Leukofiltration plus pathogen reduction prevents alloimmune platelet refractoriness in a dog transfusion model.

Human lymphocyte antigen alloimmunization to filter leukoreduced (F-LR) platelets occurs in about 18% of immunosuppressed thrombocytopenic hematology/oncology patients and represents a significant challenge for effective chemotherapy. In a dog platelet transfusion model, we have evaluated other methods of preventing alloimmune platelet refractoriness and demonstrated that successful methods in our dog model are transferable to man. In the present study, donor/recipient pairs were dog lymphocyte antigen DR-B incompatible (88% of the pairs), and recipient dogs received up to 8 weekly treated transfusions from a single donor (a highly immunogenic stimulus), or until platelet refractoriness. Continued acceptance of F-LR platelets occurred in 6 of 13 recipients (46%), but neither γ-irradiation (γ-I; 0 of 5) nor Mirasol pathogen reduction (MPR; 1 of 7) treatment of donor platelets prevented alloimmune platelet refractoriness. Combining γ-I with F-LR was associated with only 2 of 10 (20%) recipients accepting the transfused platelets. Surprisingly, F-LR platelets that then underwent MPR were accepted by 21 of 22 (95%) recipients (P < .001 vs F-LR + γ-I recipients). Furthermore, 7 of 21 (33%) of these accepting recipients demonstrated specific tolerance to 8 more weekly donor transfusions that had not been treated. In addition, platelet concentrates prepared from F-LR + MPR whole blood were also nonimmunogenic; that is, 10 of 10 (100%) recipients accepted donor platelets. Overall, 31 of 32 (97%) recipients accepted F-LR + MPR platelets; none developed antibodies to donor lymphocytes. These data are the highest rate of acceptance for platelet transfusions reported in either animals or man. This approach to platelet transfusion may be particularly important when supporting patients with intact immune systems, such as in myelodysplastic syndromes.

Ultraviolet B irradiation in the prevention of alloimmunization in a dog platelet transfusion model.

BACKGROUND: Alloimmune platelet (PLT) refractoriness remains a significant problem for chronically transfused patients with thrombocytopenia. STUDY DESIGN AND METHODS: In a dog PLT transfusion model, we evaluated ultraviolet B irradiation (UV-B) of donor PLTs-either alone or in combination with centrifuge leukoreduction (C-LR) or filtration leukoreduction (F-LR)-to prevent refractoriness to donor PLTs and to induce tolerance to standard (STD) PLTs from the same donor or to tertiary donors. RESULTS: Recipient acceptance rates for C-LR donor PLT transfusions were 14%, F-LR were 33%, and UV-B irradiated were 45% with no significant differences among the treatments given to the donor's PLTs. Adding UV-B irradiation to C-LR or F-LR PLTs increased acceptance rates to 50 and 68% (p = 0.02 and p = 0.05), respectively, comparing single treatments to the combined treatments. After a recipient had accepted any type of UV-B-treated donor PLTs, specific tolerance to subsequent transfusions of the same donor's STD PLTs averaged 65%. Nonspecific tolerance to third-party donor's STD PLTs averaged 36% if they had accepted their initial donor's treated PLTs but was only 4% (p < 0.001) if they had rejected these PLTs. CONCLUSION: Combining UV-B irradiation with a method of leukoreduction produces additive effects on prevention of alloimmune PLT refractoriness.

Masked and overt autoantibodies specific to the DPD epitope of 65-kDa glutamate decarboxylase (GAD65-DPD) are associated with preserved ß-cell functional reserve in ketosis-prone diabetes.

