A mechanism for the major histocompatibility complex-linked resistance to autoimmunity.

Certain major histocompatibility complex (MHC) class II haplotypes encode elements providing either susceptibility or dominant resistance to the development of spontaneous autoimmune diseases via mechanisms that remain undefined. Here we show that a pancreatic beta cell-reactive, I-Ag7-restricted, transgenic TCR that is highly diabetogenic in nonobese diabetic mice (H-2(g7)) undergoes thymocyte negative selection in diabetes-resistant H-2(g7/b), H-2(g7/k), H-2(g7/q), and H-2(g7/nb1) NOD mice by engaging antidiabetogenic MHC class II molecules on thymic bone marrow-derived cells, independently of endogenous superantigens. Thymocyte deletion is complete in the presence of I-Ab, I-Ak + I-Ek or I-Anb1 + I-Enb1 molecules, partial in the presence of I-Aq or I-Ak molecules alone, and absent in the presence of I-As molecules. Mice that delete the transgenic TCR develop variable degrees of insulitis that correlate with the extent of thymocyte deletion, but are invariably resistant to diabetes development. These results provide an explanation as to how protective MHC class II genes carried on one haplotype can override the genetic susceptibility to an autoimmune disease provided by allelic MHC class II genes carried on a second haplotype.

Spontaneous autoimmune diabetes in monoclonal T cell nonobese diabetic mice.

It has been established that insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice results from a CD4+ and CD8+ T cell-dependent autoimmune process directed against the pancreatic beta cells. The precise roles that beta cell-reactive CD8+ and CD4+ T cells play in the disease process, however, remain ill defined. Here we have investigated whether naive beta cell-specific CD8+ and CD4+ T cells can spontaneously accumulate in pancreatic islets, differentiate into effector cells, and destroy beta cells in the absence of other T cell specificities. This was done by introducing Kd- or I-Ag7-restricted beta cell-specific T cell receptor (TCR) transgenes that are highly diabetogenic in NOD mice (8.3- and 4.1-TCR, respectively), into recombination-activating gene (RAG)-2-deficient NOD mice, which cannot rearrange endogenous TCR genes and thus bear monoclonal TCR repertoires. We show that while RAG-2(-/-) 4.1-NOD mice, which only bear beta cell-specific CD4+ T cells, develop diabetes as early and as frequently as RAG-2+ 4.1-NOD mice, RAG-2(-/-) 8.3-NOD mice, which only bear beta cell-specific CD8+ T cells, develop diabetes less frequently and significantly later than RAG-2(+) 8.3-NOD mice. The monoclonal CD8+ T cells of RAG-2(-/-) 8.3-NOD mice mature properly, proliferate vigorously in response to antigenic stimulation in vitro, and can differentiate into beta cell-cytotoxic T cells in vivo, but do not efficiently accumulate in islets in the absence of a CD4+ T cell-derived signal, which can be provided by splenic CD4+ T cells from nontransgenic NOD mice. These results demonstrate that naive beta cell- specific CD8+ and CD4+ T cells can trigger diabetes in the absence of other T or B cell specificities, but suggest that efficient recruitment of naive diabetogenic beta cell-reactive CD8+ T cells to islets requires the assistance of beta cell-reactive CD4+ T cells.

Major histocompatibility complex class I-restricted infiltration and destruction of pancreatic islets by NOD mouse-derived beta-cell cytotoxic CD8+ T-cell clones in vivo.

NOD mouse-derived beta-cell-specific cytotoxic T-cell (beta-CTL) clones are diabetogenic in adult NOD mice, but only if co-injected with splenic CD4+ T-cells from diabetic animals. This investigation was initiated to determine whether infiltration of pancreatic islets by beta-CTL is a major histocompatibility complex (MHC) class I-restricted response, and whether beta-CTL has a direct cytopathic effect on beta-cells in vivo. Pancreatic islets from BALB/c (H-2d) or B6 (H-2b) mice were transplanted under the renal capsule of streptozotocin (STZ)-induced diabetic (NOD x BALB/c) F1 (H-2Kd, H-2Dd,b) or NOD x B6) F1 (H-2Kd,b, H-2Db) mice, respectively. H-2Kd-restricted beta-CTL clones from NOD mice were transfused into euglycemic mice within 3 days after transplantation. In all of the H-2d islet-grafted (NOD x BALB/c) F1 mice that received the beta-CTL clones, the beta-CTLs homed into the grafts, recruited host Mac-1+ cells and CD4+ and CD8+ T-cells, and caused diabetes within 7 days. In contrast, none of the H-2b islet-grafted (NOD x B6) F1 mice who received the beta-CTL clones and none of the H-2d islet-grafted (NOD x BALB/c) F1 mice who received a non-beta-cell cytotoxic CTL clone (N beta-CTL) developed graft inflammation or diabetes. Depletion of CD4+ T-cells in H-2d islet-grafted (NOD x BALB/c) F1 mice did not prevent beta-CTL clone-induced diabetes but reduced its severity. In contrast, when the beta-CTL clones were injected > 8 days after transplantation, none of the H-2d islet-grafted (NOD x BALB/c) F1 mice became diabetic or developed graft inflammation. We conclude that (1) islet-derived beta-CTLs can destroy beta-cells in vivo; (2) infiltration of grafted islets by beta-CTLs is an MHC class I-restricted response; (3) beta-CTLs can recruit naive CD4+ T-cells to the site, leading to further beta-cell damage; and (4) revascularized islet grafts are, like pancreatic islets of irradiated adult NOD mice, "sequestered" from circulating beta-CTLs.

