 0.7).

Effects of ZK-807834, a novel inhibitor of factor Xa, on arterial and venous thrombosis in rabbits.

Inhibition of factor Xa (FXa) may interrupt thrombus progression. This study compared the antithrombotic activity of a novel FXa inhibitor, ZK-807834 [MW, 527 D; Ki (human FXa), 0.11 nM], with recombinant tick anticoagulant peptide [rTAP; MW, 6,685 D; Ki, (human FXa) = 0.28 nM], and DX-9065a [MW 445 D, Ki (human FXa), 40 nM] in rabbits with arterial thrombosis induced by electrical vascular injury. ZK-807834 also was compared with low molecular weight heparin (LMWH; MW, 5,500 D) during venous thrombosis induced by placing a copper wire and threads in the vena cava. Inhibitors were administered as an i.v. bolus and 2-h infusion. Total dosages of ZK-807834, > or =0.7 micromol/kg (n = 18); rTAP, > or =1 micromol/kg (n = 18); or DX-9065a, > or =11 micromol/kg (n = 18) decreased the incidence of arterial thrombotic occlusion compared with control animals (p < 0.05). However, five of six animals given the lowest effective dosage of rTAP and four of six animals given DX-9065a bled from a surgical incision >5 min, but only two of six animals given ZK-807834 bled >5 min. Venous clot weights were reduced compared with controls for dosages of ZK-807834 > or =0.007 micromol/kg (n = 36) or LMWH > or =0.2 micromol/kg (n = 18). Prothrombin time (PT) and activated partial thromboplastin time (aPTT) were unchanged from baseline at the minimally effective dose of ZK-807834, whereas aPTT was increased twofold at the effective dose of LMWH. Thus ZK-807834 may be useful to attenuate thrombosis at lower dosages and with less perturbation of systemic hemostasis compared with available agents.

Identification and characterization of a potent, selective, and orally active antagonist of the CC chemokine receptor-1.

The CC chemokine receptor-1 (CCR1) is a prime therapeutic target for treating autoimmune diseases. Through high capacity screening followed by chemical optimization, we identified a novel non-peptide CCR1 antagonist, R-N-[5-chloro-2-[2-[4-[(4-fluorophenyl)methyl]-2-methyl-1-piperazinyl ]-2-oxoethoxy]phenyl]urea hydrochloric acid salt (BX 471). Competition binding studies revealed that BX 471 was able to displace the CCR1 ligands macrophage inflammatory protein-1alpha (MIP-1alpha), RANTES, and monocyte chemotactic protein-3 (MCP-3) with high affinity (K(i) ranged from 1 nm to 5.5 nm). BX 471 was a potent functional antagonist based on its ability to inhibit a number of CCR1-mediated effects including Ca(2+) mobilization, increase in extracellular acidification rate, CD11b expression, and leukocyte migration. BX 471 demonstrated a greater than 10,000-fold selectivity for CCR1 compared with 28 G-protein-coupled receptors. Pharmacokinetic studies demonstrated that BX 471 was orally active with a bioavailability of 60% in dogs. Furthermore, BX 471 effectively reduces disease in a rat experimental allergic encephalomyelitis model of multiple sclerosis. This study is the first to demonstrate that a non-peptide chemokine receptor antagonist is efficacious in an animal model of an autoimmune disease. In summary, we have identified a potent, selective, and orally available CCR1 antagonist that may be useful in the treatment of chronic inflammatory diseases.

Identification of selective estrogen receptor modulators by their gene expression fingerprints.

Clinical studies have shown that estrogen replacement therapy (ERT) reduces the incidence and severity of osteoporosis and cardiovascular disease in postmenopausal women. However, long term estrogen treatment also increases the risk of endometrial and breast cancer. The selective estrogen receptor (ER) modulators (SERMs) tamoxifen and raloxifene, cause antagonistic and agonistic responses when bound to the ER. Their predominantly antagonistic actions in the mammary gland form the rationale for their therapeutic utility in estrogen-responsive breast cancer, while their agonistic estrogen-like effects in bone and the cardiovascular system make them candidates for ERT regimens. Of these two SERMs, raloxifene is preferred because it has markedly less uterine-stimulatory activity than either estrogen or tamoxifen. To identify additional SERMs, a method to classify compounds based on differential gene expression modulation was developed. By analysis of 24 different combinations of genes and cells, a selected set of assays that permitted discrimination between estrogen, tamoxifen, raloxifene, and the pure ER antagonist ICI164384 was generated. This assay panel was employed to measure the activity of 38 compounds, and the gene expression fingerprints (GEFs) obtained for each compound were used to classify all compounds into eight groups. The compound's GEF predicted its uterine-stimulatory activity. One group of compounds was evaluated for activity in attenuating bone loss in ovariectomized rats. Most compounds with similar GEFs had similar in vivo activities, thereby suggesting that GEF-based screens could be useful in predicting a compound's in vivo pharmacological profile.

Allosteric inhibitors of inducible nitric oxide synthase dimerization discovered via combinatorial chemistry.

