Dharam P. Singal

Role of blood transfusions on the induction of antibodies againsts recognition sites on T lymphocytes in renal transplant patients

We have tested sera from 23 renal allograft recipients to study the effects of blood transfusions on the induction of antibodies directed against recognition sites on T lymphocytes. The results demonstrate that antibodies capable of inhibiting responses in MLC could be induced by blood transfusion. This inhibition in MLC is observed by treatment of responder lymphocytes with serum plus rabbit complement and is mediated by IgG antibodies. Also, the inhibitory effect is specific for certain responder cells and is not mediated by antibodies against common surface antigens of either the responder or the stimulator lymphocytes. The antibodies inhibiting proliferative responses in MLC against antigens present on the kidney donor were demonstrable in renal transplant recipients with functional allografts, but not in patients who had rejected the graft. The data suggest that antibodies directed against recognition sites on T lymphocytes could be induced by blood transfusions and these antibodies may be associated with prolonged graft survival.

Genetics of rheumatoid arthritis (RA): two separate regions in the major histocompatibility complex contribute to susceptibility to RA

We analyzed HLA-DR antigens and microsatellite Bat2 alleles in 97 adult caucasian patients with classical seropositive rheumatoid arthritis (RA) and 95 normal healthy controls. The results demonstrate that the prevalence of microsatellite Bat2 138 allele was significantly higher in RA-susceptibility DRB1 QKRAA/QRRAA epitope-negative patients as compared with normal controls. Analysis of the data suggested that Bat2 138 allele has primary association with RA-susceptibility in QKRAA/QRRAA epitope-negative patients. The Bat2 138 allele thus provides an additional risk in RA-susceptibility. In addition, microsatellite Bat2 138 allele showed a highly significant positive association with microsatellite D6S273 138 allele, which has similar (identical) association with RA development in DRB1 QKRAA/QRRAA epitope-negative patients. The present data demonstrate that DRB1 QKRAA/QRRAA epitope and microsatellite Bat2 138/D6S273 138 alleles more completely define the risk for development of RA. The results in the present study therefore suggest that two regions in MHC, class II (DRB1) and class III (Bat2 and D6S273 in HSP70-Bat2 region), contribute to susceptibility to RA.

Molecular basis for lack of expression of HLA class I antigens in human small‐cell lung carcinoma cell lines

HLA class I molecules present antigenic peptides to cytotoxic T lymphocytes and thus play an important role in immune surveillance of cells infected with virus or altered by malignant transformation. Immunochemical studies have demonstrated a marked deficiency or lack of expression of class I molecules on the surface of many different types of tumor cells. It is likely that this allows these cells to escape immune surveillance. In the present study, we examined the molecular basis for lack of expression of class I antigens in small‐cell lung carcinoma cell lines. Our results demonstrate that these cell lines also lacked products of MHC‐encoded proteasome subunit LMP2 and the putative peptide transporter TAP1. In contrast, LMP7 and TAP2 genes were expressed in these cell lines. Pulse‐chase experiments showed that class I molecules were unstable and thus not transported to the cell surface from endoplasmic reticulum. Our results suggest that antigenic peptides were not available for binding to class I &agr: chains due to genes showed that the tumor cells lacked trans‐regulatory nuclear protein(s), which binds to the interferon‐γ (IFN‐γ) response element (ISRE) in the TAP I, LMP2 bidirectional intergenic promoter. Treatment of tumor cells with IFN‐γ induced ISRE‐binding nuclear protein(s) and resulted in expression of TAP 1 and LMP2 genes with a concomitant increase in cell‐surface expression of class I molecules. Our data provide credence for a role of TAP and LMP genes in immune response.

Peptide transport in human lymphoblastoid and tumor cells: effect of transporter associated with antigen presentation (TAP) polymorphism.