CONTEXT: Ketosis-prone diabetes (KPD), defined by presentation with diabetic ketoacidosis (DKA), comprises 4 subgroups based on the presence or absence of islet cell autoantibodies (A(-) or A(+)) and ß-cell functional reserve (ß(-) or ß(+)). Among A(+) KPD, autoantibody epitope reactivity to 65-kDa glutamate decarboxylase (GAD65), defined by monoclonal GAD65Ab(DPD), was associated with greater ß-cell functional reserve. In a majority of healthy individuals, GAD65Ab are present in the sera but are masked by anti-idiotypic antibodies; in contrast, overtly GAD65Ab-positive patients with autoimmune type 1 diabetes patients lack these anti-idiotypic antibodies. OBJECTIVE: Our objective was to determine the presence of masked and overt GAD65Ab(DPD) in relation to ß-cell function and genetic risk factors in KPD patients. DESIGN: We investigated the associations of masked and overt GAD65Ab(DPD) with ß-cell functional reserve, and their relationship with human leukocyte antigen (HLA) class II haplotypes linked to autoimmune diabetes susceptibility or resistance, in a large KPD cohort. PATIENTS: Adult KPD patients (n = 384) were followed longitudinally in a research clinic. MAIN OUTCOME MEASURES: ß-Cell function, autoantibody status, GAD65Ab epitopes, and HLA class II haplotypes were evaluated. RESULTS: Overall, KPD patients with ß-cell functional reserve (ß(+) subgroups) showed significantly higher frequency of masked GAD65Ab(DPD) than patients without ß-cell functional reserve (ß(-) subgroups): 112 of 144 (79%) compared with 59 of 100 (59%), respectively (P = .002). Masked or overt GAD65Ab(DPD) were also more frequent among autoantibody-positive patients with preserved ß-cell functional reserve (A(+)ß(+) KPD) than those lacking ß-cell function (A(+)ß(-) KPD): 77% compared with 55% (P = .01). The susceptibility HLA haplotypes DQA1*0301/DQB1*0302 and DQA1*0301/DQB1*0201 were associated with absence of overt or masked GAD65Ab(DPD) (odds Ratios 2.3 and 2.2, respectively). CONCLUSIONS: Masked GAD65Ab(DPD) are strongly associated with preserved ß-cell functional reserve among patients with KPD. Absence of GAD65Ab(DPD) reactivity is associated with 2 HLA class II susceptibility haplotypes for autoimmune type 1 diabetes.

A novel JK null allele associated with typing discrepancies among African Americans.

The Jknun (Jk-3) phenotype, attributable to null or silenced alleles, has predominantly been found in persons of Polynesian descent. With the increased use of molecular genotyping, many new silencing mutations have been identified in persons of other ethnic backgrounds. To date, only two JK null alleles have been reported in African Americans, JK*01N.04 and JK*OlN.OS.A comparative study was undertaken to determine whether JK mutations were present in the regional African American population. Results of donor genotyping were compared with previously recorded results of serologic tests, and discrepant results were investigated. Although the two previously identified polymorphisms were not detected in the discrepant samples, a novel allele (191G>A) was identified and was assigned the ISBT number JK*02N.09. This study illustrates a limitation of using single-nucleotide polymorphisms for prediction of blood group antigens.

Presence or absence of a known diabetic ketoacidosis precipitant defines distinct syndromes of "A-ß+" ketosis-prone diabetes based on long-term ß-cell function, human leukocyte antigen class II alleles, and sex predilection.

