Endomyocardial biopsies in the diagnosis of myocardial involvement in systemic lupus erythematosus.

BACKGROUND: Endomyocardial biopsy (EMB) is considered the gold standard for diagnosing myocardial involvement in most inflammatory conditions, including systemic lupus erythematosus (SLE). However, EMBs are rarely performed, and most of the myocardial histopathology reports in SLE consist of postmortem data. We therefore sought to describe the histopathologic findings of contemporary EMBs in SLE performed in clinical practice. METHODS: A retrospective review of histopathology reports from SLE patients who underwent EMB from 1994 to 2017 was performed. A total of 41 SLE patients had cardiac pathology reports. Of these, 11 histopathology reports were EMBs, and the remaining were valvular specimens. RESULTS: A total of 11 SLE EMBs were reviewed. It was found that 45% of the patients had hypertension, 27% had coronary artery disease, 9% had hyperlipidemia, and 36% had end-stage renal disease. None had diabetes or smoked. The mean left ventricular ejection fraction was 37%. On histopathology, 10 had mild interstitial fibrosis, 9 had myocyte hypertrophy, 3 had organized blood clots, and 3 had a mild infiltration of lymphocytes and macrophages without clear evidence of myocarditis. None had vasculitis, endocarditis, ischemia, amyloid deposition, or lamellar or curvilinear inclusions. CONCLUSION: EMBs are rarely performed in SLE. In this case series, nonspecific interstitial fibrosis and myocyte hypertrophy were the most common findings, suggesting EMBs have limited value in the diagnosis of cardiac involvement in SLE and rather rule out competing conditions. These data support the need for diagnostic methods with high sensitivity and specificity for SLE heart disease.

Origin of Enriched Regulatory T Cells in Patients Receiving Combined Kidney-Bone Marrow Transplantation to Induce Transplantation Tolerance.

We examined tolerance mechanisms in patients receiving HLA-mismatched combined kidney-bone marrow transplantation (CKBMT) that led to transient chimerism under a previously published nonmyeloablative conditioning regimen (Immune Tolerance Network study 036). Polychromatic flow cytometry and high-throughput sequencing of T cell receptor-ß hypervariable regions of DNA from peripheral blood regulatory T cells (Tregs) and CD4 non-Tregs revealed marked early enrichment of Tregs (CD3+ CD4+ CD25high CD127low Foxp3+ ) in blood that resulted from peripheral proliferation (Ki67+ ), possibly new thymic emigration (CD31+ ), and, in one tolerant subject, conversion from non-Tregs. Among recovering conventional T cells, central memory CD4+ and CD8+ cells predominated. A large proportion of the T cell clones detected in posttransplantation biopsy specimens by T cell receptor sequencing were detected in the peripheral blood and were not donor-reactive. Our results suggest that enrichment of Tregs by new thymic emigration and lymphopenia-driven peripheral proliferation in the early posttransplantation period may contribute to tolerance after CKBMT. Further, most conventional T cell clones detected in immunologically quiescent posttransplantation biopsy specimens appear to be circulating cells in the microvasculature rather than infiltrating T cells.

Constitutive production of inflammatory and mitogenic cytokines by rheumatoid synovial fibroblasts.

Conditioned media obtained from fibroblasts cultured from rheumatoid and certain other inflammatory synovia were observed to stimulate [3H]thymidine incorporation in an indicator murine fibroblast line. Synovial fibroblasts derived from the joints of patients with osteoarthritis did not display this property. This effect persisted in culture for many weeks and occurred in the absence of co-stimulatory immune cells. Antibody neutralization studies implicated a role for basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-beta), granulocyte/macrophage colony-stimulating factor (GM-CSF), and interleukin 1 beta (IL-1 beta) in the increased proliferative activity of synovial fibroblast-conditioned media. Synovial cell synthesis of bFGF, TGF beta 1, GM-CSF, IL-1 beta, and IL-6 was confirmed by 35S-methionine labeling and immunoprecipitation. The constitutive production of inflammatory and mitogenic cytokines by synovial fibroblasts may represent the result of long-term, phenotypic changes that occurred in vivo. Persistent cytokine production by synovial fibroblasts may play an important role in the continued recruitment and activation of inflammatory cells in chronic arthritis and in the formation of rheumatoid pannus.