CD8+ T-cells recognize antigenic peptides presented by major histocompatibility complex (MHC) class I molecules. These peptides bind to MHC class I molecules in the endoplasmic reticulum (ER) lumen. Antigenic peptides are translocated from the cytosol to the lumen of ER by transporter associated with antigen presentation (TAP) proteins. In this study, it is shown that TAP1 polymorphism influences the peptide substrate specificity in human B-lymphoblastoid and tumor cell lines. TAP1A and 1C alleles specifically enhance translocation of model peptides containing basic C-terminal amino acid residue. However, TAP1B allele does not show specificity for the peptide C-terminus. Human basophilic leukemia (Ku812), and hepatocellular carcinoma (PLC/PRF/5) cells express TAP1 molecules and exhibit TAP-mediated allele-specific peptide uptake after gamma-interferon (gamma-IFN) treatment. Ku812 cells express TAP1A and preferentially take up antigenic peptides with a basic C-terminus, however, PLC/PRF/5 cells with the TAP1B allele take up low but equivalent levels of peptides regardless of basic, acidic, or hydrophobic C-termini. Moreover, TAP2 polymorphisms have no influence on the peptide translocation in normal or tumor cell lines. In addition, Daudi, a beta2-microglobulin (beta2m) deficient human Burkitt lymphoma, cell line also showed TAP-dependent peptide uptake. Taken together, these results suggest that human TAP1 but not TAP2 polymorphisms influence the antigenic peptide transport and that this transport is independent of beta2m in this system.

Markedly decreased expression of TAP1 and LMP2 genes in HLA class I-deficient human tumor cell lines

HLA class I antigens of the human major histocompatibility complex play an important role in immune response. These molecules present foreign antigenic peptides to cytotoxic T lymphocytes and thereby play a role in the immune surveillance of cells infected with virus or other intracellular pathogens or altered by malignant transformation. A marked deficiency or lack of expression of these antigens has been reported in a variety of human neoplasms. In the present study, we examined the expression of class I alpha chain, beta 2-microglobulin, TAP (TAP1 and TAP2) and LMP (LMP2 and LMP7) genes in a number of human tumor cell lines including small-cell lung carcinoma, hepatocellular carcinoma, colon adenocarcinoma and basophilic leukaemia. These cell lines were deficient in expression of both class I alpha chain and beta 2-microglobulin gene products. In addition, these cell lines lacked the products of MHC-encoded proteasome subunit LMP2 as well as the putative peptide transporter TAP1 genes. In contrast, TAP2 and LMP7 genes were expressed in these cell lines. Treatment of cells with gamma-IFN markedly enhanced the expression of class I alpha chain, beta 2-microglobulin, TAP1 and LMP2 genes with a concomitant increase in cell-surface expression of class I molecules. The upregulation of TAP1 and LMP2 expression is associated with increased class I expression, suggesting that endogenous antigens, e.g. tumor antigens, could be presented by class I molecules following treatment of tumor cells with gamma-IFN.

Halothane and isoflurane enhance melanoma tumour metastasis in mice

PurposeTo investigate the incidence of tumour metastasis from B16 melanoma tumour cells in expenmental animals following exposure to equipotent concentrations of halothane or isoflurane, and to differentiate if exposure to one anaesthetic resulted in greater metastases than the other.MethodsExperimental animals (C57B1 mice), were randomized to receive 1.3 MAC hours of halothane or isoflurane anaesthesia. The control group of animals received oxygen alone under identical conditions. Fifteen minutes after completion of anaesthesia, control and experimental groups were given 1 × 105 B16 melanoma cells intravenously. After 21 days, all animals were autopsied, and the metastatic nodules in their lungs were counted. The difference in the numbers of metastatic nodules between control and expenmental groups of animals was analyzed for significance by the Mann Whitney “U test”.ResultsMore metastases were observed in the animals exposed to halothane (37.28±5.08, P< 0.0001), or isoflurane anaesthesia (28.24±4.07, P< 0.0014) than in the control animals (12.22± 1.52).ConclusionExposure to halothane or isoflurane anaesthesia increased the number of pulmonary metastases in C57B1 mice compared with the control groups but there was no difference in metastases among animals treated with halothane or isoflurane.RésuméObjectifRechercher l’incidence des métastases de cellules tumorales mélanomateuses B16 sur des animaux de laboratoire après l’exposition à des concentrations équipotentes d’halothane et d’isoflurane, et vérifier si l’exposition à un anesthésique produisait un plus grand nombre de métastases qu’à l’autre.MéthodesDes animaux de laboratoires (souris 57BI) étaient réparties aléatoirement pour recevoir 1, 3 MAC heures d’anesthésie à l’halothane ou à l’isoflurane. Le groupe contrôle ne recevait que de l’oxygène dans des conditions expérimentales identiques. Quinze minutes après l’arrêt de l’anesthésie, les souris des groupes contrôle et expérimentaux recevaient 1 × 105 de cellules mélanomateuses B16 par la voie intraveineuse. Après 21 jours, tous les animaux étaient autopsiés et les nodules pulmonaires métastatiques dénombrés. La signification statistique de la différence entre le nombre nodules métastatiques dénombrés entre le groupe contrôle et les groupes expénmentaux était déterminée avec le test U de Mann Whitney.RésultatsOn a observé plus de métastases chez les animaux exposés à l’halothane (37, 28±5, 08, P< 0, 0001) ou à l’isoflurane (28, 24±4, 07, P< 0.0014) que dans le groupe contrôle (12, 22± 1, 52).ConclusionL’exposition à l’halothane ou à l’isoflurane augmente le nombre de métastases pulmonaires chez de souris C57BI comparativement au groupe contrôle mais il n’y a aucune différence entre les animaux anesthésiés que soit à l’halothane ou à l’isoflurane.