Ketosis-prone diabetes (KPD) is heterogeneous. Longitudinal follow-up revealed that patients with "A-ß+" KPD (absent autoantibodies and preserved ß-cell function) segregated into 2 subgroups with distinct evolution of ß-cell function and glycemic control. Generalized linear analysis demonstrated that the variable that most significantly differentiated them was presence of a clinically evident precipitating event for the index diabetic ketoacidosis (DKA). Hence, we performed a comprehensive analysis of A-ß+ KPD patients presenting with "provoked" compared with "unprovoked" DKA. Clinical, biochemical, and ß-cell functional characteristics were compared between provoked and unprovoked A-ß+ KPD patients followed prospectively for 1 to 8 years. Human leukocyte antigen class II allele frequencies were compared between these 2 groups and population controls. Unprovoked A-ß+ KPD patients (n = 83) had greater body mass index, male preponderance, higher frequency of women with oligo-/anovulation, more frequent African American ethnicity, and less frequent family history of diabetes than provoked A-ß+ KPD patients (n = 64). The provoked group had higher frequencies of the human leukocyte antigen class II type 1 diabetes mellitus susceptibility alleles DQB1*0302 (than the unprovoked group or population controls) and DRB1*04 (than the unprovoked group), whereas the unprovoked group had a higher frequency of the protective allele DQB1*0602. ß-Cell secretory reserve and glycemic control improved progressively in the unprovoked group but declined in the provoked group. The differences persisted in comparisons restricted to patients with new-onset diabetes. "Unprovoked" A-ß+ KPD is a distinct syndrome characterized by reversible ß-cell dysfunction with male predominance and increased frequency of DQB1*0602, whereas "provoked" A-ß+ KPD is characterized by progressive loss of ß-cell reserve and increased frequency of DQB1*0302 and DRB1*04. Unprovoked DKA predicts long-term ß-cell functional reserve, insulin independence, and glycemic control in KPD.

HLA class II alleles specify phenotypes of ketosis-prone diabetes.

OBJECTIVE: Ketosis-prone diabetes (KPD) comprises four subgroups based on the presence or absence of beta-cell autoantibodies (A+ or A-) and beta-cell functional reserve (beta+ or beta-). Genetic factors could contribute to their distinctive phenotypes. Our aim was to specify the role of HLA class II alleles associated with susceptibility or resistance to autoimmune type 1 diabetes in determining KPD phenotypes. RESEARCH DESIGN AND METHODS: A total of 185 adults presenting with diabetic ketoacidosis were followed longitudinally for a mean of 5.5 years, with measurements of autoantibodies, beta-cell functional reserve, insulin sensitivity, and insulin requirement. Frequencies of susceptibility and resistance alleles at HLA DQA1, DQB1, and DRB1 loci were correlated with clinical and phenotypic features of KPD subgroups and compared with those of ethnic-specific population control subjects. RESULTS: Susceptibility alleles were more frequent (P < 0.0001) in the two A+ than the two A- KPD subgroups; in the latter, the frequency was no greater than in population control subjects (except for DQB1*0302). Susceptibility alleles differentiated the two clinically similar beta- subgroups (more frequent in A+beta- than A-beta- KPD; P < 0.01). Resistance alleles were more frequent in the two beta+ than the two beta- KPD subgroups (P < 0.01). The frequencies of certain susceptibility (e.g., DQB1*02) and resistance (DQB1*0602) alleles were higher in African-American A-beta+ KPD patients than in African-American control subjects. DQB1*0302 was more frequent in all KPD subgroups compared with control subjects. CONCLUSIONS: HLA class II alleles associated with susceptibility or resistance to autoimmune type 1 diabetes help specify the four subgroups of KPD. Inheritance of these alleles may influence long-term beta-cell functional reserve.

Accuracy and predictive value of classification schemes for ketosis-prone diabetes.

OBJECTIVE: Ketosis-prone diabetes (KPD) is an emerging, heterogeneous syndrome. A sound classification scheme for KPD is essential to guide clinical practice and pathophysiologic studies. Four schemes have been used and are based on immunologic criteria, immunologic criteria and insulin requirement, BMI, and immunologic criteria and beta-cell function (Abeta classification). The aim of the present study is to compare the four schemes for accuracy and predictive value in determining whether KPD patients have absent or preserved beta-cell function, which is a strong determinant of long-term insulin dependence and clinical phenotype. RESEARCH DESIGN AND METHODS: Consecutive patients (n = 294) presenting with diabetic ketoacidosis and followed for 12-60 months were classified according to all four schemes. They were evaluated longitudinally for beta-cell autoimmunity, clinical and biochemical features, beta-cell function, and insulin dependence. beta-Cell function was defined by peak plasma C-peptide response to glucagon >or=1.5 ng/ml. The accuracy of each scheme to predict absent or preserved beta-cell function after 12 months of follow-up was tested using multiple statistical analyses. RESULTS: The "Abeta" classification scheme was the most accurate overall, with a sensitivity and specificity of 99.4 and 95.9%, respectively, positive and negative likelihood ratios of 24.55 and 0.01, respectively, and an area under the receiver operator characteristic curve of 0.972. CONCLUSIONS: The Abeta scheme has the highest accuracy and predictive value in classifying KPD patients with regard to clinical outcomes and pathophysiologic subtypes.

