IgM ANCA in healthy individuals and in patients with ANCA-associated vasculitis.

Low levels of IgM auto-antibodies have been reported in health and disease. IgM anti-neutrophil cytoplasmic antibodies (ANCA) have been reported in patients with ANCA-associated vasculitis (AAV). We sought to investigate if healthy individuals may have IgM ANCA in their sera. The first aim of the study was to determine whether IgM ANCA was present in healthy individuals and in patients with ANCA-associated vasculitis. The second aim was to determine what happens to IgM ANCA levels over time. The third aim was to determine whether bacterial infections affected IgM ANCA levels in non-AAV patients. Sera from healthy individuals and patients with AAV were tested for IgM ANCA by immunofluorescence on fixed neutrophils, immunoprecipitation, Western blot and ELISA. Peripheral blood mononuclear cells were isolated and tested by ELISpot for circulating IgM ANCA B cells. To determine whether infection affected IgM ANCA levels, we studied non-AAV patients with bacterial endocarditis or Staphylococcus aureus bacteraemia and measured IgM ANCA levels over time. IgM ANCA is detectable in both healthy individuals and patients with AAV and the titres decreased with increasing age. Circulating IgM ANCA B cells were identified by ELISpot. In the presence of infection, we could not find a significant change in IgM ANCA levels. We report the presence of low-level specific IgM ANCA in the sera of healthy individuals and in patients with ANCA-associated vasculitis. Bacterial infection did not affect the level of IgM ANCA in this small study.

Transmission of toxoplasmosis in two renal allograft recipients receiving an organ from the same donor.

Toxoplasma gondii is a ubiquitous protozoan parasite. After acute infection it continues to exist as cysts in the muscles and brain. Recipients of organ allografts are susceptible to the disease as a result of reactivation of quiescent infection either by transmission from the organ donor or by consumption of undercooked meat. We describe 2 cases of fatal toxoplasmosis in renal allograft recipients who received their organs from the same cadaveric donor. Both recipients died 5 weeks after renal transplantation, within days of each other. Multiorgan involvement with toxoplasmosis was demonstrated at autopsy. No evidence of the parasite was found in the transplanted kidney, either at the time of insertion or at autopsy. Neither recipient had serologic evidence of previous exposure to T. gondii. The donor had positive IgG but indeterminate IgM antibodies suggesting acute infection at the time of death; there was no clinical suspicion that the donor died from acute toxoplasmosis. We conclude that toxoplasmosis was transmitted by the donor kidneys. In an attempt to minimize the possibility of future transmission, donors are now tested for anti-toxoplasma IgM antibodies and recipients are treated with trimethoprim/sulfamethoxazole for the first 6 months after renal transplantation.

Rituximab use in systemic lupus erythematosus pneumonitis and a review of current reports.

Rituximab is a chimeric monoclonal antibody specific for human CD20 that causes selective transient depletion of the CD20+ B-cell subpopulation. We report the first case of systemic lupus erythematosus (SLE) pneumonitis resistant to conventional treatments that responded well to rituximab and review current reports on the use of rituximab in SLE.

The cell biology of MHC class I antigen presentation.

MHC class I antigen presentation refers to the co-ordinated activities of many intracellular pathways that promote the cell surface appearance of MHC class I/beta2m heterodimers loaded with a spectrum of self or foreign peptides. These MHC class I peptide complexes form ligands for CD8 positive T cells and NK cells. MHC class I heterodimers are loaded within the endoplasmic reticulum (ER) with peptides derived from intracellular proteins. Alternatively, MHC class I molecules may be loaded with peptides derived from extracellular proteins in a process called MHC class I cross presentation. This pathway is less well defined but can overlap those pathways operating in classical MHC class I presentation and has recently been reviewed elsewhere (1). This review will address the current concepts regarding the intracellular assembly of MHC class I molecules with their peptide cargo within the ER and their subsequent progress to the cell surface.

Quantitative and qualitative influences of tapasin on the class I peptide repertoire.

