Levels of Art2+ cells but not soluble Art2 protein correlate with expression of autoimmune diabetes in the BB rat.

ART2a and ART2b are isoenzymes expressed on the surface of mature T cells and intraepithelial lymphocytes (IELs) in the rat. They exhibit both adenosine diphosphoribosyltransferase and nicotine adenine dinucleotide (NAD) glycohydrolase activities, and both can generate a transmembrane signal that modulates T cell activation. The presence or absence of ART2+ T cells modulates the expression of autoimmune diabetes in the BB rat. ART2 also circulates in a soluble form whose function is unknown. We tested the hypothesis that circulating ART2 protein regulates the expression of autoimmunity. We compared the kinetics, regulation, and source of soluble ART2 in normal rats and in rats with autoimmune diabetes. Basal levels of soluble ART2 varied greatly among strains of rats and were lowest in the diabetes-prone BB (BBDP/Wor) rat. In diabetes-resistant BB (BBDR/Wor) rats, administration of anti-ART2a antibody, which is known to induce diabetes, resulted in transient clearing of soluble ART2a that was followed rapidly by a rebound increase. Repeated treatment of BBDR/Wor rats with anti-ART2a antibody resulted in sustained supraphysiologic levels of soluble ART2a. Although the number of peripheral ART2a+ T cells is known to correlate with the expression of diabetes in BBDR/Wor rats, the level of soluble ART2a protein did not. The source of the soluble ART2 protein in the rat appeared to be the gut. The results suggest that ART2+ T cells and soluble ART2 protein may subserve different immunomodulatory functions.

BB rat thymocytes cultured in the presence of islets lose their ability to transfer autoimmune diabetes.

Thymocytes from adult BB rats can adoptively transfer autoimmune diabetes to athymic recipients. It is also known that the development of BB rat T-cells is recapitulated in adult thymus organ cultures (ATOCs). Based on these observations, we tested the hypothesis that cells capable of the adoptive transfer of diabetes would be present in long-term ATOCs but could be rendered nondiabetogenic by co-culture with appropriate antigens. We observed that cells recovered from adult diabetes-resistant BB (BBDR) rat thymi cultured for up to 14 days can adoptively transfer disease to athymic WAG-rnu/rnu rats treated with polyinosinic: polycytidylic acid and a monoclonal antibody to preclude development of ART2a+ regulatory T-cells. Co-culture of adult BBDR thymi in the presence of BBDR thyrocytes had no effect on the ability of recovered cells to induce diabetes in 70-80% of adoptive recipients. In contrast, co-culture in the presence of islets prevented transfer of diabetes, on average, in >90% of recipients. Fresh islets, frozen islets, and islets pretreated with streptozotocin to deplete insulin were equally effective in preventing diabetes, but none prevented insulitis in nondiabetic recipients. Co-culture in the presence of islets was not associated with detectable alterations in phenotype or in the secretion of gamma-interferon or interleukin-4, either in cultures or in cells recovered from adoptive recipients. We conclude that islet antigens involved in the initiation of autoimmune diabetes in BB rats may be absent or deficient in BB rat thymi. Exposure of ATOCs to exogenous islets may lead to deletion or anergy of diabetogenic T-cells or to the positive selection of regulatory T-cells.

Fetal thymi from diabetes-prone but not diabetes-resistant BB/Wor rats fail to generate mature ART2+ T-cells in organ culture.

Diabetes-prone (BBDP) BB rats develop spontaneous autoimmune diabetes mellitus. They are lymphopenic and severely deficient in ART2+ T-cells. Diabetes-resistant BB (BBDR) rats do not develop spontaneous diabetes and have normal numbers of ART2+ T-cells. T-cell lymphopenia in BBDP rats results from hematopoietic stem cell defects leading to abnormal intrathymic T-cell maturation. To study this process, we established rat fetal thymic organ cultures (FTOC). Like mouse FTOC, cultures of BBDR rat thymi yielded approximately 10(5) cells per lobe. The majority of cells were CD8+ART2+ T-cells. In contrast, BBDP rat FTOC yielded 60% fewer cells (approximately 0.3 x 10(5)/lobe), a smaller percentage of CD8+ and TcRalphabeta+ T-cells, and almost no detectable ART2+ T-cells. ART2 mRNA was detectable in BBDR but not BBDP FTOC. In contrast, expression of mRNAs encoding bcl-2 and a panel of cytokines was comparable in BBDP and BBDR FTOC. Addition of anti-ICAM-1 (CD54) antibody reduced T-cell number in BBDR rat FTOC by approximately 70%, but addition of IL-7 or IL-1beta had no effect. The data demonstrate that BBDP thymocytes fail to generate mature ART2+ T-cells in rat FTOC, a system that can now be used to study the mechanism of this process.

Recapitulation of normal and abnormal BioBreeding rat T cell development in adult thymus organ culture.