The use of CD 34(+) mobilized peripheral blood as a donor cell source does not improve chimerism after in utero hematopoietic stem cell transplantation in non-human primates.

In utero hematopoietic stem cell transplantation is a therapeutic procedure that could potentially cure many developmental diseases affecting the immune and hematopoietic systems. In most clinical and experimental settings of fetal hematopoietic transplantation the level of donor cell engraftment has been low, suggesting that even in the fetus there are significant barriers to donor cell engraftment. In postnatal hematopoietic transplantation donor cells obtained from mobilized peripheral blood engraft more rapidly than cells derived from marrow. We tested the hypothesis that use of donor hematopoietic/stem cells obtained from mobilized peripheral blood would improve engraftment and the level of chimerism after in utero transplantation in non-human primates. Despite the potential competitive advantage from the use of CD 34(+) from mobilized peripheral blood, the level of chimerism was not appreciably different from a group of animals receiving marrow-derived CD 34(+) donor cells. Based on these results, it is unlikely that this single change in cell source will influence the clinical outcome of fetal hematopoietic transplantation.

Evaluation of different methods of leukoreduction of donor platelets to prevent alloimmune platelet refractoriness and induce tolerance in a canine transfusion model.

The effectiveness of different methods of leukoreduction in preventing alloimmune platelet refractoriness was evaluated in a canine model. Platelets from a random donor dog were administered for up to 8 weeks or until platelet refractoriness. Standard (STD; unmodified) platelets were accepted by 14% of recipients (n = 7) compared with 14% for centrifuge leukoreduced (C-LR) platelets (n = 21) and 31% for filter leukoreduced (F-LR) platelets (n = 13; no significant differences). Surprisingly, using both F-LR and C-LR platelets was highly effective (87% acceptance, n = 15). Transfusing F-LR/C-LR red blood cells (n = 4) or F-LR/C-LR plasma (n = 4), along with F-LR/C-LR platelets, did not affect platelet acceptance (100% acceptance). Overall acceptance of F-LR/C-LR platelets was 91% (n = 23; P < or = .05 versus STD, C-LR, or F-LR platelets). F-LR/C-LR transfusions also induced tolerance to subsequent STD platelet transfusions from the same donor (82% acceptance, n = 19) as well as to donor skin grafts without recipient immunosuppression (57% acceptance, n = 7). To evaluate mechanisms of tolerance induction, F-LR/C-LR platelets were gamma-irradiated. Although the gamma-irradiated F-LR/C-LR platelets were uniformly accepted (n = 6), tolerance to STD platelets was lost. These data suggest that some allostimulatory white cells are filter adherent, whereas others escape filtration but can be removed by centrifugation and tolerance requires a residual functioning white cell.

Fetal immune suppression as adjunctive therapy for in utero hematopoietic stem cell transplantation in nonhuman primates.