Tapasin is critical for efficient loading and surface expression of most HLA class I molecules. The high level surface expression of HLA-B*2705 on tapasin-deficient 721.220 cells allowed the influence of this chaperone on peptide repertoire to be examined. Comparison of peptides bound to HLA-B*2705 expressed on tapasin-deficient and -proficient cells by mass spectrometry revealed an overall reduction in the recovery of B*2705-bound peptides isolated from tapasin-deficient cells despite similar yields of B27 heavy chain and beta(2)-microglobulin. This indicated that a proportion of suboptimal ligands were associated with B27, and they were lost during the purification process. Notwithstanding this failure to recover these suboptimal peptides, there was substantial overlap in the repertoire and biochemical properties of peptides recovered from B27 complexes derived from tapasin-positive and -negative cells. Although many peptides were preferentially or uniquely isolated from B*2705 in tapasin-positive cells, a number of species were preferentially recovered in the absence of tapasin, and some of these peptide ligands have been sequenced. In general, these ligands did not exhibit exceptional binding affinity, and we invoke an argument based on lumenal availability and affinity to explain their tapasin independence. The differential display of peptides in tapasin-negative and -positive cells was also apparent in the reactivity of peptide-sensitive alloreactive CTL raised against tapasin-positive and -negative targets, demonstrating the functional relevance of the biochemical observation of changes in peptide repertoire in the tapasin-deficient APC. Overall, the data reveal that tapasin quantitatively and qualitatively influences ligand selection by class I molecules.

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.

Distinct functions of tapasin revealed by polymorphism in MHC class I peptide loading.

Peptide assembly with class I molecules is orchestrated by multiple chaperones including tapasin, which bridges class I molecules with the TAP and is critical for efficient Ag presentation. In this paper, we show that, although constitutive levels of endogenous murine tapasin apparently are sufficient to form stable and long-lived complexes between the human HLA-B*4402 (B*4402) and mouse TAP proteins, this does not result in normal peptide loading and surface expression of B*4402 molecules on mouse APC. However, increased expression of murine tapasin, but not of the human TAP proteins, does restore normal cell surface expression of B*4402 and efficient presentation of viral Ags to CTL. High levels of soluble murine tapasin, which do not bridge TAP and class I molecules, still restore normal surface expression of B*4402 in the tapasin-deficient human cell line 721.220. These findings indicate distinct roles for tapasin in class I peptide loading. First, tapasin-mediated bridging of TAP-class I complexes, which despite being conserved across the human-mouse species barrier, is not necessarily sufficient for peptide loading. Second, tapasin mediates a function which probably involves stabilization of empty class I molecules and which is sensitive to structural compatibility of components within the loading complex. These discrete functions of tapasin predict limitations to the study of HLA molecules across some polymorphic and species barriers.

The human leucocyte antigens and clinical medicine: an overview.

The Major Histocompatibility Complex (MHC) occupies 4-6 megabases on the short arm of chromosome 6 and is the most intensively studied segment of the human genome. This region was first discovered through its influence on transplantation rejection and on antigen-specific immune responses. The most important genes for managing these functions encode the HLA molecules (human leucocyte antigens) which are highly polymorphic in human populations. HLA typing for these polymorphisms is widely used in clinical medicine when identifying optimal organ donors or recipients and in assessing the risk of diseases such as narcolepsy, hereditary hemochromatosis, ankylosing spondylitis and certain autoimmune disorders. As new genes are identified in the MHC, the clinical impact of this genetic region is likely to assume further importance as outlined in this review.

HLA-B27-restricted antigen presentation in the absence of tapasin reveals polymorphism in mechanisms of HLA class I peptide loading.

Tapasin is a resident ER protein believed to be critical for antigen presentation by HLA class I molecules. We demonstrate that allelic variation in MHC class I molecules influences their dependence on tapasin for peptide loading and antigen presentation. HLA-B*2705 molecules achieve high levels of surface expression and present specific viral peptides in the absence of tapasin. In contrast, HLA-B*4402 molecules are highly dependent upon human tapasin for these functions, while HLA-B8 molecules are intermediate in this regard. Significantly, HLA-B*2705 like HLA-B*4402, requires tapasin to associate efficiently with TAP (transporters associated with antigen processing). The unusual ability of HLA-B*2705 to form peptide complexes without associating with TAP or tapasin confers flexibility in the repertoire of peptides presented by this molecule. We speculate that these properties might contribute to the role of HLA-B27 in conferring susceptibility to inflammatory spondyloarthropathies.