OCCURRENCE OF gamma-GLOBULIN COMPLEXES IN SERUM AND JOINT FLUID OF RHEUMATOID ARTHRITIS PATIENTS: USE OF MONOCLONAL RHEUMATOID FACTORS AS REAGENTS FOR THEIR DEMONSTRATION.

gammaG globulin complexed in an unusual form has been demonstrated in the serum of many patients with rheumatoid arthritis. Such complexes have been detected and isolated principally through precipitation reactions with monoclonal gammaM rheumatoid factors. These monoclonal rheumatoid factors exhibited a greater sensitivity to react with small complexes or aggregates of gamma-globulin than polyclonal rheumatoid factor from rheumatoid arthritis sera or isolated C1q. The serum complexes consisted in large part of high molecular weight but acid-dissociable 7S gammaG globulin molecules They however differed from the complexes in the joint fluid by not yielding precipitates with C1q and were not found in association with evidence of marked serum complement fixation or activation. A small number of systemic lupus erythematosus sera, primarily those forming cryoprecipitates, also gave reactions with monoclonal rheumatoid factor. Sera from patients with a variety of nonrheumatic diseases gave a low incidence of reactions. The exact nature of the complexes in the rheumatoid arthritis sera remains somewhat in doubt although gammaG rheumatoid factors appear partly involved.

Human B cell activation. Evidence for diverse signals provided by various monoclonal anti-IgM antibodies.

Seven murine monoclonal antibodies (mAb) with different binding characteristics for human IgM varied markedly in their ability to induce proliferation of T cell-depleted human splenocytes. Two mAb (HB57 and 5D7) that bound to distinct epitopes on IgM were highly effective initiators of B cell proliferation at very low concentrations, in the presence of a T cell factor source. In the absence of T cell supernatant, both HB57 and 5D7 mAbs produced a markedly reduced degree of stimulation at all concentrations. Two additional anti-IgM mAb (VIIIE11 and Mu53) were distinctive in that, even at high concentrations, only limited proliferation was observed compared with the first group of mAb. This proliferation depended on the presence of T cell supernatant. Competitive-binding studies revealed that the epitope recognized by mAb Mu53 may be identical or very proximate to that recognized by HB57. Three other mAb (1G6, XG9, and P24) induced little or no proliferation. 1G6 bound to a unique epitope on the IgM molecule, whereas XG9 shared a determinant with VIIIE11 mAb. Regulatory influences of Fc receptor binding cannot account for all the diversity in proliferation observed with the individual anti-IgM mAb. Markedly augmented proliferation was obtained when B cells were cultured with certain combinations of anti-IgM mAb in the presence of exogenous T cell supernatant. The proliferation induced in the absence of T cell supernatant by high concentrations of mAb mixtures that included 1G6 approached that observed for the same mixtures in the presence of T cell supernatant. The data suggest that certain signals delivered through membrane IgM can bypass the need for T cell supernatant in the activation of human B lymphocytes.

Occurrence of surface IgM, IgD, and free light chains of human lymphocytes.

An analysis was made of the immunoglobulin surface markers of the cells of patients with chronic lymphatic leukemia (CLL) in view of previous evidence of their monoclonal B-cell character. The simultaneous presence of IgM and IgD on the surface of the majority of lymphocytes was demonstrated by both immunofluorescence and hemagglutination inhibition in most cases. However, cases were observed with surface IgM without IgD as well as cases with IgD without IgM. IgG and IgA were absent. Studies of the light chains indicated only a single class in a given case. In addition to bound light chains, free light chains were readily demonstrated in most cases through the use of antisera specific for "free chain" determinants. It thus appeared that there are three major types of surface Ig on CLL lymphocytes, IgM, IgD, and free light chains.