In utero hematopoietic stem cell transplantation could potentially be used to treat many genetic diseases but rarely has been successful except in severe immunodeficiency syndromes. We explored two ways to potentially increase chimerism in a nonhuman primate model: (a) fetal immune suppression at the time of transplantation and (b) postnatal donor stem cell infusion. Fetal Macaca nemestrina treated with a combination of the corticosteroid betamethasone (0.9 mg/kg) and rabbit thymoglobulin (ATG; 50 mg/kg) were given haploidentical, marrow-derived, CD34+ -enriched donor cells. Animals treated postnatally received either donor-derived T cell-depleted or CD34+ -enriched marrow cells. Chimerism was determined by traditional and real-time polymerase chain reaction from marrow, marrow progenitors, peripheral blood, and mature peripheral blood progeny. After birth, the level of chimerism in the progenitor population was higher in the immune-suppressed animals relative to controls (11.3% +/- 2.7% and 5.1% +/- 1.5%, respectively; p = .057). Chimerism remained significantly elevated in both marrow (p = .02) and fluorescence-activated cell sorted and purified CD34+ cells (p = .01) relative to control animals at > or = 14 months of age. Peripheral blood chimerism, both at birth and long term, was similar in immune-suppressed and control animals. In the animals receiving postnatal donor cell infusions, there was an initial increase in progenitor chimerism; however, at 6-month follow-up, the level of chimerism was unchanged from the preinfusion values. Although fetal immune suppression was associated with an increase in the level of progenitor and marrow chimerism, the total contribution to marrow and the levels of mature donor progeny in the peripheral blood remained low. The level of long-term chimerism also was not improved with postnatal donor cell infusion.

Nonhuman primate models for islet transplantation in type 1 diabetes research.

Insulin-dependent diabetes mellitus is an autoimmune disease that causes a progressive destruction of the pancreatic beta cells. As a result, the patient requires exogenous insulin to maintain normal blood glucose levels. Both the pancreas and the islets of Langerhans have been transplanted successfully in humans and in animal models, resulting in full normalization of glucose homeostasis. However, insulin independence, transient or persistent, was documented in only a small fraction of cases until recently. The chronic immunosuppression required to avoid immunological rejection appears to be toxic to the islets and adds the risk of lymphoproliferative disease reported earlier. For islet transplantation to become the method of choice, it is essential first to identify islet-friendly immunosuppressive regimens and/or to develop methods that induce donor-specific tolerance and improve islet isolation and transplantation protocols. Indeed, researchers have already successfully allografted islets in the presence of nonsteroidal immunosuppression in a process known as the Edmonton protocol. An alternative method, gene therapy, could replace these other methods and better meet the insulin requirement of an individual without requiring pancreatic or islet transplantation. This alternative, however, requires animal models to develop and test clinical protocols and to demonstrate the feasibility of preclinical trials. Nonhuman primates are ideally suited to achieve these goals. The efforts toward developing a nonhuman primate diabetic model with demonstrable insulin dependence are discussed and include pancreatic and islet transplant trials to reverse the diabetic state and achieve insulin independence. Also described are the various protocols that have been tested in primates to circumvent immunosuppression by using tolerance induction strategies in lieu of immunosuppression, thus exploring the field of donor-specific tolerance that extends beyond islet transplantation.

HLA-DRB1, -DQA1, and -DQB1 subtypes or ACE gene polymorphisms do not seem to be risk markers for severe retinopathy in younger Type 1 diabetic patients.

The aim of this study was to test the hypothesis that HLA-DRB1, -DQA1, and -DQB1 subgroups or angiotensin-converting enzyme (ACE) gene polymorphisms are associated with severe retinopathy in younger Type 1 diabetic patients. Twenty-four Type 1 diabetic patients who had received panretinal photocoagulation for severe nonproliferative or proliferative diabetic retinopathy were compared with 24 Type 1 diabetic patients (participating in a photographic screening program with regular fundus examinations) with no or minimal retinopathy, matched for age at onset and duration of diabetes. The HLA-DRB1-DQA1-B1 haplotype 04-03-0302 represented 22/48 (46%) in the severe and 21/48 (44%) in the no/minimal retinopathy group, respectively (n.s.). The most common genotype, 03-0501-0201/04-03-0302, occurred in 8/24 (33%) with severe and 10/24 (42%) with no/minimal retinopathy, respectively (n.s.). There were no statistical differences between patients with severe and no/minimal retinopathy whether DRB1, DQA1, or DQB1 alleles, haplotypes, or genotypes were analysed. The ACE gene polymorphism was almost identical between patients with severe and no/minimal retinopathy, and serum ACE levels did not differ. Thus, in the present study on a small group with carefully characterised diabetic retinopathy phenotypes, there was no indication that HLA-DRB1, -DQA1, and -DQB1 subtypes or ACE gene polymorphisms were associated with severe retinopathy in younger Type 1 diabetic patients.

