Beneficial effects of parenteral GLP-1 delivery by cell therapy in insulin-deficient streptozotocin diabetic mice.

Parenteral delivery of long-acting glucagon-like peptide-1 (GLP-1) mimetics has received much attention as a therapeutic option for diabetes. However, cell therapy-based GLP-1 treatments may provide a more physiological regulation of blood glucose. The present study assessed the effects of chronic GLP-1 delivery by cell therapy, using the GLP-1-secreting GLUTag cell line, in normoglycemic and streptozotocin-induced diabetic mice. GLUTag cell aggregates were transplanted into the subscapular region of mice. Over 30 days, cellular transplantation gave rise to encapsulated and well-vascularized growths, which contained immunoreactive GLP-1. Cell implantation was well tolerated and had no appreciable metabolic effects in normal mice. However, transplantation significantly (P<0.001) countered excessive food and fluid intake in diabetic mice and maintained normal body weight. Circulating glucose (P<0.01) and glucagon (P<0.05) were significantly reduced and plasma insulin and GLP-1 dramatically increased. This was associated with significantly (P<0.01) improved glucose tolerance in diabetic mice. Histological examination of the pancreata of these mice revealed elevations (P<0.001) in islet and ß-cell area, with reduced (P<0.001) α-cell area. Increased ß-cell mass reflected the enhanced proliferation relative to apoptosis. These studies emphasize the potential of chronic GLP-1 delivery by cell therapy as a potential therapeutic option for diabetes.

Rapid screening for the detection of HLA-B57 and HLA-B58 in prevention of drug hypersensitivity.

HLA-B57 and HLA-B58 are major histocompatibility class (MHC)-I allotypes that are potentially predictive of important clinical immune phenotypes. HLA-B*5701 is strongly associated with hypersensitivity to the HIV drug abacavir, liver toxicity from the antibiotic flucloxacillin and is a marker for slow progression of HIV AIDS. HLA-B*5801 is associated with hypersensitivity to allopurinol used to treat hyperuricaemia and recurrent gout. Here we describe a monoclonal antibody (mAb) specific for HLA-B57 and HLA-B58 that provides an inexpensive and sensitive screen for these MHC-I allotypes. The usefulness of HLA-B57 screening for prediction of abacavir hypersensitivity was shown in three independent laboratories, including confirmation of the mAb sensitivity and specificity in a cohort of patients enrolled in the PREDICT-1 trial. Our data show that patients who test negative by mAb screening comprise 90%-95% of all individuals in most human populations and require no further human leukocyte antigen (HLA) typing. Patients who test positive by mAb screening should proceed to high-resolution typing to ascertain the presence of HLA-B*5701 or HLA-B*5801. Hence, mAb screening provides a low-cost alternative to high-resolution typing of all patients and lends itself to point-of-care diagnostics and rapid ascertainment of low-risk patients who can begin immediate therapy with abacavir, flucloxacillin or allopurinol.

Changes at peptide residues buried in the major histocompatibility complex (MHC) class I binding cleft influence T cell recognition: a possible role for indirect conformational alterations in the MHC class I or bound peptide in determining T cell recognition.

Recent crystallographic studies on two peptide complexes with the mouse Kb molecule have shown that peptide binding appears to alter the conformation of the class I alpha-helical regions that flank the antigen binding cleft. Given that this study also showed that much of the foreign peptide is buried within the class I binding cleft with only a small portion accessible for direct interaction with the components of the T cell receptor, this finding suggests that at least some component of T cell specificity may arise as a consequence of peptide-induced conformational changes in the class I structure. To assess this possibility, we have made systematic substitutions at residues within the Kb-restricted determinant from ovalbumin (OVA257-264) that are thought to be buried on binding to the class I molecule. We have found that changes in this determinant at the completely buried second residue (P2) can influence T cell recognition without affecting binding to Kb, suggesting that the substitutions may indirectly determine T cell recognition by altering the conformation of the class I molecule or the bound peptide.

Determinant selection of major histocompatibility complex class I-restricted antigenic peptides is explained by class I-peptide affinity and is strongly influenced by nondominant anchor residues.