Congenitally lymphopenic diabetes-prone (DP) BioBreeding (BB) rats develop spontaneous T cell-dependent autoimmunity. Coisogenic diabetes-resistant (DR) BB rats are not lymphopenic and are free of spontaneous autoimmune disease, but become diabetic in response to depletion of RT6+ T cells. The basis for the predisposition to autoimmunity in BB rats is unknown. Abnormal T cell development in DP-BB rats can be detected intrathymically, and thymocytes from DR-BB rats adoptively transfer diabetes. The mechanisms underlying these T cell developmental abnormalities are not known. To study these processes, we established adult thymus organ cultures (ATOC). We report that cultured DR- and DP-BB rat thymi generate mature CD4 and CD8 single-positive cells with up-regulated TCRs. DR-BB rat cultures also generate T cells that express RT6. In contrast, DP-BB rat cultures generate fewer CD4+, CD8+, and RT6+ T cells. Analysis of the cells obtained from ATOC suggested that the failure of cultured DP-BB rat thymi to generate T cells with a mature phenotype is due in part to an increased rate of apoptosis. Consistent with this inference, we observed that addition of the general caspase inhibitor Z-VAD-FMK substantially increases the number of both mature and immature T cells produced by DP-BB rat ATOC. We conclude that cultured DR-BB and DP-BB rat thymi, respectively, recapitulate the normal and abnormal T cell developmental kinetics and phenotypes observed in these animals in vivo. Such cultures should facilitate identification of the underlying pathological processes that lead to immune dysfunction and autoimmunity in BB rats.

DR-BB rat thymus contains thymocyte populations predisposed to autoreactivity.

We have induced autoimmune insulin-dependent diabetes mellitus (IDDM) in athymic WAG rats by transfusing thymocytes from histocompatible phenotypically normal rats of the DR-BB strain. DR-BB rats rarely develop spontaneous IDDM, but readily become hyperglycemic if depleted in vivo of regulatory T-cells that express the RT6.1 maturational alloantigen. Successful adoptive transfer of IDDM by DR-BB thymocytes required that the athymic recipients be depleted of emerging populations of donor-origin RT6.1+ T-cells. Thymocytes from both normal and RT6-depleted diabetic DR donors were equally capable of transferring autoimmunity. In contrast, thymocytes from normal histocompatible YOS rats failed to transfer IDDM. The autoreactive potential of DR-BB rat thymocytes was minimal from birth to 4 weeks of age and then increased substantially at 8-9 weeks of age. These results demonstrate that the DR-BB rat thymus harbors abnormal cell populations predisposed to autoreactivity. The data localize the developmental defect leading to diabetes in the BB rat to an abnormal intrathymic selection process.

Adoptive transfer of autoimmune diabetes mellitus to athymic rats: synergy of CD4+ and CD8+ T cells and prevention by RT6+ T cells.

We describe the induction and prevention of autoimmune insulin dependent diabetes mellitus (IDDM), and its pathological substrate, insulitis, in congenitally athymic nude rats following injections of major histocompatibility complex (MHC) compatible lymph node T cells. The cells capable of adoptive transfer of autoimmunity were obtained from diabetes resistant (DR) BB rats that had been rendered hyperglycemic by in vivo depletion of the RT6+ regulatory T cell subset. We first established that our adoptive transfer assay system is cell dose- and time dependent and therefore amenable to quantitative analysis. It was also observed that both CD4+ and CD8+ T cells are required for efficient transfer of autoimmunity. The data indicate that, as in the NOD mouse, a synergistic interaction between CD4+ and CD8+ T cells is important for beta cell destruction. Finally, we demonstrated that the admixture of equal numbers of lymph node T cells, 60% of which were RT6+, from intact, non-diabetic DR rats prevented the adoptive transfer of IDDM mediated by diabetogenic T cells from RT6-depleted DR-BB rats. We conclude that an equilibrium between autoreactive and regulatory cells determines the expression of autoimmunity in the DR-BB rat and in the adoptive transfer of diabetes in quantitative analytical systems.

Identification and characterization of B cell precursors in rat lymphoid tissues. I. Adoptive transfer assays for precursors of TI-1, TI-2, and TD antigen-reactive B cells.

