Prognosis and incidence of immunological and oncological complications after direct-acting antiviral therapy for chronic hepatitis C.

Background and study aims: The long-term comprehensive prognosis of chronic hepatitis C after direct-acting antiviral (DAA) therapy is unclear. This study aimed to investigate the prognosis and incidence of immunological and oncological complications after DAA therapy. Patients and methods: The study included a total of 1461 patients who received DAA therapy in our university hospital and affiliated hospitals between September 3, 2014 and September 30, 2018. Results: The incidence rates of total malignancies in overall or female patients after DAA therapy were significantly greater than expected in the corresponding general population. The same was true for lung malignancies. Predictive risk factors associated with the occurrence and recurrence of hepatic malignancies after DAA therapy in patients with sustained virological response were cirrhosis and insulin use, protein induced by vitamin K absence or antagonist-II level, and albumin-bilirubin score, respectively. Eight (0.5%) patients were diagnosed with autoimmune diseases after starting DAA therapy. Importantly, the attending physician considered a possible causal relationship between DAA therapy and these autoimmune diseases in five cases (four rheumatoid arthritis and one membranoproliferative glomerulonephritis). The 5-year overall survival rate was 91.6%. The most frequent primary cause of death was malignancy in 41 (60.2%) patients, including 25 with hepatic malignancies. Lung and colorectal cancers were the next most common. Conclusions: Given that the incidence of total and lung cancers might increase and DAA-related autoimmune diseases might emerge after DAA therapy, we should be alert for the development of these diseases as well as hepatic malignancies.

Proliferation of peritoneal mast cells in the skin of W/Wv mice that genetically lack mast cells.

Presence of mast cell precursors in the mouse peritoneal cavity was demonstrated, and the precursors were characterized. When a cell suspension, containing mast cell precursor(s), was directly injected into the skin of genetically mast cell-deficient WBB6F1 (WB X C57BL/6)-W/Wv mice, a cluster composed of approximately 2,000 mast cells appeared at the injection site. By determining the proportion of injection sites at which the mast cell cluster appeared, the concentration of mast cell precursors can be calculated by limiting dilution analysis. The concentration in the peritoneal cavity was about five times as great as the concentration in the bone marrow. Although peritoneal mast cell precursors were shown to originate from the bone marrow, physical characterization revealed that the peritoneal precursors differed from the marrow precursors. The peritoneal precursors were less susceptible to irradiation than the marrow precursors; the former were heavier than the latter. When a 95% pure mast cell suspension was prepared from the peritoneal cells by the removal of phagocytes and the density gradient centrifugation, 1 out of 16 cells had the potentiality to make a mast cell cluster in the skin of the W/Wv mice. Moreover, when a single mast cell was identified under the phase contrast microscope and picked up with the micromanipulator, 1 out of 17 mast cells made the cluster. This indicated that some peritoneal mast cells kept extensive proliferative potentiality even after morphological differentiation. In other words, some peritoneal mast cells themselves may function as the committed precursors.

Fate of bone marrow-derived cultured mast cells after intracutaneous, intraperitoneal, and intravenous transfer into genetically mast cell-deficient W/Wv mice. Evidence that cultured mast cells can give rise to both connective tissue type and mucosal mast cells.