The contribution of major histocompatibility complex (MHC) class I-peptide affinity to immunodominance of particular peptide antigens (Ags) in the class I-restricted cytotoxic T lymphocyte (CTL) response is not clearly established. Therefore, we have compared the H-2Kb-restricted binding and presentation of the immunodominant ovalbumin (OVA)257-264 (SIINFEKL) determinant to that of a subdominant OVA determinant OVA55-62 (KVVRFDKL). Immunodominance of OVA257-264 was not attributable to the specific T cell repertoire but correlated instead with more efficient Ag presentation. This enhanced Ag presentation could be accounted for by the higher affinity of Kb/OVA257-264 compared with Kb/OVA55-62 despite the presence of a conserved Kb-binding motif in both peptides. Kinetic binding studies using purified soluble H-2Kb molecules (Kbs) and biosensor techniques indicated that the Kon for association of OVA257-264-C6 and Kbs at 25 degrees C was integral of 10-fold faster (5.9 x 10(3) M-1 s-1 versus 6.5 x 10(2) M-1 s-1), and the Koff approximately twofold slower (9.1 x 10(-6) s-1 versus 1.6 x 10(-5) s-1), than the rate constants for interaction of OVA55-62-C6 and Kbs. The association of these peptides with Kb was significantly influenced by multiple residues at presumed nonanchor sites within the peptide sequence. The contribution of each peptide residue to Kb-binding was dependent upon the sequence context and the summed contributions were not additive. Thus the affinity of MHC class I-peptide binding is a critical factor controlling presentation of peptide Ag and immunodominance in the class I-restricted CTL response.

Hierarchical self-tolerance to T cell determinants within the ubiquitous nuclear self-antigen La (SS-B) permits induction of systemic autoimmunity in normal mice.

Systemic autoimmune diseases are frequently associated with clustering of high titer autoantibody responses towards nuclear self-antigens. Little is known, however, about the extent of immune tolerance to the target nuclear antigens or the events leading to the complex autoantibody responses that are characteristic of systemic autoimmunity. To address these issues, we have examined the mouse immune response to La autoantigen (mLa) and the homologous human La antigen (hLa), which are components of the La(SS-B)/Ro(SS-A) ribonucleoprotein (RNP) complex targeted in systemic lupus erythematosus and primary Sjögren's syndrome. The findings reveal the presence of hierarchical T cell tolerance involving multiple autodeterminants within the La autoantigen expressed by normal H-2k and H-2a mice. At one end of this spectrum, there was no detectable T or B cell autoimmunity observed in mice that were immunized with the immunodominant mLa287-301 determinant, which differed by a single residue in its core sequence from the homologous but highly immunogenic human La288-302 determinant. Interestingly, the mLa287-301 peptide acted as an altered peptide ligand that specifically antagonized the activation of an hLa288-302-specific T cell hybridoma. In contrast to the tolerogenic mLa287-301 determinant, a range of autoimmune potential was identified among poorly tolerizing, subdominant self-peptides present within mouse La autoantigen. Notably, immunization of normal mice with the autologous subdominant La25-44 and La106-129 determinants resulted in limited or no detectable autoantibody response. In contrast, immunization with the subdominant mouse La13-30 determinant induced a proliferative T cell response associated with the appearance of specific autoantibodies recognizing multiple intrastructural (La) and intermolecular components (Ro) of the murine La/Ro RNP. The findings suggest how diversified autoimmunity might follow initiation of immunity to simple peptide mimics of poorly tolerogenic determinants that are present within ubiquitous self-antigens.

A divergent transcriptional landscape underpins the development and functional branching of MAIT cells.

MR1-restricted mucosal-associated invariant T (MAIT) cells play a unique role in the immune system. These cells develop intrathymically through a three-stage process, but the events that regulate this are largely unknown. Here, using bulk and single-cell RNA sequencing-based transcriptomic analysis in mice and humans, we studied the changing transcriptional landscape that accompanies transition through each stage. Many transcripts were sharply modulated during MAIT cell development, including SLAM (signaling lymphocytic activation molecule) family members, chemokine receptors, and transcription factors. We also demonstrate that stage 3 "mature" MAIT cells comprise distinct subpopulations including newly arrived transitional stage 3 cells, interferon-γ-producing MAIT1 cells and interleukin-17-producing MAIT17 cells. Moreover, the validity and importance of several transcripts detected in this study are directly demonstrated using specific mutant mice. For example, MAIT cell intrathymic maturation was found to be halted in SLAM-associated protein (SAP)-deficient and CXCR6-deficient mouse models, providing clear evidence for their role in modulating MAIT cell development. These data underpin a model that maps the changing transcriptional landscape and identifies key factors that regulate the process of MAIT cell differentiation, with many parallels between mice and humans.

