Engagement of the B cell receptor for antigen differentially affects B cell responses to Toll-like receptor-7 agonists and antagonists in BXSB mice.

Nucleic acid sensors of the Toll-like receptor (TLR) family play a well-established role in the pathogenesis of lupus. This is particularly true for a single-stranded RNA-sensing TLR-7 receptor, as lupus mice lacking TLR-7 show ameliorated disease. Cytosine-guanosine dinucleotide (CpG)-DNA-sensing TLR-9, conversely, has a complex regulatory role in systemic lupus erythematosus (SLE). Much less is known about whether signals through the B cell receptor for antigen (BCR) may affect the ability of B cells to respond to suboptimal TLR-7 agonists and antagonists. We studied this question in prediseased BXSB male and female B cells. We found that male B cells responded more vigorously to numerous TLR-7 ligands and this responsiveness was enhanced further upon co-engagement of the BCR. This synergy was seen primarily with the interleukin (IL)-6 secretion. A number of 32-mer inhibitory oligonucleotides (INH-ODNs) with a nuclease-resistant phosphorothioate backbone were capable of blocking TLR-7, but not BCR-induced B cell activation, with an inhibitory concentration (IC)(50) of approximately 100 nm. Surprisingly, while the presence of a single TGC motif at the 5' end of an ODN did not increase its inhibitory capacity, INH-ODNs containing multiple TGC motifs had greater inhibitory potency. When BCR and TLR-7 were co-engaged, INH-ODNs showed a differential effect on B cell activation. Whereas apoptosis protection and G1-M entry completely escaped suppression, IL-6 secretion remained sensitive to inhibition, although with a 10-fold lower potency. Our results suggest that while TLR-7 antagonists may be considered as lupus therapeutics, simultaneous co-engagement of the TLR-7 and BCR might favour autoreactive B cell survival. This hypothesis needs further experimental validation.

B-cell receptor for antigen modulates B-cell responses to complex TLR9 agonists and antagonists: implications for systemic lupus erythematosus.

The capacity to make secondary structures significantly affects the ability of Toll-like receptor 9 (TLR9) agonists and antagonists to either induce or block TLR9-dependent activation in B cells. However, it has a minor impact on TLR9-induced activation in interferon alpha (IFNα)-producing dendritic cells. Based on the ability of inhibitory oligodeoxynucleotides to form predictable secondary structures, we have classified TLR9-antagonists into Class R ('restricted', palindromic) and Class B ('broadly reactive', linear) oligodeoxynucleotides. In non-autoreactive B cells, Class R oligodeoxynucleotides are at least 10-fold less potent TLR9-inhibitors. We wanted to determine whether engagement of the B-cell receptor for antigen could overcome this restriction. Here we show that in non-autoreactive mouse B cells, B-cell receptor for antigen engagement increased the potency of Class R oligodeoxynucleotides for TLR9 activation at least 10-fold, making it equal in potency to linear oligodeoxynucleotides. However, this enhanced potency was selective for TLR9-induced B-cell cycling and apoptosis protection while TLR9-induced IL-6, an event that strongly depends on signaling via late endosomes, still required 10 times more Class R oligodeoxynucleotides. Thus, pathway-specific effects of Class R oligodeoxynucleotides for TLR9/B-cell receptor for antigen co-stimulated B cells may have therapeutic advantages over non-selective targeting of B cells, a strategy that may be seen as a potential therapy for human systemic lupus erythematosus.

Serologic testing for celiac disease in the United States: results of a multilaboratory comparison study.

The aim of this study was to compare the efficiencies of six reference laboratories for serologic testing for celiac disease. Serum from 20 patients with untreated celiac disease and from 20 controls was thawed, divided, and distributed to each participating laboratory, which performed endomysial antibody tests. Five laboratories also performed antigliadin antibody tests. Sensitivity for endomysial antibody immunoglobulin A (IgA) varied from 57 to 90%. In all laboratories, the specificity for celiac disease was 100%. The sensitivity and specificity for both IgA and IgG antigliadin antibody varied significantly. When results from all three tests were combined in each laboratory, sensitivity was 90 to 100%. The specificity for endomysial antibody was 100% in the laboratories. Sensitivity was less than reported previously. Standardization of these tests is needed in the United States.

Guidelines for clinical and laboratory evaluation patients with monoclonal gammopathies.

This guideline provides the recommendations of an expert panel for the clinical and laboratory evaluation of patients suspected of having a clinical condition that produces a monoclonal protein in serum or urine. The recommendations describe the clinical conditions in which a monoclonal protein should be sought, the optimal sequence of testing to diagnose and monitor these patients, and the most effective laboratory procedures.

Autoimmune reactivity in inflammatory bowel disease.