Both connective tissue mast cells and mast cells grown in vitro are derived from multipotential hematopoietic stem cells, but these two mast cell populations exhibit many differences in morphology, biochemistry, and function. We investigated whether the phenotype of cultured mast cells or their progeny was altered when the cells were transferred into different locations in vivo. Cultured mast cells were immature by ultrastructure, and stained with alcian blue but with neither safranin or berberine sulfate, a fluorescent dye that binds to the heparin of connective tissue mast cell granules. By contrast, mast cells recovered from the peritoneal cavity of congenitally mast cell-deficient (WB X C57BL/6)F1-W/Wv (WBB6F1-W/Wv) mice 10 wk after intraperitoneal injection of cultured WBB6F1-+/+ or C57BL/6-bgJ/bgJ mast cells stained with both safranin and berberine sulfate. Staining with berberine sulfate was prevented by treatment of the cells with heparinase but not chondroitinase ABC, suggesting that the adoptively transferred mast cell population had acquired the ability to synthesize and store heparin. Furthermore, the recovered mast cells were indistinguishable by ultrastructure from the normal mature peritoneal mast cells of WBB6F1-+/+ mice, and contained substantially more histamine than mast cells studied directly from culture. Intravenous injection of cultured mast cells resulted in the development of safranin-and berberine sulfate-positive mast cells in the peritoneal cavity, spleen, skin, and glandular stomach muscularis propria. Mast cells also developed on the glandular stomach mucosa, but these cells stained with alcian blue rather than safranin, and did not stain with berberine sulfate. This result suggests that cultured mast cells can give rise to mast cells of either the connective tissue type or mucosal phenotype, depending on anatomical location. Furthermore, transplantation of cultured mast cells into WBB6F1-W/Wv mice had no measurable effect on the anemia of the recipient mice, suggesting a possible strategy for repairing the mast cell deficiency of WBB6F1-W/Wv mice without affecting other bone marrow-derived populations such as erythrocytes. Intravenous injection of representative connective tissue type mast cells (30-50% pure peritoneal mast cells derived from WBB6F1-+/+ mice) gave results similar to those obtained with cultured mast cells: mast cells developing in the peritoneal cavity, skin, spleen, and glandular stomach muscularis propria of WBB6F1-W/Wv recipients stained with safranin and berberine sulfate, whereas mast cells developing in the mucosa of the glandular stomach stained only with alcian blue.(ABSTRACT TRUNCATED AT 400 WORDS)

Induction of programmed cell death in human hematopoietic cell lines by fibronectin via its interaction with very late antigen 5.

Extracellular matrix (ECM) molecules such as fibronectin (FN), collagens, and laminin have important roles in hematopoiesis. However, little is known about the precise mechanisms by which ECM molecules regulate proliferation of human hematopoietic progenitor cells. In this study, we have investigated the effects of ECM molecules, particularly of FN, on the proliferation of a myeloid leukemia cell line, M07E, which proliferates in response to either human granulocyte/macrophage colony-stimulating factor (GM-CSF) or stem cell factor (SCF). The [3H]thymidine incorporation and cell enumeration assays showed that FN strikingly inhibited GM-CSF- or SCF-induced proliferation of M07E cells in a dose-dependent manner, whereas little or no inhibition was induced by collagen types I and IV. The growth suppression of M07E cells was not due to the inhibitory effect of FN on ligand binding or very early events in the signal transduction pathways from the GM-CSF or SCF receptors. DNA content analysis using flow cytometry after staining with propidium iodide revealed that the treatment of M07E cells with FN did not block the entry of the cells into the cell cycle after stimulation with GM-CSF or SCF, whereas the treatment resulted in the appearance of subdiploid peak. Furthermore, FN was found to induce oligonucleosomal DNA fragmentation and chromatin condensation in the cells even in the presence of GM-CSF or SCF, suggesting the involvement of programmed cell death (apoptosis) in the FN-induced growth suppression. The growth suppression or apoptosis induced by FN was rescued by the addition of either anti-FN antibody, anti-very late antigen 5 monoclonal antibody (anti-VLA5 mAb), or GRGDSP peptide, but not by that of anti-VLA4 mAb or GRGESP peptide, suggesting that the FN effects on M07E cells were mediated through VLA5. In addition, the FN-induced apoptosis was detectable in VLA5-positive human hematopoietic cell lines other than M07E cells, but not in any of the VLA5-negative cell lines. These results suggest that FN is capable of inducing apoptosis via its interaction with VLA5, and also raise the possibility that the FN-VLA5 interaction may contribute, at least in part, to negative regulation of hematopoiesis.

Overexpression of the Arabidopsis thaliana EDS5 gene enhances resistance to viruses.