Immunodominance hierarchies and gender bias in direct T(CD8)-cell alloreactivity.

Allogeneic solid organ transplantation often occurs across multiple donor-recipient HLA mismatches with consequent risk of allograft rejection. However, there is growing evidence that not all HLA mismatches are equivalent in their stimulation of allogeneic T cells making it important to determine which of these might be more significant as predictors of allograft rejection. To this end, we used defined antigen-presenting cell (APC) transfectants expressing single MHC-I allotypes as target cells that could discriminate the relative contribution of individual mismatched MHC-I allotypes to direct T-cell alloreactivity. We demonstrate remarkably reproducible patterns of immunodominance in reactivity across mismatched MHC-I allotypes. These patterns are HLA context-dependent, partly reflecting alloantigenic competition in responder cell responses. In strong alloresponses, we also observed an increased percentage of alloreactive T(CD8) cells in female responders, regardless of the stimulator gender, highlighting HLA-independent factors in the potency of the alloresponse. This approach provides a potential measure of specific alloreactive T cells that could be used in clinical practice for selection of donors, assessment of posttransplant outcomes, modulation of immunosuppression and detection of rejection episodes.

Daily administration of the GIP-R antagonist (Pro3)GIP in streptozotocin-induced diabetes suggests that insulin-dependent mechanisms are critical to anti-obesity-diabetes actions of (Pro3)GIP.

AIM: Glucose-dependent insulinotropic polypeptide-receptor (GIP-R) antagonism using (Pro3)GIP improves glucose tolerance and ameliorates insulin resistance and abnormalities of islet structure and function in a commonly used model of obesity-diabetes, namely ob/ob mice. The effect of GIP-R antagonism in a streptozotocin (STZ)-induced model of insulin deficiency has not been evaluated. The present study has investigated the effects of daily administration of (Pro(3))GIP to STZ-treated mice. METHODS: Swiss TO mice received once-daily injection of (Pro3)GIP (25 nmol/kg body weight) or saline 4 days prior to and 16 days after injection of STZ, and effects on metabolic parameters and islet architecture were assessed. RESULTS: (Pro3)GIP treatment had no significant effect on hyperphagia or body weight loss. However, hyperglycaemia and glycated haemoglobin were worsened, glucose tolerance further decreased and insulin sensitivity was impaired by (Pro3)GIP. These effects were observed on an STZ-induced background characterized by severe reductions of circulating insulin, beta-cell mass and pancreatic insulin stores. CONCLUSIONS: These data indicate that the beneficial actions of the GIP-R antagonist, (Pro3)GIP, in obesity-diabetes appear to be largely mediated through insulin-dependent mechanisms that merit further investigation.

Identification of a human-specific epitope in a conserved region of the La/SS-B autoantigen.

Human anti-La/SS-B autoantibodies are known to react with highly conserved epitopes suggested to be functional or active sites on the La/SS-B polypeptide. This study was designed to determine whether the autoantibodies also react with poorly conserved regions of La/SS-B as predicted by an antigen-driven autoimmune response. Binding of human autoantibodies to purified human, mouse, and bovine recombinant fragments representing immunodominant regions of the La/SS-B polypeptide was compared using Western blotting and ELISA. A cross-reactive epitope was located in the highly conserved NH2-terminal region of La/SS-B. Significantly, human-specific epitopes were identified in both the conserved RNA-recognition motif and a poorly conserved COOH-terminal fragment, providing direct evidence for an autoantigen-driven response. The lack of autoantibody cross-reactivity with a conserved domain of mouse and bovine La/SS-B implies that a small number of residues in human autoepitopes may be critical for autoimmunogenicity.

Clinical and microbiological parameters of naturally occurring periodontitis in the non-human primate Macaca mulatta.