Inflammatory bowel disease remains a poorly understood chronic inflammatory condition of the bowel. No etiologic agents, or agreed upon pathogenesis is currently known. Recent findings that perinuclear antineutrophil cytoplasmic antibodies (P-ANCA) are present in the vast majority of patients with this disease have proven to be of use in epidemiologic studies. Its clinical use is currently being explored by several investigators.

Celiac disease in an adult population with insulin-dependent diabetes mellitus: use of endomysial antibody testing.

OBJECTIVE: Studies from Europe and North Africa suggest an association between type 1 diabetes mellitus (IDDM) and celiac disease (CD). Although IDDM is as common in the United States as it is in Europe, CD is diagnosed much less often in this country than in Europe. The purpose of our study was to determine the frequency with which CD occurs in patients with IDDM in the United States. METHODS: Several serological tests are used for CD screening. The most specific and sensitive of these, the antiendomysial antibody, is the indirect immunofluorescence test which uses monkey esophagus smooth muscle as substrate. This test, which correlates closely with actual enteropathy, was used to screen 185 unselected patients with IDDM who attended the Diabetic Clinic or were housed on the Diabetic Unit of the University of Iowa Hospitals and Clinics. RESULTS: Nine of 185 patients had positive IgA antiendomysial antibody tests. Antibody positivity did not correlate with the presence of diabetic complications, age, sex, or duration of IDDM. Five of nine antibody-positive patients underwent subsequent small intestinal biopsy. Enteropathy was confirmed in four of these patients. CONCLUSIONS: These data suggest that CD is more common in American patients with IDDM than was previously suspected.

Autoimmune retinopathy in the absence of cancer.

PURPOSE: To report the clinical and immunologic features of two patients with progressive retinal degeneration and circulating antiretinal antibodies without systemic malignancy. METHODS: Two patients were followed up for 5 to 7 years. Comprehensive medical and ophthalmic examinations and visual function testing included manual perimetry and standardized electroretinography. Patient sera were tested for antiretinal antibodies by Western blot and immunoperoxidase indirect cytochemistry techniques. RESULTS: Two patients had family history of autoimmune disease. Each had severe monocular visual loss with photopsia, a ring scotoma, and abnormal electroretinogram despite a normal-appearing ocular fundus. One had a flat electroretinogram; the other had inner retina dysfunction, with selective b wave loss and abnormal oscillatory potentials. Both patients' sera had antiretinal antibodies that specifically labeled the inner plexiform layer of donor retina by indirect immunoperoxidase testing. Neither had any sign of cancer. CONCLUSIONS: In two patients without systemic malignancy, the symptoms, perimetric findings, and normal fundus appearance resembled cancer associated retinopathy. Electroretinography and antibody findings indicating dysfunction of the inner retina are distinct from those of cancer-associated retinopathy. These two cases raise the possibility of an autoimmune mechanism for retinal degeneration that is not cancer associated. Further study is necessary to determine the role of antiretinal antibodies in these patients.

Primary germinoma of the spinal cord: a case report with 28-year follow-up and review of the literature.

Germ cell neoplasms occur in extra-gonadal midline locations of the retroperitoneum, mediastinum, pineal gland, areas of the suprasellar cistern, and rarely in the spinal cord. We recently reviewed a case of an unresectable lumbar spinal cord tumor in a 16-year-old female previously diagnosed as "metastatic poorly differentiated carcinoma." An extensive evaluation for a primary neoplasm at that time was unsuccessful and the patient was treated with local radiation therapy. Recently, additional histochemical and immunocytochemical studies were performed on the archival formalin-fixed paraffin-embedded material and the clinical history was reviewed. These ancillary studies (including positive immunohistochemical staining for placental alkaline phosphatase) support a diagnosis of intramedullary germinoma of the conus medullaris. This patient has enjoyed 28 years of disease free survival which reflects the radiosensitive nature of this neoplasm. These data lend support to the existence of a primary germinoma in the spinal cord and illustrate the utility of using histochemical stains and immunohistochemistry to assist in diagnosing this treatable neoplasm.

Necrotizing medullary lesions in patients with ANCA associated renal disease.

One hundred and five renal specimens from patients with antineutrophil cytoplasmic antibody and antiglomerular basement membrane antibody-associated diseases were reviewed for necrotizing lesions involving the renal medulla. Necrotizing medullary lesions were identified in eight of 56 cases in which medullary tissue was present. All eight were in patients with antineutrophil cytoplasmic antibody (ANCA) associated disease (seven, C-ANCA; one P-ANCA). Four types of medullary lesions were identified; necrotizing capillaritis (seven cases), necrotizing arteriolitis (two cases), pathergic granulomas (three cases) and papillary tip necrosis (one case). Both medullary arteriolitis and medullary peritubular capillaritis developed without corresponding cortical arteriolitis or cortical peritubular capillaritis. Although necrotizing glomerulonephritis was present in seven of eight patients, its activity did not parallel the severity of the medullary lesions. We conclude that several forms of necrotizing medullary vascular lesions may develop in ANCA-associated disease and that there is discordance between state of activity and types of vessel affected between cortical and medullary vascular compartments.

