Characterization of human monocytic cell line, U937, in taking up acetylated low-density lipoprotein and cholesteryl ester accumulation. A flow cytometric and HPLC study.

The uptake of LDL and acetylated LDL and the ability of cholesteryl ester accumulation by cells of a human monocytic cell line, U937, has been characterized by flow cytometric assay using a fluorescent probe, DiI, and by high-performance liquid chromatography (HPLC). The increase of mean fluorescence intensity of U937 incubated with DiI-labeled lipoproteins demonstrates that this cell line could incorporate DiI-AcLDL, as well as DiI-labeled LDL. Competition and saturation studies indicate that the manner of taking up DiI-AcLDL is receptor-mediated. While differentiated U937 incubated with 16 nM phorbol myristate acetate for 24 h took up little DiI-AcLDL, HPLC analysis confirmed that intracellular free and esterified cholesterols significantly increase in the U937 cells incubated with AcLDL or LDL. The ability of mouse peritoneal macrophage to abundantly accumulate at least five kinds of cholesteryl ester were also shown in this analysis. In contrast, in U937 cells, free fatty acids are incorporated into various substances rather than into cholesteryl esters (as revealed by HPLC analysis), so that the cholesterol in AcLDL taken up by U937 cells is not synthesized into cholesteryl esters to any great extent.

Enhanced induction of thyroid cell MHC class II antigen expression in rats highly responsive to thyroglobulin.

Initial experiments demonstrated that the degree of autoantibody and proliferative T cell responses to syngeneic rat thyroglobulin differed markedly between Buffalo (high responder) and Fisher (low responder) rats after classical immunization schedules. While varying immune responsiveness may be due to qualitative and quantitative T and B cell differences, the role of thyroid cell MHC class II antigens may be pivotal to the onset of autoimmune thyroiditis in such animal models. We, therefore, examined the induction of MHC class II antigens in thyroid monolayers derived from Buffalo and Fisher rats treated with methimazole (0.1% in their water) for 4 weeks to induce mild thyroid hyperplasia. After thyroidectomy, thyroid cell monolayers were prepared and exposed to recombinant rat gamma-interferon (gamma IF; 10-1000 U/ml) for 1-7 days in the presence and absence of TSH (1 mU/ml). Both Buffalo and Fisher thyroid monolayers responded to gamma IF with MHC class II antigen expression when assessed by laser flow cytometry using MRC OX-6 monoclonal anti-RT1.B. In both types of culture, TSH enhanced MHC class II antigen expression in the presence of gamma IF to the same degree. However, there was a consistently earlier and greater degree of MHC class II antigen expression in Buffalo thyroid monolayers compared to Fisher monolayers, a phenomenon not explicable on the basis of fibroblast contamination as assessed by cytokeratin staining. These data demonstrate that end-organ sensitivity to MHC class II antigen expression may be important in the pathogenesis of autoimmune thyroid disease.

Measurement of spontaneous and stimulated anti-microsomal antibody synthesis in vitro by avidin-biotin enzyme immunoassay.

The spontaneous and stimulated anti-microsomal (anti-Mic) antibody synthesis in vitro by peripheral blood lymphocytes (PBL) from patients with Hashimoto's thyroiditis (HT) was studied by a highly sensitive and thyroid microsome-specific enzyme immunoassay using an avidin-biotin system (A-B EIA). Since the amount of the synthesized anti-Mic antibody by PBL in vitro is very small, it is difficult to study its kinetics and response to mitogens or the specific antigen by conventional assay systems. We applied the avidin-biotin system to conventional indirect EIA and established an assay system which was about four times as sensitive as indirect EIA. PBL from patients with HT synthesized significant amount of IgG anti-Mic antibody spontaneously but those from normal individuals and patients with rheumatoid arthritis did not. IgG anti-Mic antibody synthesis with pokeweed mitogen stimulation was increased in all HT patients and that with thyroid microsome stimulation was increased in three out of five patients. These results indicate that A-B EIA is a useful system to study the mechanism of anti-Mic antibody synthesis in vitro.

Pretreatment of human vascular smooth muscle cells with interleukin-1 enhances interleukin-6 production and cell proliferation (action of IL-1 on vascular smooth muscle cells).