Background: Non-human primates appear to represent the most faithful model of human disease, but to date the oral microbiome in macaques has not been fully characterized using next-generation sequencing. Objective: In the present study, we characterized the clinical and microbiological features of naturally occurring periodontitis in non-human primates (Macaca mulatta). Design: Clinical parameters of periodontitis including probing pocket depth (PD) and bleeding on probing (BOP) were measured in 40 adult macaques (7-22 yrs), at six sites per tooth. Subgingival plaque was collected from diseased and healthy sites, and subjected to 16S rDNA sequencing and identification at the species or higher taxon level. Results: All macaques had mild periodontitis at minimum, with numerous sites of PD ≥ 4 mm and BOP. A subset (14/40) had moderate-severe disease, with >2 sites with PD ≥ 5mm, deeper mean PD, and more BOP. Animals with mild vs moderate-severe disease were identical in age, suggesting genetic heterogeneity. 16S rDNA sequencing revealed that all macaques had species that were identical to those in humans or closely related to human counterparts, including Porphyromonas gingivalis which was present in all animals. Diseased and healthy sites harboured distinct microbiomes; however there were no significant differences in the microbiomes in moderate-severe vs. mild periodontitis. Conclusions: Naturally occurring periodontitis in older macaques closely resembles human adult periodontitis, thus validating a useful model to evaluate novel anti-microbial therapies.

Mucosal-associated invariant T-cell activation and accumulation after in vivo infection depends on microbial riboflavin synthesis and co-stimulatory signals.

Despite recent breakthroughs in identifying mucosal-associated invariant T (MAIT) cell antigens (Ags), the precise requirements for in vivo MAIT cell responses to infection remain unclear. Using major histocompatibility complex-related protein 1 (MR1) tetramers, the MAIT cell response was investigated in a model of bacterial lung infection employing riboflavin gene-competent and -deficient bacteria. MAIT cells were rapidly enriched in the lungs of C57BL/6 mice infected with Salmonella Typhimurium, comprising up to 50% of αß-T cells after 1 week. MAIT cell accumulation was MR1-dependent, required Ag derived from the microbial riboflavin synthesis pathway, and did not occur in response to synthetic Ag, unless accompanied by a Toll-like receptor agonist or by co-infection with riboflavin pathway-deficient S. Typhimurium. The MAIT cell response was associated with their long-term accumulation in the lungs, draining lymph nodes and spleen. Lung MAIT cells from infected mice displayed an activated/memory phenotype, and most expressed the transcription factor retinoic acid-related orphan receptor γt. T-bet expression increased following infection. The majority produced interleukin-17 while smaller subsets produced interferon-γ or tumor necrosis factor, detected directly ex vivo. Thus the activation and expansion of MAIT cells coupled with their pro-inflammatory cytokine production occurred in response to Ags derived from microbial riboflavin synthesis and was augmented by co-stimulatory signals.

Donor methylenetetrahydrofolate reductase genotype is associated with graft-versus-host disease in hematopoietic stem cell transplant patients treated with methotrexate.

Methotrexate (MTX), used as a graft-versus-host disease (GvHD) prophylactic agent in hematopoietic stem cell transplantation (HSCT), exerts its effect via folate cycle inhibition. A critical enzyme involved in folate metabolism is 5,10-methylenetetrahydrofolate reductase (MTHFR). We examined the association of a single nucleotide polymorphism (SNP) at position 677 in the MTHFR gene on GvHD outcomes in allogeneic HSCT patients administered MTX. MTHFR genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) on 193 HSCT patients and donors. A total of 140 patients were transplanted with an HLA-matched related donor and 53 with an unrelated donor. GvHD outcomes were compared between genotypes by univariate and multivariate analysis. The combined donor 677CT and TT genotypes were associated with a decreased incidence of GvHD (acute and chronic combined) in HSCT recipients with an HLA-matched related donor (75% at 1 year in the CT and TT group compared with 91% in the wild type CC group, P=0.01), increased time to onset of first GvHD (P=0.001) and time to first GvHD treated with systemic therapy (P=0.022). Unrelated donor MTHFR genotype was not associated with outcome parameters and no associations of recipient genotype in either related or unrelated donor cohorts were observed.

