CD4+ T Cells Reactive to Enteric Bacterial Antigens in Spontaneously Colitic C3H/HeJBir Mice: Increased T Helper Cell Type 1 Response and Ability to Transfer Disease

C3H/HeJBir mice are a new substrain that spontaneously develop colitis early in life. This study was done to determine the T cell reactivity of C3H/HeJBir mice to candidate antigens that might be involved in their disease. C3H/HeJBir CD4⁺ T cells were strongly reactive to antigens of the enteric bacterial flora, but not to epithelial or food antigens. The stimulatory material in the enteric bacteria was trypsin sensitive and restricted by class II major histocompatibility complex molecules, but did not have the properties of a superantigen. The precursor frequency of interleuken (IL)-2–producing, bacterial-reactive CD4⁺ T cells in colitic mice was 1 out of 2,000 compared to 1 out of 20,000–25,000 in noncolitic control mice. These T cells produced predominately IL-2 and interferon γ, consistent with a T helper type 1 cell response and were present at 3–4 wk, the age of onset of the colitis. Adoptive transfer of bacterial-antigen–activated CD4⁺ T cells from colitic C3H/HeJBir but not from control C3H/HeJ mice into C3H/HeSnJ scid/scid recipients induced colitis. These data represent a direct demonstration that T cells reactive with conventional antigens of the enteric bacterial flora can mediate chronic inflammatory bowel disease.The inflammatory bowel diseases (IBD)¹, encompassing Crohn''s disease and ulcerative colitis, are complex chronic inflammatory diseases of the intestine whose etiology and pathogenesis remain unknown. There are multiple etiologic theories, one of which is that a dysregulated CD4 T cell response to the abundant antigens in the lumen may be responsible (1, 2). This hypothesis is based on theoretical grounds and there is only limited supporting data in humans as yet (3, 4). However, support for this hypothesis has come from the results of studies done in a number of recently developed experimental models of IBD, some of which have been the unexpected result of gene deletions by selective gene targeting (5–9). In a number of such models, CD4⁺ T cells have been found to mediate colitis, and most commonly this has involved an exaggerated Th1 response manifested by excessive IFN-γ production in the lesions (10–12). The localization of inflammatory disease to the colon of mice that have global deficiencies of an immune molecule suggests that the bacterial flora is the major immune stimulant leading to chronic intestinal inflammation. Indeed, in some models, animals that are raised germ-free no longer develop colitis (5, 13), and in others rederivation with a defined flora (6, 12) or antibiotic treatment (14) ameliorates the disease. Moreover, reconstitution of intestinal bacteria into germ-free animals can restore intestinal inflammation (15). However, it has remained unclear how the bacterial flora generates chronic intestinal inflammation.Humans with IBD do not have absolute deficiencies of the immune molecules whose deletion in mice has resulted in colitis. For this reason, we have derived and studied a new strain of mice which develop colitis spontaneously, namely the C3H/HeJBir strain (16). C3H/HeJBir mice develop a predominantly right-sided colitis early in life that largely resolves by 3 mo of age. Previous studies on the immunopathogenesis of disease in this mouse strain have found that C3H/HeJBir mice, but not mice of the parenteral C3H/HeJ strain, have high titer serum IgG antibodies to a selected subset of antigens of the enteric bacterial flora (17). These antibodies are mainly of the IgG2a subclass, compatible with a predominant Th1 response to these bacterial antigens. This study was undertaken to define the CD4⁺ T cell response of C3H/HeJBir mice to enteric bacterial antigens. Compatible with our earlier results analyzing antibody responses, strong CD4⁺ Th1 T cell reactivity to protein antigens of the enteric bacterial flora was identified. Moreover, disease could be induced by transfer of enteric bacterial antigen-activated C3H/HeJBir CD4⁺ T cells, but not by control C3H/HeJ T cells, into C3H/ HeSnJ scid/scid recipients. These results demonstrate for the first time that CD4⁺ T cells reactive with conventional antigens of the bacterial flora are able to mediate chronic intestinal inflammation.     