Systemic vasculitis is an inflammatory disorder of blood vessels characterized by a perivascular mononuclear cell infiltration around the vessel and fibrinoid necrosis within vessel walls. Interleukin-1 (IL-1) is a multipotent inflammatory mediator and affects several properties of vascular cells. To determine whether IL-1 could contribute to the pathogenesis of vascular diseases, we examined the effect of IL-1 on B cell stimulatory factor-2/interleukin-6 (IL-6) production by cultured human vascular smooth muscle cells (SMC) and the proliferation of these cells. Supernatants of SMC stimulated IgM synthesis of human B cell line. SKW6-CL4 cells. This activity was increased (1.7 to 2.6-fold) when SMC were pretreated with IL-1 or calcium ionophore A23187 for 48 h, and was completely blocked by rabbit anti-human IL-6 antibodies. These IL-6 activities of the SMC supernatants were also assessed by using an IL-6 dependent murine hybridoma cell line. MH-60. BSF-2. In addition, we observed that pretreatment of SMC with IL-1 for 48 h stimulated growth of SMC during the 96 h incubations, as assessed by cell number (p less than 0.05). These results suggest that IL-1 may contribute to the pathogenesis of inflammatory and immunological vasculitis by the augmentation of IL-6 release and growth of SMC.

Studies on prolyl endopeptidase from shakashimeji (Lyophyllum cinerascens): purification and enzymatic properties.

High prolyl endopeptidase (post-proline cleaving enzyme) [EC 3.4.21.26] activity was detected in fruit bodies of shakashimeji (Lyophyllum cinerascens), tsukuritake (mushroom: Agaricus bisporus), hirohachichitake (Lactarius hygrophoroides), and yaburebenitake (Russula lepida) which belong to the genus Basidiomycetes. Cell-free extract of shakashimeji showed high activities of proline iminopeptidase and arylamidase as well as prolyl endopeptidase. The prolyl endopeptidase was purified from the extract of shakashimeji by sequential chromatographies on DEAE-Toyopearl, DEAE-Sephadex and hydroxyapatite, and high-performance liquid chromatography with a DEAE-5PW column. The purified enzyme was homogeneous as judged by disc gel electrophoresis. The enzyme was most active at pH 6.8 as checked with Z-Gly-Pro-beta-naphthylamide as a substrate and was stable in the range of pH 5.8-7.4. The isoelectric point of the enzyme was 5.2 and the molecular weight was estimated to be 76,000 by gel filtration on Sephadex G-150 and by sodium dodecyl sulfate (SDS) gel electrophoresis, suggesting that the enzyme was a monomer. The enzyme was completely inhibited by diisopropyl fluorophosphate (DFP), Z-Gly-Pro-CH2Cl, and Z-Pro-prolinal, while it was not inhibited by p-chloromercuribenzoate (PCMB), phenylmethylsulfonyl fluoride (PMSF), or metal chelators. It was estimated that at least five subsites were concerned with the enzyme-substrate binding. Among them, the S1, S2, and S1' sites showed high stereospecificity, as in mammalian, microbial, and plant enzymes. The enzyme hydrolyzed TRH at the carboxyl side of the proline residue. The mushroom enzyme, that was sensitive to DFP, Z-Pro-prolinal, and Z-Gly-Pro-CH2Cl, but not to PCMB, were quite similar in characteristics to the Flavobacterium enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

[An autopsy case of Sjögren syndrome with organized and fresh subdural hemorrhage (hematoma)].

An autopsy case of Sjögren syndrome with organized and fresh subdural hemorrhages (hematoma) is reported. A 49-year-old woman who had been suffering from Sjögren syndrome had gradually lost her consciousness and was taken to the hospital where she died several hours later. Subsequently a doctor found the subdural hematoma of unknown origin on her Brain CT. At autopsy, her skin was dry and all of her teeth were missing. There were sporadic cutaneous purpura and subcutaneous hemorrhages in her trunk and limbs. The histopathological examination revealed that the submandibular gland had no normal acini, and was replaced by fibrous and adipose tissues with numerous lymphocytes. There were signs of fibrosis with inflammation in her liver, kidneys and lungs. The thyroid gland showed thyroiditis. Serological findings showed a significant high level of antinuclear antibody, positive RA factor and high gamma-globulinemia. The autopsy revealed that her cause of death was acute subdural hematoma and uncal herniation. There were no external injuries on her head or face. It is suggested that her acute subdural hematoma according to the hemorrhagic tendency, affected by her Sjögren syndrome.

[An autopsy case of the Reye's syndrome].

A case of sudden unexpected natural death in a 6-year-old girl was reported. She was found in dead with vomiting on her bed in the morning on 24. December. Autopsy did not reveal any injuries and abnormalities on her appearance. Small amount of light yellowish mucus in her nose, trachea and bronchus was found. Brain was slightly edematous (1395 g in weight). Liver was 750 g in weight and focal yellowish lesions were observed on its cut surfaces. Microscopically fatty degeneration (granulous fatty deposits) was observed in hepatocytes. Immunohistochemical staining of mitochondria in heart showed no staining microscopically, and degeneration and breakdown of mitochondria were found electromicroscopically. From the results of autopsy and histopathological findings, her cause of death was diagnosed as the Reye's syndrome.