Coexistent anti-neutrophil cytoplasmic antibody and antiglomerular basement membrane antibody associated disease = report of six cases.

From a series of 95 patients biopsied for rapidly progressive glomerulonephritis, twelve patients were identified with anti-glomerular basement membrane-mediated renal disease who were also tested for antineutrophil cytoplasmic antibody (ANCA). Six patients had both anti-glomerular basement membrane and ANCA antibodies. Three of the latter six patients had significant extrarenal disease, including severe hemoptysis, while the remaining three patients had only renal disease. The three patients with extrarenal disease had either a myeloperoxidase-positive perinuclear-ANCA (two patients) or a proteinase-3-positive cytoplasmic-ANCA (one patient). Two patients with renal disease alone had a myeloperoxidase-negative and proteinase-3-negative perinuclear-ANCA, and one patient had a proteinase-positive cytoplasmic-ANCA. Renal biopsy in all six patients showed a severe necrotizing and crescentic glomerulonephritis involving 94 to 100% of glomeruli. Renal arteritis was also noted in one perinuclear-ANCA patient. Despite aggressive therapy with steroids, cyclophosphamide, and plasma exchange, two of the six double-antibody patients died and four are on dialysis. We conclude that ANCA is commonly present in anti-glomerular basement membrane-associated disease and believe that this observation may have implications in the serologic evaluation of ANCA- and anti-glomerular basement membrane-positive patients.

Minimally differentiated acute leukemia.

We have studied 35 adult patients with morphologically undifferentiated peroxidase-negative acute leukemia that failed to meet the criteria for acute lymphoblastic leukemia and compared them to patients with FAB M1-M7 seen by the same physicians. The diagnosis of minimally differentiated acute leukemia (MD-AL) was associated with a higher incidence of prior hematologic disease, lower WBC, fewer blood blasts, lower marrow cellularity and a tendency towards older age. Of all patients treated with AML since January 1983, those with MD-AL were less likely to get a complete remission than those with other subtypes (35 vs 64%, p = 0.03). Treatment failure was usually due to resistant disease. Analysis of outcome as a function of drugs used during induction therapy showed an advantage for regimens containing vincristine and prednisone. The leukemic blast cells of nine patients were immunophenotyped for myeloid, lymphoid and megakaryoblast/platelet antigens. Although there were too few for a full statistical analysis as was applied to the larger group of 35 patients with MD-AL, these patients had a lower bone marrow cellularity as compared to FAB M1-M7 and a low remission rate. Eight of these were found to have positive myeloid markers and met the criteria for FAB M0. We conclude that patients with MD-AL form a distinct group with characteristic presenting features and a low response rate. Outcome data suggest that vincristine and prednisone should be included in experimental induction programs.

Antineutrophil cytoplasmic antibody in inflammatory bowel and hepatobiliary diseases. High prevalence in ulcerative colitis, primary sclerosing cholangitis, and autoimmune hepatitis.

The prevalence of antineutrophil cytoplasmic antibodies was evaluated in patients with ulcerative colitis, primary sclerosing cholangitis, and various other gastrointestinal and hepatobiliary diseases to define the sensitivity and specificity of the test. The presence of antineutrophil cytoplasmic antibodies was detected in alcohol-fixed cytospin preparations of peripheral blood neutrophils with an indirect immunofluorescence technique. A perinuclear staining pattern was considered positive. Thirty-six of 50 patients (72%) with ulcerative colitis and/or primary sclerosing cholangitis had positive results. Twenty-two of 210 patients (10%) in the control group had positive findings, including a significant proportion of patients with autoimmune hepatitis (50%) and non-A, non-B and non-C hepatitis (27%). This test for antineutrophil cytoplasmic antibodies has a sensitivity of 72% and specificity of 90% for either ulcerative colitis or primary sclerosing cholangitis. It may be useful in the differential diagnosis of Crohn's disease and ulcerative colitis and in the early diagnosis of ulcerative colitis. It also may be employed to distinguish primary biliary cirrhosis from primary sclerosing cholangitis.

Identification of apparent dual ANCA specificities in a subset of patients with systemic vasculitis and crescentic glomerulonephritis.

23% of a random selection of ANCA (+) patients had dual, non-cross reactive autoantibody specificities. We found no clinical difference in such patients as compared to those having a single antibody. A second group of ANCA (+) patients had neither anti-MPO nor anti-PR3 autoantibody specificity. These findings suggest that multiple, coincident ANCA specificities occur more commonly than previously reported, and that additional, unidentified ANCA specificities are present in some patients.