Potent and selective inhibitors of inducible nitric oxide synthase (iNOS) (EC ) were identified in an encoded combinatorial chemical library that blocked human iNOS dimerization, and thereby NO production. In a cell-based iNOS assay (A-172 astrocytoma cells) the inhibitors had low-nanomolar IC(50) values and thus were >1,000-fold more potent than the substrate-based direct iNOS inhibitors 1400W and N-methyl-l-arginine. Biochemical studies confirmed that inhibitors caused accumulation of iNOS monomers in mouse macrophage RAW 264.7 cells. High affinity (K(d) approximately 3 nM) of inhibitors for isolated iNOS monomers was confirmed by using a radioligand binding assay. Inhibitors were >1,000-fold selective for iNOS versus endothelial NOS dimerization in a cell-based assay. The crystal structure of inhibitor bound to the monomeric iNOS oxygenase domain revealed inhibitor-heme coordination and substantial perturbation of the substrate binding site and the dimerization interface, indicating that this small molecule acts by allosterically disrupting protein-protein interactions at the dimer interface. These results provide a mechanism-based approach to highly selective iNOS inhibition. Inhibitors were active in vivo, with ED(50) values of <2 mg/kg in a rat model of endotoxin-induced systemic iNOS induction. Thus, this class of dimerization inhibitors has broad therapeutic potential in iNOS-mediated pathologies.

Increased aortic stiffness assessed by pulse wave velocity in apolipoprotein E-deficient mice.

Atherosclerosis develops and progresses spontaneously in apolipoprotein E-knockout (apoE-KO) mice. A direct consequence of atherosclerosis is an increase in vascular stiffness. Pulse wave velocity (PWV) has been used to assess the stiffness of large vessels and was found to be increased in patients with atherosclerosis. In the present study, aortic stiffness was assessed by PWV in 4- and 13-mo-old apoE-KO mice and age-matched controls (C57BL/6J). In 13-mo-old apoE-KO mice with extensive atherosclerotic lesions in the aorta (61 +/- 4%), PWV increased significantly (3.8 +/- 0.2 m/s) compared with controls (2.9 +/- 0.2 m/s). Endothelial nitric oxide (EDNO)-mediated vasorelaxation in response to ACh was markedly diminished in the aortic rings isolated from 13-mo-old apoE-KO mice compared with age-matched controls. In contrast, in 4-mo-old apoE-KO mice with only moderate atherosclerotic lesions in the aorta (23 +/- 5%), there were no significant changes in PWV and EDNO-mediated relaxation compared with controls. Blood pressure was not different among the four groups of mice. There were no significant differences in endothelium-independent vascular responses to sodium nitroprusside among different groups investigated. Histological evaluation revealed focal fragmentation of the elastic laminae in the aortic walls of 13-mo-old apoE-KO mice. These results demonstrate for the first time that aortic stiffness determined by PWV increases in 13-mo-old apoE-KO mice. Endothelial dysfunction and elastic destruction in vascular wall caused by atherosclerosis may have contributed.

Accelerated atherosclerosis and premature calcified cartilaginous metaplasia in the aorta of diabetic male Apo E knockout mice can be prevented by chronic treatment with 17 beta-estradiol.

Epidemiological data indicate that estrogens significantly reduce the risk of morbidity and mortality due to cardiovascular diseases in postmenopausal women. Although numerous animal studies demonstrated inhibition of early atheromatous lesion formation by estrogen treatment in several species, information about the potential benefits of estrogens on complex, advanced atherosclerotic lesions is still lacking. The present study was designed to test whether chronic treatment with 17 beta-estradiol affects hyperglycemia-induced premature advanced lesion formation in 40-week-old male apolipoprotein E-deficient (Apo E-KO) mice. In order to accelerate advanced lesion formation, we treated male Apo E-KO mice with streptozotocin (STZ) at the age of 6 weeks. Two weeks later the STZ-treated mice received a slow release pellet containing either 17 beta-estradiol or placebo. STZ treatment caused sustained hyperglycemia without changes in serum total cholesterol or triglyceride levels compared to citrate control mice. STZ-treated Apo E-KO mice developed significantly more lesions in some (but not all) parts of the aorta and its main branches, and caused premature calcified cartilaginous metaplasia in the lesions of the proximal aorta. Chronic treatment with 17 beta-estradiol lead to a significant decrease in blood glucose and triglyceride levels, reduced the lesion area in all vascular segments studied and prevented cartilaginous metaplasia in STZ-treated Apo E-KO mice. The results of this study show that STZ treatment leads to significant acceleration of atherosclerotic lesion formation and premature occurrence of calcified cartilaginous areas in Apo E-KO mice, which could be effectively prevented by chronic estrogen treatment.

Molecular IDDM epidemiology: international studies. WHO DiaMond Molecular Epidemiology Sub-Project Group.

The WHO DiaMond Molecular Epidemiology Sub-Project is testing the hypothesis that the geographic differences in IDDM incidence reflect population variation in the frequency of IDDM susceptibility genes (i.e., DQA1 and DQB1 alleles with sequences coding for arginine (R) in position 52 of the DQ alpha-chain, and an amino acid other than aspartic acid (ND) in position 57 of the DQ beta-chain, respectively) using a standardized case-control design. Data from twelve populations which have completed (or have nearly completed) recruitment and HLA molecular analyses are presented. There was an approximate 2-fold increase in the frequencies of DGA1*0301, DQB1*0201 and DQB11*0302 among IDDM cases compared to non-diabetic controls in most populations. Interestingly, DQA*0301 was more common in low versus moderate-high incidence countries. DQB1*0201 and DQB1*0302 were more prevalent in the moderate-high incidence areas. DQA1*R and DQB1*ND were both consistent markers of IDDM risk, with stronger associations in moderate-high versus low incidence areas. In general, individuals homozygous for both DQA1*R and DQB1*ND had the highest genotype-specific IDDM incidence rates, which approximated risk estimates for first degree relatives in several countries. These data revealed considerable variation in the frequencies of DQB1 and DQA1 alleles across countries, which likely contribute to the global patterns of IDDM incidence.