Cloning and characterization of the Neisseria meningitidis asd gene.

The asd mutants of Gram- and some Gram+ bacteria have an obligate requirement for diaminopimelic acid (DAP), an essential constituent of the cell wall of these organisms. In environments deprived of DAP, i.e., mammalian tissues, they will undergo lysis. This has previously been exploited to develop vaccine strains of Salmonella typhimurium and Streptococcus mutans. As a first step for the development of a biosafe Neisseria meningitidis laboratory strain, we have cloned the asd from wild-type strain B16B6 by complementation of an Escherichia coli asd mutant. By subcloning and insertion mutagenesis, the N. meningitidis asd was localized to a 1.5-kb DNA fragment. In a T7 RNA polymerase-T7 promoter expression system, a 38-kDa protein was strongly expressed from this DNA fragment. The N-terminal amino acid (aa) sequence was deduced from the nucleotide sequence, which was determined with the help of an in-frame Asd'::'LacZ protein fusion. A comparison of the N-terminal aa of the Asd proteins from N.meningitidis and E. coli revealed 70% identity, suggesting that the Asd protein may be highly conserved among Gram- bacteria.

Cardiovascular responses to nonrespiratory and respiratory arousals in a porcine model.

Spontaneous and provoked nonrespiratory arousals can be accompanied by a patterned hemodynamic response. To investigate whether a patterned response is also elicited by respiratory arousals, we compared nonrespiratory arousals (NRA) to respiratory arousals (RA) induced by airway occlusion during non-rapid eye movement sleep. We monitored mean arterial blood pressure (MAP), heart rate, iliac and renal blood flow, and sleep stage in 7 pigs during natural sleep. Iliac and renal vascular resistance were calculated. Airway occlusions were obtained by manually inflating a chronically implanted tracheal balloon during sleep. The balloon was quickly deflated as soon as electroencephalogram arousal occurred. As previously reported, NRA generally elicited iliac vasodilation, renal vasoconstriction, little change in MAP, and tachycardia. In contrast, RA generally elicited iliac and renal vasoconstriction, an increase in MAP and tachycardia. The frequent occurrence of iliac vasoconstriction and arterial pressure elevation following RA but not NRA suggests that sleep state change alone does not account for the hemodynamic response to airway occlusion during sleep.

Beta-cell-cytotoxic CD8+ T cells from nonobese diabetic mice use highly homologous T cell receptor alpha-chain CDR3 sequences.

Insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice results from a cell-mediated autoimmune process against pancreatic beta-cells. We have shown that beta-cell-cytotoxic CD8+ T cell clones can transfer IDDM to irradiated NOD mice if co-injected with nondiabetogenic CD4+ spleen T cells. To determine whether CTLs recruited to pancreatic islets recognize a restricted set of local Ags, we sequenced TCR-alpha and TCR-beta cDNA generated by anchor PCR from CD8+ CTL lines and clones derived from islets of 10 different NOD mice. These CTL lines were oligoclonal, but did not show skewed V alpha, V beta, J alpha, or J beta gene usage when compared with CD8+ spleen T cells. However, of the 26 different CTL-derived TCR-alpha sequences from all of these CTL lines and clones, 17 (65%) used one of three highly related, N region-encoded, CDR3 motifs. Motifs 1 and 2 (7 clonotypes each) contained a hydrophobic amino acid followed by Arg and a negatively charged or a polar residue (Asn or Gly), respectively. Motif 3 (3 clonotypes) was x-Arg-Gly. In 12 of these 17 rearrangements, the core sequence was followed by Tyr or Ser. By contrast, none of 31 different TCR-alpha rearrangements used by CD8+ spleen T cells encoded motifs 1 or 2, and only one encoded motif 3. Different TCR-beta rearrangements within individual lines also used homologous CDR3 sequences, but these sequences varied between lines. Skewed TCR-alpha-CDR3 usage by islet-derived CTLs was substantiated further by isolation of CTL clones transcribing highly homologous TCR-alpha, but different TCR-beta, rearrangements. These data suggest that CTLs recruited to pancreatic islets during spontaneous IDDM recognize a restricted set of beta-cell autoantigenic determinants.