The occurrence of the HL-B alloantigens on the cells of unclassified acute lymphoblastic leukemias.

Six cases of acute lymphoblastic leukemia were studied by a variety of T- and B-lymphocyte surface markers. Two appeared to represent T-cell leukemias with the lymphoblasts forming sheep erythrocyte rosettes. The other four lacked all the usual membrane markers. However, indirect immunofluorescence with alloantisera detected the presence of the Ia-related HL-B antigens on the cells of the latter four cases; these antigens were absent in the first two cases. The primary association of the HL-B antigens with B cells raises the possibility that the positive group of cases are of B-cell lineage.

Association of certain Ia allotypes with the occurrence of chronic lymphocytic leukemia. Recognition by a monoclonal anti-Ia reagent of a susceptibility determinant not in the DR series.

The Ia antigen allospecificities of individuals with B type chronic lymphocytic leukemia differed significantly from those of a control population. A monoclonal antibody, IVD12, directed to a MB3-1ike determinant, reacted with 92.5 percent of the individuals with leukemia and yielded the greatest positive relative risk, 13.5. A lower degree of positive association was found with the presence of the MT2 determinant. In contrast, the low observed frequency of the MT1/MB1 determinant among leukemic individuals was associated with the most significant negative relative risk, -8.1. Among HLA- DR specificities, the relative risk associated with the presence of DR5 was positive while that with DR2 was negative.

Parallel synthesis of immunoglobulins and J chain in pokeweed mitogen-stimulated normal cells and in lymphoblastoid cell lines.

The synthesis of intracellular J chains was found to be closely associated with that of intracellular immunoglobulin, regardless of its class, during the process of B-cell differentiation. This parallelism between the synthesis of J chain and immunoglobulin was particularly evident in their coincident appearance in serial observations of pokeweed mitogen (PWM)-stimulated lymphocytes. The intensity of J-chain staining by fluorescent reagents in the stimulated cells synthesizing IgG was similar to that found in cells synthesizing IgA or IgM. Evidence was obtained that the presence of J chain in the IgG-producing cells did not reflect antecedent synthesis of IgA or IgM. T cells stimulated by phytohemagglutinin and PWM failed to show J-chain synthesis. Observations on lymphoid cell lines showed a similar parallelism between intracellular Ig and J-chain synthesis; no relation to surface Ig was found.

Anti-IgM-mediated B cell signaling. Molecular analysis of ligand binding requisites for human B cell clonal expansion and tolerance.

The ligand binding requisites for membrane IgM-mediated signaling of human B lymphocyte clonal expansion and B cell tolerance were investigated with a well-characterized set of soluble murine anti-human IgM mAbs. Evaluation of the impact of mu chain domain specificity, affinity, and binding stoichiometry for membrane IgM on antibody-induced regulation of normal and leukemic B cell DNA synthesis revealed that the ligand binding requisites for inducing or, alternatively, suppressing B cell DNA synthesis are significantly different. First, while the induction of S phase entry required micrograms/ml concentrations of ligand, orders of magnitude lower concentrations of ligand sufficed for inhibitory signaling. Second, while an upper affinity threshold for achieving maximal stimulation of B cell DNA synthesis was never detected, inhibitory signaling by bivalent ligands appeared to become relatively affinity independent at Fab binding affinities greater than 7.0 x 10(6) M-1. Third, while a C mu 1-specific mAb with an enhanced incidence of monogamous binding to mIgM was ineffective at inducing B cell DNA synthesis, the antibody was not significantly compromised in ability to initiate inhibitory signals. These differences could be observed in a clonal B cell population which positively or negatively responded to mIgM ligation depending upon its state of activation. The accumulated observations indicate that the ligand binding requisites for inhibitory signal transduction in human B lymphocytes are much less rigorous than those for stimulatory signal transduction and suggest that many physiologically relevant anti-Ig antibodies are more likely to function in the negative feedback regulation of B cell responses than in the direct triggering of human B cell clonal expansion.

