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The lymphocyte transformation response to the mitogen phytohemagglutinin (PHA) was determined in forty well controlled insulin-dependent diabetics, forty matched normal subjects and fourteen poorly controlled insulin-dependent diabetics. There was no significant difference in the PHA responses of normal subjects and well controlled diabetics, but poorly controlled diabetics showed a marked depression of lymphocyte transformation.Peripheral blood T and B lymphocyte subpopulations were also measured in fifteen normal subjects, fifteen well controlled diabetics and ten poorly controlled diabetics. The results showed no significant difference between normal and diabetic subjects, whether well or poorly controlled.The depressed PHA response in poorly controlled diabetics would seem to reflect inadequately corrected metabolic disturbance rather than an inherent, genetically determined immunologic abnormality. DIABETES 23:708-12, August, 1974.Recent studies have examined the role of cellmediated immune mechanisms in diabetes mellitus. For example, the leucocyte migration test (LMT) 1 has reportedly shown cellular hypersensitivity in diabetics against both nonspecific antigens (human and rat liver mitochondria 2 ) and an antigen derived from the microsomal fraction of the islets of Langerhans (porcine pancreatic antigen 3 ). In the latter study a positive correlation was found between the LMT response and delayed-type skin hypersensitivity to the antigen. 3Other investigators have used the lymphocyte transformation response to the mitogen phytohemagglutinin (PHA) 4 as an in vitro test of cell-mediated immune function: some record the PHA response to be impaired with diabetic lymphocytes, 5 suggesting immunologic abnormality, while others report it to be normal. 6 To re-examine these results we have measured the lymphocyte transformation response to PHA in well controlled and poorly controlled insulindependent diabetics and in normal subjects; in addition erythrocyte rosette 7 " 9 and in indirect immunofluorescence 7>9 technics have been used to compare the numbers of circulating T and B lymphocytes in peripheral blood from these three groups. PATIENTS AND METHODS PatientsPHA responses were studied in forty well controlled diabetic and forty normal subjects who were carefully matched for age and sex. The diabetic subjects (twenty-two women, eighteen men; mean age 42.4 years) were insulin-dependent, attended an outpatient clinic, and were free from infection on the day of study. The control subjects were healthy volunteers, mainly laboratory personnel or hospital outpatients not known to have endocrine disease or immunologic abnormality.PHA responses were also studied in a group of fourteen poorly controlled insulin-dependent diabetics. These patients had been brought to an outpatient clinic for routine or emergency review and their disorder was judged to be poorly controlled by the following criteria: midmorning blood glucose exceeding 350 708 DIABETES, VOL. 2 3 , NO. 8

Allograft bone, in the form of fresh-frozen human femoral head, gives clinical results at least as good as autograft bone in instrumented posterolateral lumbar spinal fusion and completely avoids any donor site complications.

RhD is a major blood group and the most important target antigen in hemolytic disease of the newborn (HDN). The aims of this study were to establish a humanized mouse model of responses to the RhD protein and to test whether these could be prevented by the induction of immune tolerance. HLA-DR15 is a major restricting element for human T-helper (Th) cells specific for RhD protein, and expression of this HLA-DR transgene was found to confer on mice the ability to respond to immunization with purified RhD protein. Synthetic peptides containing dominant IntroductionThe RhD antigen is a highly immunogenic and clinically important human blood group. Approximately 10% of pregnancies are at risk for hemolytic disease of the newborn (HDN) caused by RhD incompatibility, 1 and current prophylaxis is dependent on passive RhD immune globulin. Fuller understanding of the immune response to RhD will enable the design of alternative strategies to prevent HDN.Most immunoglobulin G (IgG) antibody responses are dependent on T-cell help, and the production of antibodies specific for red blood cells (RBCs), 2 including anti-D, 3 is no exception. T-helper (Th) cells recognize short antigen-derived peptides displayed by specialized antigen-presenting cells (APCs), and it is now clear that the context in which such recognition takes place determines whether a specific immune response is activated or tolerized. 4,5 Methods to manipulate Th recognition of the RhD protein to favor tolerance are of particular interest because this would permit antigen-specific intervention to prevent HDN, without the risks associated with the use of blood products. Mucosal administration of helper epitopes is an approach that has been used to induce systemic tolerance to antigens of pathogenic relevance in models of transplantation, 6 autoimmunity, 4,5 and asthma. 7 In many cases, the induction of such tolerance has been attributed to active mechanisms of immune regulation, particularly the stimulation of regulatory T cells. 6,8,9 Dominant epitopes from the RhD protein that induce the proliferation of Th cells from alloimmunized donors in vitro have previously been mapped by us. 3 In particular, peptides RhD 52-66 , RhD [97][98][99][100][101][102][103][104][105][106][107][108][109][110][111] were each able to stimulate Th cells in vitro from more than 50% of alloimmunized donors, with responses to at least 1 sequence in every donor. The question arises as to whether administering these peptides by a tolerogenic route could prevent responses to the entire RhD protein in vivo. To address this possibility, we first developed a humanized model of responsiveness to the RhD protein. The HLA-DRB1*1501 allele is significantly overrepresented (47.6%; 2 ϭ 4.269; P ϭ .039) in RhD-negative donors who have produced anti-D antibodies in response to exposure to RhD-positive RBCs, 10 and HLA-DR is the major restricting locus for Th cells specific for RhD protein epitopes. 3 Mice transgenic for HLA-DR15 were selected for this study, predicting that the RhD protein would...