Gene studies in newborn males with Duchenne muscular dystrophy detected by neonatal screening.

18,000 newborn males were screened for Duchenne muscular dystrophy (DMD) by creatine kinase (CK) analysis of filter paper blood spots between Jan 1, 1986, and Dec 31, 1987. 5 affected boys have been identified, and in 3 of 5 probands molecular deletions or duplications have been found. 3 of 5 mothers were judged highly likely to be carriers of DMD because of repeatedly raised CK levels, identified gene rearrangements, or both abnormalities. 1 mother has a very low probability of being a carrier and 1 is at an intermediate risk. The use of DNA analyses in new DMD probands identified by neonatal screening has allowed confirmation of the diagnosis and accurate assignment of carrier status in mothers and female relatives in over half the cases studied, and may help to reduce the population incidence of DMD by avoiding delay before clinical diagnosis.

Acceleration of spontaneous diabetes in TCR-beta-transgenic nonobese diabetic mice by beta-cell cytotoxic CD8+ T cells expressing identical endogenous TCR-alpha chains.

The role of target cell autoantigens and their repertoire vs those of foreign Ags, superantigens, or non-Ag-specific stimuli in the activation and recruitment of effector T cells in most spontaneous models of autoimmune diseases remains elusive. Here we report on the use of single TCR-beta transgenic mice to study the mechanisms that drive the accumulation of pathogenic T cells in the pancreatic islets of nonobese diabetic (NOD) mice, a model for insulin-dependent diabetes mellitus. Expression of the V(beta)8.1+ TCR-beta rearrangement of a diabetogenic H-2Kd-restricted beta cell cytotoxic CD8+ T cell (beta-CTL) clone in NOD mice caused a 10-fold increase in the peripheral precursor frequency of beta-CTL and a selective acceleration of the recruitment of CD8+ T cells to the pancreatic islets of prediabetic animals. This resulted in an earlier onset and a faster progression of beta cell depletion, and led to a dramatic acceleration of the onset of diabetes. Most islet-derived beta-CTL from diabetic transgenic NOD mice expressed an endogenously-derived TCR-alpha sequence identical to that of the clonotype donating the TCR-beta transgene, and a TCR-alpha-CDR3 sequence homologous to those expressed by most islet-derived beta-CTL from nontransgenic NOD mice. TCR-beta transgene expression did not change the peripheral frequency of beta cell-specific CD4+ T cells, the rate at which these cells accumulated in the pancreatic islets, or the incidence of diabetes. Taken together, our data indicate that retention of CD8+ and CD4+ T cells in the pancreatic islets of NOD mice is driven by beta cell autoantigens, rather than by local superantigens or non-Ag-specific stimuli, and that beta-CTL are major effectors of beta cell damage in spontaneous insulin-dependent diabetes mellitus.

Ultrasonography by emergency physicians in patients with suspected cholecystitis.

This article investigates the use of bedside abdominal ultrasonography (BAU) performed by emergency physicians (EPs) to screen patients for cholelithiasis and cholecystitis. In this prospective study EPs performed BAU on 116 patients. Agreement between BAU and formal abdominal ultrasound (FUS) performed in the radiology department for detecting cholelithiasis and cholecystitis was determined using Kappa statistics. Test characteristics of BAU for detecting cholelithiasis and acute cholecystitis were calculated. Agreement between BAU and FUS was 0.71 for cholelithiasis and 0.46 for acute cholecystitis. Test characteristics of BAU for cholelithiasis were sensitivity 92%, specificity 78%, positive predictive value (PPV) 86%, negative predictive value (NPV) 88%. Test characteristics of BAU for acute cholecystitis compared with clinical follow-up were sensitivity 91%, specificity 66%, PPV 70%, NPV 90%. BAU may be used to exclude cholelithiasis and is sensitive for cholecystitis. However, when EPs with limited experience identify cholecystitis a confirmatory test is warranted before cholecystectomy.