The maize transposable element system Ac/Ds as a mutagen in Arabidopsis: identification of an albino mutation induced by Ds insertion.

A two-component transposon system based on the Ac element of maize was used as a mutagen in Arabidopsis thaliana. Transposition of a Ds element marked with a hygromycin-resistance gene was activated from four different locations in the Arabidopsis genome. The progeny of 201 plants carrying independent transposition events were screened for mutants with severe, visible phenotypes. Seven mutants were identified and four of them were analyzed genetically. Three mutations were shown to be very closely linked to a transposed copy of the element. Moreover, a mutation (alb3) causing an albino phenotype was conclusively shown to be caused by insertion of the Ds element: somatic and germinal reversion of the mutation occurred in the presence of the transposase gene but not in its absence, and in three revertants the Ds had excised from its position in the mutant line. The DNA adjacent to Ds in the mutant was isolated and it was demonstrated that revertants retained part of the 8-bp duplication caused by insertion of Ds. These experiments indicate that the Ac/Ds system can be used as an insertional mutagen in the heterologous host Arabidopsis, which will permit the isolation of genes from this species by transposon tagging.     

Improved first-trimester Down syndrome screening performance by lowering the false-positive rate: a prospective study of 9941 low-risk women.

OBJECTIVE: To determine the performance of screening for Down syndrome (DS) and other major chromosomal abnormalities using nuchal translucency (NT), free beta-human chorionic gonadotropin (beta-hCG) and pregnancy-associated plasma protein-A (PAPP-A) in a prospective study of a non-selected population. METHODS: Of 9941 women with an early ultrasound examination, NT was measured in 8622 singleton pregnancies with a gestational age between 10 + 3 and 13 + 6 weeks. beta-hCG and PAPP-A were analyzed in 6441 cases. Detection rates (DR) and false-positive rates (FPR) for the NT screening, the double test (beta-hCG and PAPP-A) and the combined test (NT and the double test) were calculated using a 1 : 250 cut-off. RESULTS: NT could be measured in 97.5% of cases. The DR for DS with NT screening alone was 75% with a FPR of only 1.8%. The double test detected 73% and the combined test 91%, for FPRs of 8.8% and 2.1%, respectively. We detected 80% of fetuses with other major chromosomal abnormalities with a combination of NT screening and other ultrasound findings. Low beta-hCG and PAPP-A values (below 0.4 MoM) were observed in 0.5% of the women including all cases of triploidy and trisomy 18 and 13. CONCLUSIONS: The performance of a screening strategy for DS using a combination of NT and the double test was superior to that using either NT or the double test alone due to a very low FPR and a higher DR.     

A bio-effect directed fractionation study for toxicological and chemical characterization of organic compounds in bottom sediment.

The major aim of this study was to characterize toxic organic compounds in bottom sediments from a PCB polluted bay. To overcome difficulties in pinpointing toxicants in complex environmental samples we applied a bio-effect directed (BED) fractionation approach and investigated the relationships between aromaticity, teratogenicity, and aryl hydrocarbon receptor (AhR) mediated toxicity. Hepatic ethoxyresorufin O-deethylase (EROD) activities and malformations were investigated in rainbow trout (Oncorhynchus mykiss) larvae exposed by injecting sediment extract and fractions (separated by their degree of aromaticity) thereof into newly fertilized eggs. Our results imply that non-additive effects get more pronounced the more complex the exposure. The fraction mainly composed of dicyclic aromatic compounds (DACs), including PCBs, was surprisingly less teratogenic than the fraction mainly composed of polycyclic aromatic compounds (PACs). A major part of the latter potential was isolated in a subfraction mainly composed of three- and four-ring compounds (including alkylated and sulphur-heterocyclic compounds). Though no clear relationship between aromaticity and EROD induction was observed, both the DAC- and the PAC-fractions contributed equally to the EROD induction potential. A major part of the PAC-fraction's induction potential came from a subfraction containing compounds with more than five rings. No clear relationship between teratogenicity and EROD induction was observed, underlining the need for a battery of biomarkers in estimating environmental risk. Two specific malformations not previously described in literature-asymmetric yolk sac and fin edema-could be tracked through the fractionation steps, suggesting that this BED-fractionation strategy is a reliable tool for pinpointing toxic compounds in the environment.     