The structural influence of individual residues located within peptide antigen depends upon their sequence context.

Individual residues derived from antigenic peptides are believed to control certain mAb epitopes on MHC class I molecules. To further evaluate this influence we studied the antibody recognition of H-2Kb complexed with the antigenic peptides OVA257-264, OVA55-62 and their reciprocally substituted analogs. The mAb 20.8.4, Y-3, EH144 and AF6 reacted strongly with Kb complexed to both OVA peptides and their analogs. However, mAb 28.8.6 bound to Kb/OVA257-264 but not Kb/OVA55-62 complexes. Recognition of Kb/OVA55-62 by mAb 28.8.6 was restored by a single OVA55-62-->OVA257-264 substitution at position 1 (OVA55-62P1K-->S). Recognition by mAb 5F1 was also peptide-dependent in that Kb complexed to either the P4 substituted OVA257-264-->OVA55-62 peptide (OVA257-264P4N-->R) or the P1 substituted OVA55-62-->OVA257-264 peptide (OVA55-62P1K-->S) was not permissive for 5F1 recognition even though Kb complexed to the parental peptides OVA55-62 (P4R) and OVA257-264 (P1S) reacted with this mAb. These data demonstrate that the same peptide residues located within defined positions can exert a negative influence on mAb recognition in one sequence context but be permissive for antibody binding in another context. These findings might be explained by conformational effects on class I heavy chain structure, the extended structure of the peptide or altered orientation of specific peptide side chains located at the same position but within different sequence contexts. Therefore the sequence context of defined amino acids within peptide antigen can strongly influence the fine structural contributions of these residues to MHC-peptide conformation.

Molecular basis of IgE-recognition of Lol p 5, a major allergen of rye-grass pollen.

Grass pollen, especially of rye-grass (Lolium perenne). represents an important cause of type I allergy. Identification of IgE-binding (allergenic) epitopes of major grass pollen allergens is essential for understanding the molecular basis of interaction between allergens and human IgE antibodies and therefore facilitates the devising of safer and more effective diagnostic and immunotherapy reagents. The aim of this study was to identify the allergenic epitopes of Lol p 5, a major allergen of rye-grass pollen, immunodissect these epitopes further so that the amino acid residues critical for antibody binding can be determined and investigate the conservation and nature of these epitopes within the context of the natural grass pollen allergens. Peptides, 12-13 amino acid residues long and overlapping each other by 4 amino acid residues, based on the entire deduced amino acid sequence of the coding region of Lol p 5, were synthesised and assayed for IgE-binding. Two strong IgE-binding epitopes (Lol p 5 (49-60) and (265-276), referred to as peptides 7 and 34, respectively) were identified. These epitopes were further resolved by truncated peptides and amino acid replacement studies and the amino acid residues critical for IgE-binding determined (Lol p 5 (49-60) residue Lys57 and (265-276) residue Lys275). Sequences of these epitopes were conserved in related allergens and may form the conserved allergenic domains responsible for the cross-reactivity observed between pollen allergens of taxonomically related grasses. Furthermore, due to its strong IgE-reactivity, synthetic peptide Lol p 5 (265-276) was used to affinity-purify specific IgE antibodies which recognised proteins of other clinically important grass pollens. further indicating presence of allergenic cross-reactivity at the level of allergenic epitope. Moreover, Lol p 5 (265 276) demonstrated a strong capacity to inhibit IgE-binding to natural rye-grass pollen proteins highlighting the antibody accessibility to these sequences within the context of the natural allergens. Strong IgE-binding epitopes of Lol p 5 have been identified down to single critical amino acid residues and are shown to occur as linear or continuous domains in the natural conformation of natural Lol p 5 and other group 5 grass pollen allergens. The fact that such an allergenic synthetic epitope has the capacity to strongly inhibit IgE-binding to natural allergens highlight its potential for use as a candidate in future therapeutics to treat pollen-associated allergies.

Electroporation and commercial liposomes efficiently deliver soluble protein into the MHC class I presentation pathway. Priming in vitro and in vivo for class I-restricted recognition of soluble antigen.