Ia determinants on stimulated human T lymphocytes. Occurrence on mitogen- and antigen-activated T cells.

Human T-cell blasts were generated by stimulation with mitogens and antigens. A proportion of these blasts expressed Ia antigens detectable by immunofluorescence with both allo- and hetero-antiserums. The maximal expression of Ia antigens was delayed and usually occurred after the peak of blastogenesis. Among the three mitogens used, pokeweed mitogen (PWM) was most effective in giving a high percentage and intense Ia staining of T-cell blasts. Phytohemagglutinin and concanavalin A blasts gave weaker and lower percentages of Ia staining. Activation by alloantigens and soluble antigens such as tetanus toxoid and purified protein derivative resulted in Ia expression on T cells comparable to PWM stimulation. Depletion of Ia+ cells from freshly isolated T cells with anti-Ia and complement decreased subsequent Ia expression, suggesting that a proportion of Ia+ blasts were derived from Ia-bearing peripheral blood T cells. When the specificities of the Ia antigens on T-cell blasts were examined with alloantiserums, it was evident that the T blasts expressed similar HLA-DR determinants to those on B cells from the same donor; occasional minor differences between stimulated T cells and autologous B-cell lines or fresh B cells were encountered.

Two types of Ia-positive T cells. Synthesis and exchange of Ia antigens.

Two distinct types of Ia-positive T cells have been described. One type represents a blastoid T cell responding from stimulation by mitogens, antigens, and in allogeneic and autologous mixed lymphocyte culture reactions. This is a large cell that is strongly positive for Ia antigens as measured by a variety of different antisera. The other general type is a smaller cell with a lower expression of Ia antigens that is found at low levels in normal peripheral blood and is markedly elevated in various pathological states. It also rises rapidly after inoculation with tetanus toxoid and PPD in sensitized individuals. This cell does not incorporate thymidine and is enriched in the Tgamma fraction; it can be markedly concentrated from normal lymphocytes, and current evidence indicates that it is a T cell. The marked elevation of this cell in the blood of patients with rheumatoid arthritis is of special interest. Considerable evidence indicates that, at least in certain instances, the Ia antigens are synthetized by the cells that carry them. Incorporation of labeled amino acid experiments and the in vitro translation results presented above indicate this. However, the ready exchange of Ia antigens between cells in the experiments described indicates that uptake from other cells may be a significant source.

Disease associations of the Ia-like human alloantigens. Contrasting patterns in rheumatoid arthritis and systemic lupus erythematosus.

Increasing evidence has been obtained of the special value of Ia-like B-cell alloantisera for demonstrating disease associations with histocompatibility antigens. This was particularly evident for the study of the immunogenetics of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), two conditions frequently considered related. The profiles of antigens recognized by the alloantisera in patients from each disease group was distinctive. Two types of alloantisera were obtained that illustrated the divergence between the twod iseases. One type showed a higher than normal incidence in RA but lower than normal in SLE; the other showed a higher incidence in SLE. While these sera were not totally defined, evidence was obtained that the SLE-reactive alloantiserum related to two alleles of the major histocompatibility complex DRw2 and DRw3, while the RA-reactive alloantiserum related to a common specificity shared by cells positive for either DRw4, DRw7, or DRw10. The data indicate that immunogenetic factors are relevant to the development of both RA and SLE, but that these are distinct for each disease.

Human megakaryocytes. I. Characterization of the membrane and cytoplasmic components of isolated marrow megakaryocytes.