A case of Sjögren's syndrome complicated by nephrogenic diabetes insipidus and renal tubular acidosis.

Abstract We describe the case of a 46-year-old woman with Sjögren's syndrome (SS) presenting with a 6-year history of polyuria and polydipsia. Laboratory data revealed hyperchloremic metabolic acidosis, a normal anion gap, and an inability to acidify urine following an acid loading test and to concentrate the urine in response to water deprivation and antidiuretic hormone administration. Lymphocyte infiltration in the interstitium was found on renal biopsy. These findings allowed us to diagnose distal renal tubular acidosis (RTA) and nephrogenic diabetes insipidus (NDI). Steroid pulse therapy resulted in normalization of the blood pH, but failed to remit the inability to concentrate the urine. These observations suggest therapeutic applications for RTA in SS, and that further investigation is required to design a therapeutic strategy for NDI in SS.

Generation of thyroglobulin-specific T-cell clones derived from F1 Fisher rats.

We established T-cell clones from 'poor-responder' Fisher rats specific for thyroglobulin (Tg) with a view to examining Tg presentation by cloned Fisher rat thyroid (FRTL) cells. From the screening of 60 T-cell clones, three high-responding Tg-specific clones (B21.01, B21.04 and B21.05) were isolated from the lymph nodes of F1 generation (Fisher x 'high-responder' Buffalo) female rats immunized with murine Tg in complete Freund's adjuvant (CFA). Three T-cell clones expressed a W3/25+, OX-8- phenotype and responded specifically to murine and rat Tg in T-cell proliferation assays with Fisher rat antigen-presenting cells and secreted IL-2 as measured using a murine cytotoxic T-lymphocyte line (CTLL-2). Both B21.04 and B21.05 T-cell clones were capable of providing helper T-cell function for rat Tg antibody production in syngeneic reconstitution cultures in vitro. In contrast, clone B21.01 inhibited Tg antibody secretion. These data demonstrate that 'poor-responder' Fisher rats are capable of mounting significant T-cell responses to Tg in an F1 generation. Such Tg-specific T-cell clones will allow us to analyse their interaction with cloned Fisher rat thyroid cells and determine the role, if any, of thyroid cell antigen self-presentation to the immune system.

Hereditary recombined three-allele variant of the Gc system.

A three-allele variant with Gc 2, Gc 1F and Gc 1A2 alleles was detected in both a baby and his mother during paternity testing by isoelectric focusing. His father had a normal Gc phenotype, Gc 2-1F. Further examination of his mother's relatives revealed that his grandfather also had the same three-allele variant, while his grandmother and his aunt had normal Gc 2-1F and Gc 2-2. From these results, it was considered that the Gc 1F and Gc 1A2 alleles were on the same single chromosome. It was suggested that recombination had occurred between two chromosomes that had the Gc 1F and Gc 1A2 allele, respectively, forming the variant allele Gc 1F1A2 on a single chromosome.

Duodenal hemorrhage and dermal vasculitis associated with mixed connective tissue disease.

We describe a 23-year-old woman with mixed connective tissue disease (MCTD) who developed duodenal bleeding responsive to treatment with corticosteroid and recurrent skin eruptions due to leukocytoclastic vasculitis. The endoscopic findings revealed patchy, reddened, edematous, friable mucosa with scattered small ulcers and oozing of blood in the second portion of the duodenum. Endoscopic pictures from the onset to healing are presented. This appears to be the first recorded occurrence of such findings in MCTD.

[A case of systemic lupus erythematosus presenting with myelofibrosis as a cause of pancytopenia].