Class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocyte responses to ovalbumin (OVA) were evaluated following delivery of soluble antigen mixed with commercial liposomes or by electroporation of soluble protein into target cells. Splenic antigen presenting cells (APC) and transfected L cell lines were sensitised for recognition by OVA-specific, class I-restricted T hybridomas when antigen was introduced by either method into live cells. Delivery of soluble OVA by both electroporation and commercial liposomes proved more efficient than osmotic loading in sensitising for class I presentation. OVA-specific cytotoxic T lymphocytes (CTL) were effectively primed in naive mice following reinjection of spleen cells pulsed with soluble OVA encapsulated by liposomes or electroporated in vitro. These CTL recognised the well defined OVA257-264 determinant in association with H-2Kb and were derived under conditions where CTL activity obtained from cross priming by soluble OVA alone was undetectable. In addition, using electroporation and commercial liposomes, the loading of APC for OVA recognition required intact MHC-linked antigen presentation genes deleted in the T2-Kb cell line. Antigen delivery to APC by electroporation and commercial liposomes provides a simple and efficient way of studying class I-restricted T cell recognition of soluble protein antigens.

Class II cytoplasmic and transmembrane domains are not required for class II-mediated B cell spreading.

B cells cultured on immobilized anti-class II monoclonal antibody (mAb) change from round to flattened cells, with lamellipodia and filopodia. This change in cell morphology, termed 'spiders', occurs within 30 min upon culture and is mediated through either I-A or I-E molecules. Class II molecules that are defective in mediating protein kinase C (PKC) due to the deletions of both alpha and beta chain's cytoplasmic (Cy) domain sequences can induce spider formation. B-cell transfectants that express chimeric MHC class II/class I molecules, where the ectodomains are class II sequences and the transmembrane and Cy domains are class I sequences also form spiders when cultured on anti-class II mAb. The spider morphology is not induced by either anti-immunoglobulin (Ig) or anti-MHC class I mAb. Treatment of B cells to increase intracellular cAMP, a component of the class II signaling pathway also results in spider formation with the same kinetics and percent change in the responding population as that induced by anti-class II mAb. Cytochalasin A treatment which disrupts cytoskeletal actin filaments and the tyrosine kinase inhibitor, genistein, both inhibit spider formation. Actin redistributes from a concentric ring in round cells to the ends of the filopodia in the spiders. The mechanism of spider induction whether resultant from second messengers following class II signaling or from non-signaling-induced physical interactions of class II with intracellular cytoskeletal components only requires the extracellular domains of class II. The biologic relevance of B-cell spiders is currently not known but has been reported to be associated with class II signal transduction and efficient Ag presentation.

Endogenous and exogenous factors contributing to the surface expression of HLA B27 on mutant APC.

We have examined the expression of HLA B*2705 in the mutant cell line 721.220, which lacks endogenous HLA A and B alleles and expresses a defective tapasin molecule. Several peptide sensitive mAbs distinguish between HLA B*2705 expressed on the surface of 721.220 cells (B27.220) and 721.220 cells co-transfected with human tapasin (B27.220.hTsn). This differential staining defines subtle differences in the conformation of HLA B27, which most likely reflect changes in the repertoire of antigenic peptides bound to B27 in the presence and absence of wild type tapasin. HLA B27 molecules expressed on the surface of 721.220 display increased levels of "free" B27 heavy chain (HC-10 staining), an epitope that is dependent on TAP-translocated peptides. The conformation and stability of B27 molecules was examined by investigating the integrity of mAb epitopes and the half-lives of these complexes on cells cultured with and without serum. The decay of surface B27 epitopes occurred more rapidly in B27.220 and this effect was exaggerated in serum free media. Importantly, the decay of surface B27 molecules in B27.220.hTsn cells was characterized by an early increase in HC-10 staining when the cells were grown in serum free media. This decay of B27 molecules via HC-10 reactive intermediates was not observed in B27.220 cells, implying molecules on these cells may already have passed through this stage prior to surface expression. Taken together these observations indicate that tapasin has a significant contribution to the composition and stability of the B27-bound peptide repertoire.

Evolution in HLA-DRB1 and major histocompatibility complex class II haplotypes of Australian aborigines. Definition of a new DRB1 allele and distribution of DRB1 gene frequencies.