Human marrow megakaryocytes have been isolated with high purity and yield by processing marrow cells sequentially through density centrifugation and velocity sedimentation. Analysis of the isolated cells for various platelet-associated components by immunofluorescence demonstrated that fibrinogen, plasma factor VIII antigen (factor VIII:AGN) platelet myosin, platelet glycoproteins I and III are present on the membrane and in the cytoplasm of over 90% of marrow megakaryocytes. Parallel studies of human and mouse megakaryocytes and platelets for IgG receptor (FcR), complement receptor type one (CR1) (C3b receptor), complement receptor type two (CR2) (C3d receptor), and Ia antigen by fluorescence and (or) rosette formation methods were performed. FcR were present on most human megakaryocytes and platelets. The Ia antigen was detected on a proportion (10-15%) of human megakaryocytes but it was undetectable on human platelets. CR1 was found on 20-40% of mouse megakaryocytes and also on a proportion of mouse platelets. These differentiation markers may be of use in monitoring megakaryocyte maturation.

C1q PRECIPITINS IN THE SERA OF PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS AND OTHER HYPOCOMPLEMENTEMIC STATES: CHARACTERIZATION OF HIGH AND LOW MOLECULAR WEIGHT TYPES.

Precipitin reactions of C1q in gel diffusion proved useful in detecting unknown complexes containing gamma-globulin in the sera of patients with SLE. Using this method low molecular weight C1q reactants also have been detected in a number of sera from patients with SLE as well as other diseases. Both the circulating complexes and the unidentified low molecular weight reactants are associated with disease activity and in vivo complement depression. In some sera from patients with SLE, circulating complexes as detectable by C1q precipitation were closely associated with cryoprecipitins and an active nephritic process. Evidence is presented that both rheumatoid factors and C1q interact with circulating complexes in these patients and that the interaction is related to cryoprecipitation. The demonstration of the same rheumatoid factors in the cryoprecipitates and in the renal glomerular deposits suggests that rheumatoid factors have a special significance in the presence of circulating complexes.

Recognition by pregnancy serums of non-HL-A alloantigens selectively expressed on B lymphocytes.

A group of alloantibodies are found in pregnancy sera which react with antigens present on B lymphocytes and monocytes but are not detectable on the vast majority of unstimulated T cells. This specificity distinguishes them from HL-A antibodies which react with both cell types. They were readily recognized through indirect fluorescent antibody analysis by employing the combination of B-cell lymphoid lines and normal peripheral blood T cells. Different sera gave a variety of patterns of reactivity with a panel of 11 lymphoid lines. Similar differential patterns were also observed with normal B cells from different individuals particularly after concentrating the B cells. The antibodies were also cytotoxic to B cells and this procedure gave parallel results to the fluorescence method. The pattern of reactions obtained indicated a very heterogeneous system similar to that for HL-A. Special study of certain of the sera provided evidence that the lymphocyte-defined determinants of the mixed lymphocyte reaction system were involved. For convenience the term HL-B has been employed for these antigens.

Similarities in the light chains of anti-gamma-globulins showing cross-idiotypic specificities.

A marked homogeneity of the light chains was observed in an analysis of 17 IgM proteins with anti-gamma-globulin activity. The V region subgroups of the light chains were determined by both sequence and antigenic analysis. The latter procedure permitted large scale screening for comparisons with control proteins. The two methods showed general agreement in the determination of Kappa III proteins; all proteins positive by antigenic analysis were also positive by sequence but exceptions were noted in the opposite direction. The anti-gamma-globulins showed by antigenic analysis a 92% incidence of VK III light chains as compared to an incidence of 27% among 81 control proteins without this activity. A similar selection was observed for an antigen (VK III b) which subdivided the kappa III proteins. The major Wa group of anti-gamma-globulins which had been delineated previously on the basis of cross-idiotypic specificity was primarily responsible for the special light-chain selection. All the proteins of this group contain VK III light chains and all were of the VK III b type. Current evidence indicates that additional light-chain similarities were involved in the cross-idiotypic specificity of the Wa group. It thus appears that for the anti-gamma-globulins various levels of selection of light chains are manifest ranging from a preponderance of kappa type, of kappa III subgroup, of kappa III b sub-subgroup and perhaps of still further subdivisions to account for the cross-idiotypic specificity.

Identification, at the genomic level, of an HLA-DR restriction element for cloned antigen-specific T4 cells.