Cutaneous vasculitis in the newborn of a mother with cutaneous polyarteritis nodosa.

Vasculitis in an infant of a woman who had a long history of cutaneous polyarteritis nodosa is reported. During the neonatal period the child developed cutaneous vasculitis manifested by livedo reticularis, cutaneous nodules, and acral necrosis. The infant's vasculitis remitted by age 7 months. This is the third such report and strongly suggests the presence of a circulating factor that is capable of crossing the placenta and inducing cutaneous polyarteritis nodosa.

Renal artery stenosis modifies glomerular injury in antineutrophil cytoplasmic antibody-associated disease.

A 68-year-old man presented with renal failure, heart failure, gastrointestinal bleeding, and a pulmonary infiltrate. Serologic evaluation revealed a perinuclear antineutrophil cytoplasmic antibody (ANCA) at a titer of 1:1280, which on immunoblot and enzyme immunoassay showed antimyeloperoxidase specificity. Autopsy showed microscopic polyarteritis based on the presence of necrotizing alveolitis and crescentic glomerulonephritis. The extent and activity of the glomerular disease was modified by a right renal artery stenosis (RAS). Twenty percent of glomeruli on the right and 82% glomeruli on the left contained crescentic lesions. Furthermore, predominantly active lesions were associated with renal artery stenosis, while the contralateral kidney contained mostly organized crescents. This observation suggests that hemodynamic factors or its sequelae can influence the onset and severity of ANCA-associated disease.

Vascular smooth muscle hyperplasia underlies the formation of glomeruloid vascular structures of glioblastoma multiforme.

The origin of the vascular hyperplasia seen in glioblastoma multiforme is a matter of debate. To test the predominant hypothesis that these glomeruloid structures are of endothelial origin the following study was undertaken. Seven glioblastomas containing prominent glomeruloid vascular structures were stained with Ulex europaeus agglutinin I (UEA-1) and with antibodies against factor VIII/related antigen (fVIII/RAg), glial fibrillary acidic protein (GFAP), S-100 protein, muscle specific alpha-actin (MSA) and smooth muscle specific alpha-actin (SMSA). The GFAP and S-100 antibodies stained the neoplastic glial component of each tumor but did not bind to vascular cells. Endothelial cells lining the lumina of normal vessels and the lumina of glomeruloid vascular structures stained positively with both UEA-1 and fVIII/RAg antibody. No other cells were found to be stained by UEA-1 or by fVIII/RAg antibody. Smooth muscle cells of the normal vasculature stained positively exclusively with anti-MSA and anti-SMSA antibodies. The same pattern of positive actin antibody staining was seen in the majority of cells forming the glomeruloid structures; however, the cells lining the vascular lumina did not bind the MSA and SMSA antibodies. These data strongly suggest that the vascular proliferation resulting in glomeruloid structures is due in large measure to smooth muscle hyperplasia.

Smooth muscle can comprise the sarcomatous component of gliosarcomas.

The sarcomatous component of gliosarcomas is thought by many to originate from the vascular proliferation seen in glioblastoma multiforme and has, therefore, been assumed to be endothelial. Immunohistochemical staining of four gliosarcomas has led us to an alternate theory. Pathologically all four tumors were composed of at least two cell types; the first had a stellate, glial appearance and the second was either spindled or polygonal in shape. Polygonal cells were associated with glomeruloid vascular structures in some areas. Both components of each neoplasm were cytologically malignant. Glial fibrillary acidic protein and S-100 antibodies stained most of the glial-appearing cells and some of the spindled cells, but not the polygonal cells. Muscle specific alpha-actin and smooth muscle specific alpha-actin antibodies stained only the malignant spindled and polygonal cells and normal vascular smooth muscle. Ulex europaeus agglutinin I and anti-factor VIII/related antigen antibody stained only cells lining vascular lumina. The staining results suggest that the malignant mesenchymal component of these tumors is of smooth muscle origin. Having demonstrated elsewhere that glomeruloid vascular structures of glioblastoma multiforme contain smooth muscle cells, we propose here that gliosarcomas can represent one end of the spectrum of glioma-induced vascular smooth muscle proliferation.

Granulomatous mycosis fungoides. Clinicopathologic study of two cases.

Granulomatous mycosis fungoides is a rare form of mycosis fungoides with controversial histogenesis. Early reports seemed to indicate a favorable prognosis for these patients. We report two cases of granulomatous mycosis fungoides, both of which had other unusual clinical features. The cases were studied with routine light microscopy, immunohistochemistry, electron microscopy, and gene probe studies. Despite some clinical and histopathologic similarities, the results of the immunohistochemical and molecular biologic studies were diverse. These results suggest that granulomatous mycosis fungoides does not define a single subset of cases, immunophenotypically or biologically.