Ketosis-prone diabetes: dissection of a heterogeneous syndrome using an immunogenetic and beta-cell functional classification, prospective analysis, and clinical outcomes.

Ketosis-prone diabetes is heterogeneous. Its causes could include novel beta-cell functional defects. To characterize such defects, 103 patients with diabetic ketoacidosis were evaluated for beta-cell autoimmunity and human leukocyte antigen (HLA) class II alleles, with longitudinal measurements of beta-cell function and biochemical and clinical parameters. They were classified into four A beta groups, based on the presence of glutamic acid decarboxylase (GAD)65, GAD67, or IA-2 autoantibodies (A+ or A-) and beta-cell functional reserve (beta+ or beta-). The group distribution was: 18 A+beta-, 23 A-beta-, 11 A+beta+, and 51 A-beta+. Collectively, the two beta- groups differed from the two beta+ groups in earlier onset and longer duration of diabetes, lower body mass index, less glycemic improvement, and persistent insulin requirement. HLA class II genotyping showed that the A-beta- group differed from the A+beta- group in having lower frequencies of two alleles strongly associated with autoimmune type 1 diabetes susceptibility: DQA*03 and DQB1*02. Similarly, the A-beta+ group differed from the A+beta+ group in having a lower frequency of DQB1*02. Ketosis-prone diabetes comprises at least four etiologically distinct syndromes separable by autoantibody status, HLA genotype, and beta-cell functional reserve. Novel, nonautoimmune causes of beta-cell dysfunction are likely to underlie the A-beta+ and A-beta- syndromes.

In utero hematopoietic stem cell transplantation in nonhuman primates: the role of T cells.

In utero transplantation of hematopoietic stem cells is a promising treatment for immune and hematologic diseases of fetuses and newborns. Unfortunately, there are limited data from nonhuman primates and humans describing optimal transplantation conditions. The purpose of this investigation was to determine the effect of T-cell number on engraftment and the level of chimerism after in utero transplantation in nonhuman primates. CD34(+) allogeneic adult bone marrow cells, obtained from the sire after G-CSF and stem cell factor administration, were transplanted into female fetal recipients. The average CD34(+) cell dose was 3.0 x 10(9)/kg (range, 9.9 x 10(8) to 4.4 x 10(9)) and the T-cell dose ranged from 2.6 x 10(5) to 1.1 x 10(8)/kg. Chimerism was determined in peripheral blood subsets (CD2, CD13, and CD20) and in progenitor cell populations by using polymerase chain reaction. Chimerism was noted in seven of eight live-born animals. The level of chimerism in the progenitor population was related to the fetal T-cell dose (r = 0.64, p < 0.02). At the lowest T-cell dose (2.6 x 10(5)/kg), no chimerism was detected. As the T-cell dose increased to 10(6-7)/kg, the level of chimerism increased. Adjusting the T-cell dose to 1.1 x 10(8)/kg resulted in fatal graft-versus-host disease (GVHD). The results of this study emphasize the importance of T cells in facilitating donor cell engraftment and in producing GVHD in fetal nonhuman primates. Some animals achieved levels of chimerism in the marrow hematopoietic progenitor cell population that would likely have clinical relevance. However, the levels of chimerism in peripheral blood were too low for therapeutic benefit. Further studies are needed to test methods that are likely to enhance donor cell engraftment and peripheral blood levels of donor cells.

Immunogenetic correlates for Chlamydia trachomatis-associated tubal infertility.