Elimination of antigen-presenting cells and autoreactive T cells by Fas contributes to prevention of autoimmunity

Fas (also known as Apo-1 and CD95) receptor has been suggested to control T cell expansion by triggering T cell-autonomous apoptosis. This paradigm is based on the extensive lymphoproliferation and systemic autoimmunity in mice and humans lacking Fas or its ligand. However, with systemic loss of Fas, it is unclear whether T cell-extrinsic mechanisms contribute to autoimmunity. We found that tissue-specific deletion of Fas in mouse antigen-presenting cells (APCs) was sufficient to cause systemic autoimmunity, implying that normally APCs are destroyed during immune responses via a Fas-mediated mechanism. Fas expression by APCs was increased by exposure to microbial stimuli. Analysis of mice with Fas loss restricted to T cells revealed that Fas indeed controls autoimmune T cells, but not T cells responding to strong antigenic stimulation. Thus, Fas-dependent elimination of APCs is a major regulatory mechanism curbing autoimmune responses and acts in concert with Fas-mediated regulation of chronically activated autoimmune T cells.     

Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells

Adult bone-marrow-derived mesenchymal stem cells are immunosuppressive and prolong the rejection of mismatched skin grafts in animals. We transplanted haploidentical mesenchymal stem cells in a patient with severe treatment-resistant grade IV acute graft-versus-host disease of the gut and liver. Clinical response was striking. The patient is now well after 1 year. We postulate that mesenchymal stem cells have a potent immunosuppressive effect in vivo.     

High mobility group box chromosomal protein 1: a novel proinflammatory mediator in synovitis

OBJECTIVE: High mobility group box chromosomal protein 1 (HMGB-1) is a ubiquitous chromatin component expressed in nucleated mammalian cells. It has recently and unexpectedly been demonstrated that stimulated live mononuclear phagocytes secrete HMGB-1, which then acts as a potent factor that causes inflammation and protease activation. Macrophages play pivotal roles in the pathogenesis of arthritis. The aim of this study was to determine whether synovial macrophage expression of HMGB-1 is altered in human and experimental synovitis. METHODS: Intraarticular tissue specimens were obtained from healthy Lewis rats, Lewis rats with Mycobacterium tuberculosis-induced adjuvant arthritis, and from patients with rheumatoid arthritis (RA). Specimens were immunohistochemically stained for cellular HMGB-1. Extracellular HMGB-1 levels were assessed in synovial fluid samples from RA patients by Western blotting. RESULTS: Immunostaining of specimens from normal rats showed that HMGB-1 was primarily confined to the nucleus of synoviocytes and chondrocytes, with occasional cytoplasmic staining and no extracellular matrix deposition. In contrast, inflammatory synovial tissue from rats with experimental arthritis as well as from humans with RA showed a distinctly different HMGB-1 staining pattern. Nuclear HMGB-1 expression was accompanied by a cytoplasmic staining in many mononuclear cells, with a macrophage-like appearance and an extracellular matrix deposition. Analysis of synovial fluid samples from RA patients further confirmed the extracellular presence of HMGB-1; 14 of 15 samples had HMGB-1 concentrations of 1.8-10.4 microg/ml. CONCLUSION: The proinflammatory mediator HMGB-1 was abundantly expressed as a nuclear, cytoplasmic, and extracellular component in synovial tissues from RA patients and from rats with experimental arthritis. These findings suggest a pathogenetic role for HMGB-1 in synovitis and indicate a new potential therapeutic target molecule.     

Case-Based Learning in Surgery: Lessons Learned

Background  

The aim of the study was to obtain a deepened understanding of the implementation process of case-based learning (CBL) during a surgical semester at the Undergraduate Medical Program at Karolinska Institutet. The objectives are to identify the level of success of the implementation and to identify practical and theoretical implications of importance in connection to the process.