Quantitative adoptive transfer assays were developed to detect the precursors of TI-1, TI-2, and TD antigen-reactive B cells in rat lymphoid tissues. Studies on the immune responses in normal and athymic nude rats validate the use of TNP-lipopolysaccharide as a TI-1 antigen, TNP-Ficoll as a TI-2 antigen, and SRBC as a TD antigen in rats. The precursors to these immunologically competent B cells are detected, following transfer into irradiated histocompatible recipients, by their ability to generate expanded populations of antigen-reactive B cells capable of mounting antibody responses (splenic IgM plaque-forming cells) to these antigens. Maximal numbers of antigen-reactive B cells emerge in antigenically naive rats after an interval of 7-12 days following transfer of donor lymphoid cells and decline rapidly thereafter. The delayed responses in adoptive recipients reconstituted with spleen cells are proportional to the numbers of spleen cells transferred and are shown to be primarily donor derived using histocompatible Ig kappa chain alloantigen disparate rat strain combinations. The precursors of TI-1, TI-2, and TD antigen-reactive B cells are present in both donor spleen and bone marrow. However, precursor cells to TI-1 and TD antigens are largely absent from donor lymph node cells, whereas precursors to the TI-2 antigen are as prevalent in donor lymph node as in donor spleen. These results support the hypothesis that newly formed virginal B cells represent transient populations of precursor cells that undergo further proliferation and differentiation in the spleen before acquiring immunological competence. The results also suggest that the precursors of TI-2 antigen-reactive B cells differ developmentally from those of TI-1 and TD antigen-reactive B cells, and that the antigen-reactive progeny of these precursors require additional stimulation in order to join the pool of long-lived peripheral B cells.

Characterization of the effector mechanism of help for antigen-presenting and bystander resting B cell growth mediated by Ia-restricted Th2 helper T cell lines.

The mechanism of help for resting B cell growth in MHC-restricted T-B collaboration was investigated using an in vitro polyclonal model for these T cell-B cell interactions. In the presence of rabbit anti-mouse Ig, small, size-selected B cells elicit help from syngeneic Ia-restricted Th2 cell lines specific for F(ab')2 rabbit globulin. Both Ag-presenting and bystander B cells receive signals from stimulated Th cells that lead to B cell proliferation. The results suggest that the direct activation of resting Ag-presenting and bystander B cells by Th2 cells is mediated by a similar effector mechanism. Although proliferative responses by Ag-presenting B cells are of greater magnitude, help for both Ag-presenting and bystander B cell populations is characterized by the lack of a requirement for membrane Ig cross-linking, by identical kinetics, and by the necessity for direct cell contact or close proximity with Th cells. B cell proliferation is not induced by exposure to the sequence of diffusable mediators released from a synchronized Ag-specific T-B interaction. The T cell-dependent proliferation by both B cell populations can be inhibited by excess mitomycin C-treated syngeneic "cold target" B cells, demonstrating a requirement for a short-range T cell-B cell interaction. mAb inhibition experiments fail to identify a role for class II, LFA-1, or CD4 membrane molecules in the delivery of help to bystander B cells. Antibody against H2d bystander class II molecules has no effect on bystander B cell proliferation at concentrations that completely block Ag presentation by H2d B cells to an H2d-restricted Th cell line. Antibodies against the cell adhesion molecule LFA-1 or the Th cell molecule CD4 do inhibit bystander B cell proliferation, but only to the extent that they block T cell activation and the induction of help. The inductive stimulus leading to resting B cell growth results from an early, short-range interaction with Th cells. B cell proliferation is supported by T cell soluble mediators as a consequence of this interaction, which is required for at least 8 hr after T cell recognition of Ag/Ia on the surface of Ag-presenting B cells.

Impact of a mandatory second-opinion program on medicaid surgery rates.

The effect of the Massachusetts second-opinion program on the volume of elective surgery in the Medicaid population was assessed using two approaches: a study of the program experience and surgery decisions of 2,501 program referrals, and an analysis of Medicaid surgery rates before and after program implementation. Nonconfirmation rates, which averaged 14.5 per cent, varied by procedure from 4 per cent for cholecystectomy to 26 per cent for disc surgery. The patient's surgery decision was related to the outcome of the second-opinion consultation: 85.5 per cent of the confirmed patients had the originally proposed operation, as compared with 31 per cent of the nonconfirmed patients. In the year after program implementation, the program was associated with a 20 per cent reduction in the volume of those procedures covered by the program. The greatest percentage declines were for hysterectomies, meniscectomies, hemorrhoidectomies and tonsillectomies/adenoidectomies. The decline in surgery rates was attributed both to a direct effect on patients referred to the program and to a sentinel effect whereby fewer operations were proposed. We conclude that the mandatory second-opinion program in Massachusetts saved Medicaid $3 to $4 for every dollar spent.

The effect of a thirty per cent reduction in physician fees on Medicaid surgery rates in Massachusetts.

In this paper, we use an interrupted time series analysis to assess the effect of a 30 per cent reduction in the Medicaid reimbursement fee for physician services on the rate at which eight elective surgical procedures were performed in the Massachusetts Medicaid population. Tonsillectomy/adenoidectomy is the only procedure in which there was a statistically significant decline in the rate of surgery in most areas of the state following the fee cut. There is some evidence of an increase in the rate of disc surgeries/spinal fusions. The rate of other procedures increased in some areas of the state and decreased in other areas in the period after the fee cut.