OBJECTIVE: To understand immunogenetic mechanisms of Chlamydia trachomatis infection and tubal scarring. METHODS: We measured and compared previously significant human leukocyte antigen (HLA) class II DQ alleles, their linked DRB genes, and polymorphisms in selected cytokine genes (tumor necrosis factor alpha-308 promoter; transforming growth factor beta1-10 and -25 codons; interleukin 10-1082, -819, and -592 promoters; interleukin 6-174 promoter; and interferon gamma+874 codon 1) among Kenyan women with confirmed tubal infertility with and without C trachomatis microimmunofluorescence antibody. RESULTS: Two class II alleles, HLA-DR1*1503 and DRB5*0101, were detected less commonly in C trachomatis microimmunofluorescence seropositive women than in C trachomatis microimmunofluorescence seronegative women with infertility (0% versus 20%; odds ratio [OR] 0.05; 95% confidence interval [CI] 0, 0.7, and 6% versus 26%; OR 0.2; 95% CI 0.02, 1.0, respectively). These alleles are commonly linked as a haplotype at the DRB locus. This finding could not be explained through linkage disequilibrium with the other studied HLA or cytokine genes. CONCLUSION: These alleles may lead to an immunologically mediated mechanism of protection against C trachomatis infection and associated tubal damage, or alternatively increase risk for tubal scarring due to another cause.

Diabetes duration may modify the association between genetic variation in the glycoprotein Ia subunit of the platelet collagen receptor and risk of severe diabetic retinopathy: a working hypothesis.

Genetic factors appear to contribute to the severity and progression of diabetic retinopathy. We assessed the associations of the C807T and Glu505Lys variants of the glycoprotein Ia (alpha(2) integrin) subunit of the platelet/endothelial collagen receptor and risk of retinopathy in a population-based survey of 288 diabetic patients in one Swedish community. Neither variant was associated with retinopathy risk overall. However, the 807T variant was associated with increased risk of severe retinopathy, and the association was modified by diabetes duration. Among patients with diabetes of longer duration (>/=25 years), the 807T variant was strongly associated with risk of severe retinopathy (odds ratio 7.49, 95% confidence interval 1.75 to 32.1). There was no association between the 807T variant and risk of severe retinopathy among patients with diabetes duration <25 years. The Lys505 variant of glycoprotein Ia was associated with an odds ratio for severe retinopathy of 1.88 (95% confidence interval 0.83 to 4.24). Overall, there was a significant interaction between glycoprotein Ia genotype and duration of diabetes on the risk of retinopathy (P-value for interaction = 0.019). These results suggest the hypothesis that genetic variation of platelet glycoprotein Ia may play a particularly important role during the advanced stages of diabetic retinopathy.

Genetic variants of the hemostatic system and development of transplant coronary artery disease.

BACKGROUND: The occurrence of coronary artery disease (CAD) after heart transplantation may represent an accelerated inflammatory and thrombotic response to coronary vascular endothelial cell injury. Several common mutations involving hemostasis and cellular adhesion proteins have been associated with genetic susceptibility to native coronary heart disease. The clinical setting of heart transplantation provides a unique opportunity to examine the relative contribution of circulating (i.e., recipient) vs local vascular (i.e., donor) hemostatic components to the occurrence of CAD. METHODS: We performed genotyping for several common hemostatic polymorphisms among 53 cardiac transplant patients and their heart donors. Patients were observed for an average of 43 months, and the presence of transplant CAD was determined by coronary angiography. RESULTS: The development of transplant CAD did not relate to recipient hemostatic genotype, but 2 donor polymorphisms (PAI-1 4G/5G and alpha(2) integrin C807T) were important predictors of transplant CAD. The risk ratio (RR) of transplant CAD associated with donor PAI-1 4G/4G genotype was 2.6 (95% confidence interval [CI] 1.1-6.2) and was modified by recipient cytomegalovirus status, hyperlipidemia, diabetes, and recipient factor XIII Val34Leu genotype. The RR of transplant CAD associated with donor alpha(2) integrin 807 T/T genotype was 7.4 (95% CI, 2.5-22.0). CONCLUSIONS: Genetic and metabolic factors contributed by both donor and recipient may interact at the level of the coronary vessel wall in the development of transplant-associated CAD, and this finding may provide additional support for the importance of local thrombotic response to endothelial injury in the pathogenesis of this disorder.