A 54 year-old woman who had a 6 month history of polyarthralgias, oral ulcers, weight loss and fatigue was admitted to the Urawa Municipal Hospital. She developed high fever, dyspnea and thrombocytopenia. Chest radiograph revealed massive right pleural effusion. At this time, laboratory investigations gave the following results: hemoglobin 12.7 g/dl, WBC 7700/microliters and platelet count 9.2 x 10(4)/microliters. Antibody to DNA was negative. Antinuclear antibody was positive at a titer of 320x in a centromere pattern; Anti-RNP and anti-Sm antibodies were negative. CH50 was 18.6 u/ml. C3 was 42.9 mg/dl. C4 was 11.5 mg/dl. Circulating immune complex (Clq) was 30.5 micrograms/ml. Circulating lupus anti-coagulant and anticardiolipine antibodies were positive. Thoracocentesis was performed; the material was a straw-colored exudate with over two thousands white cell per ul and showed marked reduction of complement titiers and elevated immune complex levels. She was then diagnosis as having SLE. Two weeks after admission, progressive leukopenia and anemia succeedingly occurred and resulted in severe pancytopenia. Bone marrow biopsy demonstrated marked marrow fibrosis and increased reticulin content with no evidence of malignancy. Steroid pulse therapy for 3 days started, and subsequently she was treated with 60 mg/day of prednisolone. Three weeks after starting on steroids, the massive pleural effusion was completely disappeared and complement titiers were normalized. Circulating immune complex has not been detected any more. After 8 weeks, the peripheral blood count was normalized. The dose of prednisolone was reduced progressively. On this occasion, the biopsy showed normocullular marrow with a marked reduction in the amount of reticulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Myelodysplastic syndrome associated with immunoblastic lymphadenopathy-like T-cell lymphoma: simultaneous clinical improvement with chemotherapy.

A 75-year-old woman presented with anemia, lymphadenopathy, hepatomegaly and lingual tumor, but no constitutional symptoms. The laboratory data showed pancytopenia and polyclonal hypergammaglobulinemia. A bone marrow aspirate represented an apparent myelodysplastic syndrome (MDS) feature, specifically, refractory anemia with excess of blasts. A lymph-node biopsy revealed the disappearance of normal architecture, small arborizing blood vessels, large lymphoid cells with prominent cytoplasm (so-called pale cells) and a clonal proliferation of T-lymphocytes. The patient was diagnosed as having MDS associated with immunoblastic lymphadenopathy (IBL)-like T-cell lymphoma. She was subsequently treated with cyclophosphamide, adriamycin, vincristine and prednisolone for lymphoma which successfully induced a remission of not only the T-cell lymphoma but also the MDS. The case suggested that MDS might be a paraneoplastic complication of IBL-like T-cell lymphoma.

Isolated adrenocorticotropic hormone deficiency: an autopsy case of adrenal crisis. A case report.

We present a case of fatal adrenal crisis due to isolated adrenocorticotropic hormone (ACTH) deficiency. Autopsy revealed each adrenal gland weighed 0.9 g and the adrenal cortexes were very thin and atrophic. Additionally, cortisol could not be observed in the adrenal cortex by immunohistochemical staining. Furthermore, urine cortisol and 17-OHCS concentration had decreased to a very low level, 20 mg/L and 0.8 mg/L respectively. The anterior pituitary gland was atrophic, and showed fibrosis and lymphocytosis was suspected. Immunohistochemically growth hormone (GH)-stained pituitary gland cells were observed, but there were no cells stained with anti-ACTH antibody. From the history and pathological findings, no other deficiencies of pituitary hormones were evident. Therefore, isolated ACTH deficiency was suspected. Furthermore, as the thyroid gland showed lymphocytic thyroiditis, is was considered that isolated ACTH deficiency was associated with an autoimmune cause. Generally, as patients of chronic adrenocortical insufficiency are exposed to stress and, therefore, have an increased requirement for glucocorticoids, the blood pressure falls, leading to hypovolemic shock called " an adrenal crisis." Without treatment, patients die in crisis within several hours. In our case, the deceased had drunk alcohol without sleep for 2 days. We believe that the stress of drinking and sleeplessness induced adrenal crisis and caused his death.

Kinetic analysis of Ia expression on T cells in patients with systemic lupus erythematosus.

An increased proportion of Ia positive circulating T cells was observed in patients with systemic lupus erythematosus (SLE) (17.2 +/- 5.8%, n = 20, p less than 0.005), compared with normal subjects (3.2 +/- 0.9%, n = 10) by the analysis with flow cytometry using OKIa1. There was a significant difference (p less than 0.05) in the proportion of Ia positive T cells (Ia +T cells) between inactive patients (19.6 +/- 5.8%, n = 12) and active patients (13.6 +/- 3.7%, n = 8). The kinetic analysis in normal subjects revealed that Ia expression on T cells stimulated with Concanavalin A (ConA) was significantly increased on day 4 (10.2 +/- 6.2%, n = 8). However, when stimulated with native DNA from calf thymus (nDNA), Ia expression was not increased (1.4 +/- 1.6%, n = 8). In SLE patients, initial Ia expression faded out within 4 days without stimulus (3.9 +/- 4.0%, n = 10). In spite of the decline of Ia expression with stimulation of both Con A and nDNA, the initial proportion of Ia positive T cells was relatively well maintained with the stimulation of nDNA (16.2 +/- 4.7%). Further analysis of T-cell subpopulations in SLE patients revealed that both OKT8- (T4) and OKT4- (T8) cells expressed Ia antigens. Interestingly, in 4 out of 5 patients with active disease, the incidence of Ia expression in OKT4- cells was increased with nDNA stimulation. Thus, in patients with SLE, circulating Ia positive T cells might be activated in vivo, and be implicated in the development of autoimmunity to DNA.