T Cells from Children with IDDM are Sensitized to Bovine Serum Albumin

Epidemiological and experimental evidence suggested that denial of dietary cow milk protein early in life protects genetically susceptible children and animals from insulin‐dependent diabetes (IDDM). Bovine serum albumin (BSA) was proposed as a candidate milk‐borne mimicry antigen responsible for the diabetogenic cow milk effect. Elevated anti‐BSA antibodies have been observed in patients and diabetic rodents, and these antibodies precipitate p69 from islet cell lysates. IDDM is a T ceil mediated disorder but efforts to detect BSA‐specific T cells in diabetic children have so far failed. We describe here a culture system which allowed the detection of BSA‐specific T cells and we mapped this response to the ABBOS peptide (pre‐BSA position 152–169) previously identified as a possible mimicry epitope. ABBOS sensitized T ceils were found in 28/31 children with recent onset TDDM but not in non‐diabetic controls nor in children with SLE or JRA. T cell proliferative responses declined within the first few years of diabetes diagnosis. Although no effector cell role for BSA/ABBOS specific T lymphocytes has been demonstrated, the presence of BSA peptide‐specific T cells strengthens the postulated link between a cow milk protein and IDDM.

Gold induced thrombocytopenia: 12 cases and a review of the literature

Gold induced thrombocytopenia is immune mediated, with the production of platelet associated IgG leading to peripheral platelet destruction. An association with HLA-DR3 has been demonstrated. Corticosteroid therapy is effective in treatment, although other modes of therapy may be as efficacious.

The Fetus as an Allograft: Evidence for Antiidiotypic Antibodies Induced by Pregnancy

ABSTRACT: We tested sera from 20 women [9 parous, 8 with no children, and 3 with abortion(s)] for inhibition in mixed lymphocyte culture (MLC). In these experi‐ments, the responder (wife) lymphocytes were treated with autologous serum and rabbit complement and then tested for responses against stimulator cells from the husband and from third‐party allogeneic donors. The results demonstrate that antibodies capable of inhibiting responses of wifes lymphocytes to husbands cells in MLC are present in sera from parous women, but not in women without children and in women with abortion(s). The MLC‐inhibiting activity in parous women sera was in the IgG fraction. The results from immunofluorescence and absorption experiments suggest that the inhibition in MLC was due to antibodies directed against recognition sites on wifes T lymphocytes against husbands alloantigens. These observations suggest that antiidiotypic antibodies against husband‐specific idiotypes on wifes lymphocytes could be induced by pregnancy and that maternal tolerance to fetus may be attributable to a similar mechanism occurring in vivo.

Polymorphism in both X and Y box motifs controls level of expression of HLA-DRB1 genes

The HLA class II antigens of the human major histocompatibility complex play an important role in immune response. The quality of the immune response is determined not only by polymorphisms in their coding region, but also by the level of their cell-surface expression which affects, for example, the extent of T-cell activation. We have previously described allelic polymorphisms in the upstream regulatory regions of HLA-DRB genes, which affected DNA-protein interactions and resulted in significantly different promoter strengths. In the present study, we investigated the effect of polymorphisms in the X and Y box motifs on the transcriptional activity of DRB1 geme promoters in the DR1, DR51, and DR53 haplotype groups. We used normal, chimeric, and mutated DRB promoters and compared their relative abilities to initiate transcription of the CAT reporter gene in human B-cell lines. The results show that polymorphisms in both the X1 and Y box motifs play a dominant role in the promoter strength. In the gel mobility shift assay, we observed differential ability of nuclear proteins that bind to the polymorphic X1 and Y box elements. The results in the present study confirm earlier data in that the nucleotide variation in the X1 box affects the level of expression of DRB1 genes. In addition, the present data demonstrate that polymorphism in the Y box, which affects the inverted CCAAT sequence, also plays a dominant role in the transcriptional activity of DRB1 promoters.