Two T4 cell clones (TLC) specific for antigenic epitopes on Chlamydia trachomatis were studied. Using a panel of allogeneic antigen-presenting cells (APC), both TLC were found to be restricted by HLA class II elements closely associated with, but not identical to the DRw5S specificity, as determined by highly selected alloantisera, a monoclonal antibody (mAb), 109d6, and confirmed on the DNA level by determination of restriction fragment length polymorphisms (RFLP) with a DR beta probe. Furthermore, HLA-DR-specific mAb, including 109d6, but not other HLA class II- or class I-specific antibodies inhibited the two TLC, strongly suggesting that the restriction element is expressed by a DR molecule. Using digestion with Hind III restriction enzyme and a DR beta probe, we found a complete concordance between the appearance of a 9.3 kilobase band and the ability of allogeneic APC to restimulate the T cell clones. Thus, the restriction element for these T cell clones appear to be expressed by DR molecules, but can, at present, only be detected at the genomic level.

Cross-idiotypic specificity among monoclonal IgM proteins with anti- -globulin activity.

Through the use of absorbed idiotypic antisera prepared against single isolated monoclonal IgM anti-gamma-globulins, partial cross-idiotypic specificity was demonstrated with other IgM anti-gamma-globulins. Such antisera classified these proteins into at least three groups. The major group which included 60% of the anti-gamma-globulins was particularly homogeneous. The anti-gamma-globulin specific antigens were detected best in hemagglutination and hemagglutination inhibition systems. They were not found in monoclonal IgM proteins that lacked anti-gamma-globulin activity although related antigens were detected at low concentrations in pooled immunoglobulin preparations as well as in heterogeneous anti-Rh antibodies. Several lines of evidence were obtained indicating that the antibody combining site was involved in the specific determinants. Attempts were made to analyze the fine specificity of each anti-gamma-globulin for the Fc fragment of different subclasses of human immunoglobulins as well as those of other species. Differences were observed but these were not readily related to the cross-specificity antigens. The anti-gamma-globulin specific antigens were very analogous to those previously described for monoclonal IgM cold agglutinins. Although each protein could be distinguished from all the others on the basis of individual idiotypic antigens, the antigens common to the specific groups of proteins with each of these activities were prominent and readily detected with multiple antisera. The results indicate basic similarities between proteins of a given activity even in unrelated individuals.

The sequential appearance of Ia-like antigens and two different complement receptors during the maturation of human neutrophils.

Ia antigens and two different types of complement (C) receptors appeared on membrane surfaces in a distinct sequence during the maturation of human neutrophils. Taking advantage of the finding that neutrophil celll density increased with maturation, density gradient centrifugation was used to separate neutrophils into fractions that were greatly enriched in cells representing individual stages of differentiation. Myeloblasts, the earliest cells recognized in the myeloid series of both normal and myelogenous leukemic individuals, expressed Ia determinants, whereas Ia determinants were absent or diminished on the majority of promyelocytes and completely undetectable on more mature granulocytes. Double marker studies demonstrated that Ia determinants were lost from the membrane of developing myeloid cells before the appearance of any type of C receptor. In the next phase of maturation defined by surface markers, neutrophils acquired a CR2-type C receptor (C3d receptor) that was similar in specificity to CR2 of B lymphocytes. This stage of maturation approximately corresponded to the myelocyte-metamyelocyte stage defined by standard morphologic criteria, and preceded the third stage of surface marker maturation when developing neutrophils began to express CR1-type C receptors (immune adherence, C4b-C3b receptors) in addition to CR2. In the final stage of surface marker-defined maturation, CR2 was lost from high density polymorphonuclear neutrophils and CR1 was maximally expressed. Normal blood polymorphonuclear neutrophils contained only 17% of CR2-bearing cells and these were shown to be of lower density than the majority of neutrophils that expressed only CR1. There was some variation in the correlation of surface marker expression and maturation stage defined by morphologic criteria, but in all cases the sequence of marker appearance was the same: Ia leads to CR2 leads to CR1CR2 leads to CR1.