The distribution of HLA-DRB1 alleles was studied in Australian aborigines from different parts of Australia. There were significant differences in the frequencies of DRB1*0412, 1409, and 1410 between the Central Desert and Yuendumu populations and the previously reported Cape York and Kimberley aboriginal populations. A new DRB1 allele, DRB1*1414, present at low frequency in the Central Desert population, was identified. DRB1*1414 appears to be closely related to DRB1*1407 and is proposed to have arisen by intragenic recombination. A novel DR-DQ haplotype, DRB1*1402-DRB3*0101-DQA1*0501-DQB1*0402, was also identified. This haplotype may be ancestral to the DRB1*1409-DQB1*0402 haplotype present in these populations. The presence of alleles and haplotypes apparently confined to Australian aboriginal populations and differences in the distribution of these alleles in different populations suggests that evolution has occurred in the class II region in the period since colonization of Australia, an estimated 50,000 years ago.

Matching for HLA DPA1 and DPB1 alleles in unrelated bone marrow transplantation.

The impact of donor-recipient DPA1 and DPB1 matching was examined in 122 unrelated bone marrow transplant pairs. All pairs were serologically matched at the time of transplantation for HLA class I and II and a majority also DRB1 allele matched. Retrospective A, B, C, DRB1, DQA1, DQB1 in addition to DPA1 and DPB1 allele matching was performed by molecular techniques. The percentage of pairs that were allele matched was as follows; HLA-A = 91% (n = 80), HLA-B = 94% (n = 80), HLA-C = 78% (n = 80), HLA-DRB1 = 96% (n = 122), HLA-DQA1 = 99% (n = 80), HLA-DQB1 = 92% (n = 122). 92 recipient/donor pairs with informative clinical data were available for analysis. DPA1 identity (no incompatibility in either direction) was observed in 57% and DPA1 compatibility in 76% of pairs with no apparent beneficial effect of matching on patient survival or Graft Versus Host Disease (GVHD). DPB1 identity was observed in 11% and compatibility in 27% of pairs. A significant improvement in patient survival was observed in DPB1 matched compared to one DPB1 mismatch (p < 0.01) and combined one and two DPB1 mismatched transplants (p = 0.03). This beneficial effect remained when allele mismatches at HLA-A, B, C, DRB1, DQA1, DQB1 were excluded (p = 0.05, p = 0.03, respectively). There was a significant association of increased frequency of severe GVHD (grades III-IV) compared to mild GVHD (grades I-II) with DPB1 mismatched transplants compared to DPB1 matched transplants (p = 0.04). In DPB1 mismatched transplants an association between patient survival and matching for individual DPB1 polymorphic regions was not observed; however in the HLA-A, B, DRB1, DQA1, DQB1 allele matched transplants a non significant increase in the frequency of Grade IV GVHD was observed in recipients who were negative compared to those who were positive for DPB1 alleles coding for glutamic acid at position 69.

Stable expression of the human immunodeficiency virus type 1 envelope glycoprotein in transfected L cells.

An SV40-based expression vector was used to generate CD4-negative murine L cell lines which stably expressed the human immunodeficiency virus envelope glycoprotein (env). Despite the presence of abundant intracellular envelope glycoprotein, the expression of env gp120/41 was not detected on the cell surface. Pulse-chase studies showed that the majority of the gp120 detected at the end of a 20-h chase was in the culture medium. Therefore gp120 was shed and/or secreted from these cells. Transfected L cells (H-2k) served as targets for specific lysis by CTL raised against vaccinia virus-encoded env gp160. The discrepancy in relative levels of intracellular versus surface expression of env was probably due to the highly inefficient processing of newly synthesized gp160, as well as the apparent instability of the gp120/41 complex in the transfected cell lines. Digestion of immunoprecipitated gp120 and gp160 with endoglycosidase H and peptide N-glycosidase F revealed that the envelope glycoprotein in transfected L cells possessed both high mannose and complex N-glycans, analogous to the posttranslational modification of the mature envelope glycoprotein in infected T cells. These studies indicate that the relatively inefficient processing of env gp160 occurs in the absence of CD4, and that the stable surface expression of envelope gp120/41 complex may require additional factors not present in transfected cells.