Individually matched (rhesus-D) Rh-positive frozen/thawed red blood cells were used to immunize 28 Rh-negative male volunteers. The immunizing schedule consisted of a single unit (200 ml) of frozen/thawed red blood cells, followed by six monthly booster doses (0.5 to 1.0 ml) after a rest period of six months. A final response rate of 93 percent (26 of 28) was achieved. All responders had produced anti-D before the second booster injection (mean detection time 4.25 months). Retrospective analysis indicated that the final response rate and the level of anti-D response could be predicted as early as seven to eight months from the start of immunization. These findings have practical implications for deciding when to discontinue immunization. The Rh genotype of the immunizing cells did not appear to be an important factor in determining the anti-D response, and with the matching system used, antibodies other than anti-C, D, and/or E were not produced. The use of frozen/thawed red blood cells for immunization has the advantage of permitting optimum matching for undesirable red blood cell antigens and minimizing the risk of transmitting disease to the recipients.

Although considerable effort has been devoted to characterizing alloantibodies specific for the Rhesus D (RhD) blood group antigen, virtually nothing is known about the helper response that drives their production. Therefore, the aim of this study was to map alloreactive T-cell epitopes on the RhD protein. Peripheral blood mononuclear cells (PBMCs) were obtained from 22 RhD-negative volunteers in whom anti-D alloantibodies had developed after deliberate immunization or RhD-incompatible pregnancy. The PBMCs were stimulated with a panel of up to 68 overlapping synthetic 15-mer peptides spanning the complete sequence of the RhD protein. One or more peptides elicited proliferative responses by PBMCs from all 22 of the alloimmune volunteers but from only 2 of 8 alloantibody-negative control donors. Proliferation of PBMCs from the alloimmune donors was mediated by major histocompatibility complex class II–restricted T cells expressing the CD45RO marker of previous activation or memory. The number of peptides that induced proliferative responses was unrelated to either the frequency of, or time since, exposure to RhD-positive red blood cells, but it correlated strongly (Rs = 0.75;P < .003) with the level of anti-D antibodies in deliberately immunized donors. The patterns of stimulatory peptides varied among alloimmune volunteers, but particular sequences were commonly recognized, with 4 peptides each eliciting a response in more than 50% of these donors. Identification of such peptides containing dominant alloreactive helper epitopes is the first step in the development of improved or new approaches to preventing hemolytic disease of the newborn that are based on modulating the T-cell response to the RhD protein.

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Background/aims-Sympathetic ophthalmia (SO) is a classic example of autoimmune disease where human leucocyte antigen (HLA) genomic associations could provide further understanding of mechanisms of disease. This study sought to assess HLA genetic polymorphism in British and Irish patients with SO, and to assess whether HLA gene variants are associated with clinical phenotype or disease severity. Methods-High resolution DNA based HLA typing using polymerase chain reaction sequence specific primers was performed in 27 patients with SO and 51 matched healthy controls. Clinical phenotype and markers of disease severity were determined prospectively in 17 newly diagnosed patients and from medical record review and repeat clinical examination in 10 previously diagnosed patients. Results-HLA-Cw*03 (p=0.008), DRB1*04 (p=0.017), and DQA1*03 (p=0.014) were significantly associated with SO. For class II alleles at higher resolution, only HLA-DRB1*0404 (relative risk (RR) = 5.6, p = 0.045) was significantly associated with SO. The highest relative risk for any of the associated haplotypes was with HLA-DRB1*0404-DQA1*0301 (RR=10.9, p=0.019). Patients with the DRB1*04-DQA1*03 associated haplotype were significantly more likely to develop SO earlier, with fewer inciting ocular trauma events, and to require more systemic steroid therapy to control inflammatory activity. Conclusions-Sympathetic ophthalmia is associated with HLA-DRB1*04 and DQA1*03 genotypes in white patients, similar to Japanese patients. DiVerences in DRB1*04 gene variant associations (−0404 in Britain and Ireland and −0405 in Japan) may have implications for HLA peptide binding in disease initiation. The DRB1*04-DQA1*03 haplotype is a marker of increased SO susceptibility and severity, as in Vogt-Koyanagi-Harada disease, which also has similar clinicopathological and HLA associations. (Br J Ophthalmol 2001;85:281-286)