The Arabidopsis thaliana ENHANCED DISEASE SUSCEPTIBILITY 5 gene (EDS5) is required for salicylic acid (SA) synthesis in pathogen-challenged plants. SA and EDS5 have an important role in the Arabidopsis RCY1 gene-conferred resistance against the yellow strain of Cucumber mosaic virus [CMV(Y)], a Bromoviridae, and HRT-conferred resistance against the Tombusviridae, Turnip crinkle virus (TCV). EDS5 expression and SA accumulation are induced in response to CMV(Y) inoculation in the RCY1-bearing ecotype C24. To further discern the involvement of EDS5 in Arabidopsis defence against viruses, we overexpressed the EDS5 transcript from the constitutively expressed Cauliflower mosaic virus 35S gene promoter in ecotype C24. In comparison to the non-transgenic control, the basal level of salicylic acid (SA) was twofold higher in the 35S:EDS5 plant. Furthermore, viral spread and the size of the hypersensitive response associated necrotic local lesions (NLL) were more highly restricted in CMV(Y)-inoculated 35S:EDS5 than in the non-transgenic plant. The heightened restriction of CMV(Y) spread was paralleled by more rapid induction of the pathogenesis-related gene, PR-1, in the CMV(Y)-inoculated 35S:EDS5 plant. The 35S:EDS5 plant also had heightened resistance to the virulent CMV strain, CMV(B2), and TCV. These results suggest that, in addition to R gene-mediated gene-for-gene resistance, EDS5 is also important for basal resistance to viruses. However, while expression of the Pseudomonas putida nahG gene, which encodes the SA-degrading salicylate hydroxylase, completely suppressed 35S:EDS5-conferred resistance against CMV(Y) and TCV, it only partially compromised resistance against CMV(B2), indicating that SA-dependent and -independent mechanisms are associated with 35S:EDS5-conferred resistance against viruses.

Blood pressure regulates platelet-derived growth factor A-chain gene expression in vascular smooth muscle cells in vivo. An autocrine mechanism promoting hypertensive vascular hypertrophy.

To clarify the role of PDGF A-chain in hypertensive vascular hypertrophy of spontaneously hypertensive rats (SHRs), we studied levels of PDGF A-chain gene expression and transcription factors related to the gene in vascular smooth muscle cells (VSMCs) of SHRs in vivo. RNase protection assay and in situ hybridization showed that PDGF A-chain mRNA levels in VSMCs of SHRs were twofold higher than in those of normotensive Wistar-Kyoto rats. Gel retardation assays showed that levels of Sp1 and AP-2 in VSMCs of SHRs were twofold more abundant than in those of Wistar-Kyoto rats. Treatment with four pharmacologically different species of antihypertensive drugs for 2 wk decreased the levels of both PDGF A-chain mRNA and Sp1, but not AP-2 level in VSMCs of SHRs with regression of aortic hypertrophy, indicating that increases in levels of both PDGF A-chain mRNA and Sp1 in VSMCs of SHRs were associated with high blood pressure. These results suggest that high blood pressure is a stimulus which upregulates PDGF A-chain gene expression in VSMCs of SHRs, resulting in an autocrine enhancement in hypertensive vascular hypertrophy, and that the activation of the gene may be mediated through increases in Sp1 in these cells.

Molecular basis of CD36 deficiency. Evidence that a 478C-->T substitution (proline90-->serine) in CD36 cDNA accounts for CD36 deficiency.