Human leukocyte antigen phenotype imposes complex constraints on the antigen-specific cytotoxic T lymphocyte repertoire.

The memory response to the immunodominant Epstein-Barr virus (EBV) epitope FLRGRAYGL, which associates with HLA B8, is exceptionally restricted, being dominated by cytotoxic T lymphocytes (CTL) with a single, public T cell receptor (TCR). CTL clones that express this receptor fortuitously cross-react with the alloantigen HLA B44. However, of the two major subtypes of this HLA, B*4402 and B*4403, that differ by a single amino acid, only the former is recognized by these mature CTL clones. Individuals heterozygous for HLA B8 and B*4402 use alternative TCR for the EBV determinant since the dominant TCR is potentially self-reactive. We now demonstrate that this clonotype is also essentially absent from the repertoire of CTL directed against the viral epitope in seven from seven unrelated individuals heterozygous for HLA B8 and B*4403. Thus immune tolerance of these CTL recognizing HLA B*4402 is associated with expression of either B*4402 or B*4403. This suggests that tolerance in the human T cell compartment requires a lower threshold of recognition than for effector function, thus providing a buffer zone minimizing the risk of autoimmunity. These data also illustrate the potential for non-restricting HLA molecules to bias dramatically the T cell repertoire used for specific immune responses. Such influences may be the basis of the "protective" effects of certain HLA alleles in susceptibility to autoimmune disorders.

Recurrent mesangial IgA nephritis following renal transplantation.

Recurrence of mesangial IgA deposits in renal allografts of patients whose original disease was primary IgA nephropathy (IgAN) has been studied. Forty-six patients with primary IgAN received 51 renal allografts and have been followed for 3-183 months. A prospective study of 11 patients (11 biopsies) and a retrospective analysis of 17 patients (16 biopsies; 2 nephrectomy specimens) have been combined. Seventeen of the 29 allografts had recurrent mesangial IgA deposits and of these three patients have negative urinalysis, normal glomeruli by light microscopy, and stable renal function; six patients have microhaematuria, mesangial proliferative nephritis, but at present stable renal function; and five have mesangial proliferative glomerulonephritis with microhaematuria, heavy proteinuria, hypertension, and progressive allograft failure secondary to IgA disease alone, and one of these is now back on dialysis. Three other grafts with recurrent deposits are failing because of transplant glomerulopathy or rejection. The only predictor identified for recurrence of mesangial IgA deposits was length of time post-transplantation, with allograft tissue being studied at 45.9 +/- 10.0 versus 15.3 +/- 4.8 months (P = 0.008) post-transplantation in patients with and without recurrent deposits respectively. Cyclosporin A did not prevent recurrence. By virtue of a longer follow-up of patients post-transplantation than all other reported series, these results suggest that with increasing time post-transplantation recurrence of mesangial IgA disease will become increasingly important as a cause of progressive allograft dysfunction and failure unless effective treatment is found for the primary disease.

The effect of chlorothiazide on bone-related biochemical variables in normal post-menopausal women.

OBJECTIVE: To evaluate the effects of short-term administration of chlorothiazide on fasting urinary hydroxyproline, an index of bone resorption, and other bone-related biochemical parameters in normal post-menopausal women. DESIGN: Subjects served as their own control before and after chlorothiazide treatment. SETTING: Subjects were recruited by advertisement. PARTICIPANTS: Thirteen healthy post-menopausal women with a mean age of 65 years. INTERVENTION: Each subject was given chlorothiazide 500 mg bd po for 7 days. Fasting blood and urine samples were obtained immediately before the commencement of chlorothiazide (day 1) and 2 and 7 days after starting chlorothiazide. RESULTS: Chlorothiazide decreased the urinary calcium/creatinine (mean value day 1, 0.267; day 2, 0.143; day 7, 0.135; P < 0.001) and hydroxyproline/creatinine (day 1, 0.0192; day 2, 0.0145; day 7, 0.0139; P < 0.02) molar ratios. CONCLUSION: Chlorothiazide decreases fasting urinary hydroxyproline, a marker of bone resorption in post-menopausal women. This observation supports a potential role for thiazide diuretics in the prevention of osteoporosis. The observed fall in urinary hydroxyproline is of the same order as that seen after treatment with estrogen or calcium supplements.