Induction of donor-specific tolerance to islet allografts in nonhuman primates.

Pancreatic tissue grafting is by far the most physiological therapeutic solution to the insulin deficiency of diabetes. Recent clinical trials have indicated somewhat successful use of nonsteroidal immunosuppressive regimens and a successful nonhuman primate trial using CD154 for costimulation blockade was reported. However, these protocols need to be replaced with safe and efficacious ones in which long-term allotolerance would make these treatments routine in a clinical setting. With the specific objective of testing whether peripheral infusions of stem cells or stem cell fractions in conjunction with islet allografting would induce allograft tolerance, we have established a macaque diabetic model. The macaques were rendered diabetic by streptozotocin and required daily doses of insulin to maintain lower blood glucose levels. The diabetic macaques then received islets and stem cells from unrelated and MHC-mismatched donors without any immunosuppression. In our initial analysis, 5 of 7 macaques that received stem cell infusions at the time of islet allografting have shown allograft survival longer than the group of macaques that received islets without the stem cell infusion. One of these five macaques has been normoglycemic for 10 months, with no exogenous insulin. This macaque received stem cell population enriched for CD34+ cells with depletion of CD18 cells, which have shown low or no allostimulatory potential in mixed lymphocyte cultures. Increased levels in insulin and C-peptide levels were shown in the macaques after islet transplantation.

Induction of islet allotolerance in nonhuman primates.

Recent clinical trials have pioneered the successful use of a nonsteroidal immunosuppressive regimen and established a basis for application in a routine clinical setting. In this study, a single islet transplant was not sufficient to regulate blood glucose levels, and a second transplant became necessary. A similar observation was made in our macaque islet transplant study, where animals after the second transplantation have shown trends towards normoglycemia in the presence of mycophenolate mofetil. All five animals that received the second transplant have shown an initial rise in C peptide levels, which rapidly decreased as we tapered the MMF dose from 20 mg/kg BID to 5 mg/kg SID. Two animals of the five that were preconditioned with MMF one week prior to transplantation have shown significantly higher C peptide levels. We believe that it is very important to understand the relationship between the first graft failure and subsequent islet allograft success. Since graft success did not correlate with number of transplanted islets, the correction of blood glucose levels toward normoglycemia after the second transplantation suggests a mechanism by which the allotolerance to second transplant is facilitated by the first islet transplantation. These initial observations suggest approaches to "tolerize" the recipient to accept the second-transplant islets (a) through preconditioning the animal to improve the rate of success for the first transplant or (b) through tolerization to islets in the first transplant to facilitate better engraftment of the second-transplant islets.

ICA+ relatives with DQA1*0102/DQB1*0602 have expected 0602 sequence and DR types.

The HLA haplotype DQA1*0102/DQB1*0602 reportely confers protection from type 1 diabetes. DQA1*0102/DQB1*0602 is present in more than 7% of ICA positive relatives screened as part of the Diabetes Prevention Trial--type 1. The presence of autoantibodies in these subjects suggests that the mechanism that protects DQB1*0602 subjects from diabetes occurs after the disease process has been initiated. However, as previously suggested, the method used to type the DQB1*0602 alleles may have lacked the sensitivity to identify alleles similar, but not identical, to DQB1*0602. In addition unusual extended haplotypes may be presented that could help account for the presence of diabetes autoantibodies. We therefore sequenced and performed extended haplotyping on samples from ICA+ relatives with DQA1*0102/DQB1*0602. In this group, sequencing confirmed DQB1*0602 in 149/150, and 152/165 have the common DRB1*1501-DQB1*0602 haplotype. Thus, high resolution typing of class II alleles either by PCR-based oligotyping or nucleotide sequencing fail to indicate any unusual genetic characteristics about these antibody-positive relatives, of which few are expected to progress to clinical disease.