Abnormal production of and response to B-cell growth factor and B-cell differentiation factor in patients with systemic lupus erythematosus.

We examined the production of and the response to B-cell growth factor (BCGF) and B-cell differentiation factor (BCDF) in 21 patients with systemic lupus erythematosus (SLE) and 23 normal subjects. T cells, 2.5 X 10(6)/ml, were cultured for 24 or 72 h with 1% phytohaemagglutinin (PHA). After absorption of PHA by chicken erythrocytes (CRBC), they were used for BCGF and BCDF. In inactive SLE, BCGF activity was significantly lower than that in normal subjects. Active SLE contained two separate groups, one showing normal BCGF activity and the other showing lower activity than normal. In contrast, BCDF activity from initial culture in active SLE was elevated. The B-cell response both to BCGF and BCDF was elevated in active SLE without Staphylococcus aureus Cowan I antigen (SAC) preactivation. However, the B-cell response to SAC was markedly disturbed. Thus SLE B cells were shifted to the mature state in vivo. We also demonstrated pivotal abnormalities of monocytes in SLE B-cell growth and differentiation. These results may contribute to the understanding of the abnormalities of T-B interactions and the overproduction of antibody in SLE.

Activation of MHC-restricted rat T cells by cloned syngeneic thyrocytes.

We have previously demonstrated that rat thyrocytes express MHC class II Ag (RT1.B&D) in response to IFN-gamma. To determine whether MHC class II-positive thyrocytes can be recognized by MHC-restricted T cells, we used our clone of rat thyroid cells (1B-6) derived from the Fisher rat thyroid cell line (FRTL-5) and known to express MHC class II Ag in response to recombinant rat IFN-gamma. CD4+ and CD8+ normal syngeneic Fisher rat spleen T cells were selected by flow cytometry and averaged greater than 96% purity. We demonstrated that irradiated MHC class II-positive but not class II-negative 1B-6 thyrocytes stimulated CD4+ T cells in a primary sensitization reaction over 4 days. In contrast, CD8+ T cells had no response in similar experiments. This stimulation of CD4+ T cells was dose dependent for 1B-6 thyrocytes and was abrogated by anti-rat MHC class II mAb (MRC OX-6). Autoreactive (Fisher) and alloreactive (Buffalo) T cell lines and isolated CD4+ T cells derived from these lines, which were developed against Fisher rat spleen cells, similarly recognized MHC class II Ag expressed on 1B-6 cells but had no detectable response to 1B-6 MHC class II-negative thyrocytes or MHC class II-positive human thyroid cells. The CD4+ T cell recognition of 1B-6 cells via MHC class II Ag supports our previous data with autologous human thyroid T cell co-cultures and is indicative of an autospecific role for thyrocytes in the development of autoimmune thyroiditis.

Flow cytometric analysis of lipid droplet formation in cells of the human monocytic cell line, U937.

Active uptake of a free fatty acid, oleate, and its incorporation into triacylglycerol and phospholipid in the human monocytic cell line, U937, was identified using 14C-labelled oleate. Excess triacylglycerol accumulates in the form of lipid droplets in the cytoplasm. The extent of lipid droplet formation in each cell can be assessed in the absence of any staining by 90 degrees light scatter intensity, one of the basic parameters of flow cytometry.

Immunohistological investigations of autopsied carotid bodies and their application to diagnosing strangulation.

Using immunohistochemical staining, the histological changes and the presence of neuropeptides (enkephalin and VIP) in the carotid body have been investigated in medico-legal autopsy cases, especially asphyxia cases. Only in cases of manual and/or ligature strangulation cases that sustained a force near the carotid body, were the chief cells mainly lightly stained, indicating that they had been "active" cells. Furthermore, these cells and their nuclei were enlarged in comparison to the chief carotid body cells in other autopsy groups. It was thus felt that these changes had resulted from the force that had directly affected the carotid body. Based on these findings, it was concluded that immunohistochemical investigation of the carotid body offers a useful possibility for diagnosing manual asphyxia, especially in autopsy cases involving strangulation.