CD36 deficiency is divided into two subgroups: neither platelets nor monocytes express CD36 (type I deficiency), and monocytes express CD36 in spite of the lack of platelet CD36 (type II deficiency). We have already demonstrated that a 478C-->T substitution (proline90-->serine) in platelet CD36 cDNA predominates in type II deficiency (Kashiwagi, H., S. Honda, Y. Tomiyama, H. Mizutani, H. Take, Y. Honda, S. Kosugi, Y. Kanayama, Y. Kurata, and Y. Matsuzawa. 1993. Thromb. Haemostasis. 69:481-484). In this study, we revealed that monocyte CD36 cDNA from two type II deficient subjects was heterozygous for C478 and T478 form, while platelet CD36 cDNA of these subjects consisted of only T478 form. In a type I deficient subject, both platelet and monocyte CD36 cDNA showed only T478 form. Expression assay using C478 or T478 form of CD36 cDNA transfected cells revealed that there was an 81-kD precursor form of CD36, and that the maturation of the 81-kD precursor form to the 88-kD mature form of CD36 was markedly impaired by the substitution. The mutated precursor form of CD36 was subsequently degraded in the cytoplasm. These results indicate that the 478C-->T substitution directly leads to CD36 deficiency via defects in posttranslational modification, and that this substitution is the major defects underlying CD36 deficiency.

Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of c-kit product.

The c-kit proto-oncogene encodes a receptor tyrosine kinase. Binding of c-kit ligand, stem cell factor (SCF) to c-kit receptor (c-kitR) is known to activate c-kitR tyrosine kinase, thereby leading to autophosphorylation of c-kitR on tyrosine and to association of c-kitR with substrates such as phosphatidylinositol 3-kinase (PI3K). In a human mast cell leukemia cell line HMC-1, c-kitR was found to be constitutively phosphorylated on tyrosine, activated, and associated with PI3K without the addition of SCF. The expression of SCF mRNA transcript in HMC-1 cells was not detectable by means of PCR after reverse transcription (RT-PCR) analysis, suggesting that the constitutive activation of c-kitR was ligand independent. Sequencing of whole coding region of c-kit cDNA revealed that c-kit genes of HMC-1 cells were composed of a normal, wild-type allele and a mutant allele with two point mutations resulting in intracellular amino acid substitutions of Gly-560 for Val and Val-816 for Asp. Amino acid sequences in the regions of the two mutations are completely conserved in all of mouse, rat, and human c-kit. In order to determine the causal role of these mutations in the constitutive activation, murine c-kit mutants encoding Gly-559 and/or Val-814, corresponding to human Gly-560 and/or Val-816, were constructed by site-directed mutagenesis and expressed in a human embryonic kidney cell line, 293T cells. In the transfected cells, both c-kitR (Gly-559, Val-814) and c-kitR (Val-814) were abundantly phosphorylated on tyrosine and activated in immune complex kinase reaction in the absence of SCF, whereas tyrosine phosphorylation and activation of c-kitR (Gly-559) or wild-type c-kitR was modest or little, respectively. These results suggest that conversion of Asp-816 to Val in human c-kitR may be an activating mutation and responsible for the constitutive activation of c-kitR in HMC-1 cells.

Functional expression of interleukin 2 receptor in a human factor-dependent megakaryoblastic leukemia cell line: evidence that granulocyte-macrophage colony-stimulating factor inhibits interleukin 2 binding to its receptor.

Human interleukin 2 (IL-2) is a member of the class of crucial regulators of lymphocyte proliferation. The action of IL-2 is known to be mediated through binding to a specific IL-2 receptor (IL-2R) which comprises at least two distinct proteins: IL-2R alpha (p55) and IL-2R beta (p70-75). However, the expression and function of IL-2R are largely unknown in acute myeloblastic leukemia cells. In a human granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, or stem cell factor-dependent myeloid leukemia cell line (M07E), IL-2 was found to stimulate proliferation in a dose-dependent manner and to augment GM-CSF- and stem cell factor-induced proliferation of M07E cells. The expression of IL-2R beta on M07E cells was detectable with 125I-IL-2 binding and affinity cross-linking analyses and with a monoclonal antibody against IL-2R beta, Mik-beta 1. Although the expression of IL-2R beta was not down-regulated but somewhat up-regulated by treatment with GM-CSF in both mRNA and protein levels, GM-CSF was found to compete (75%) with radiolabeled IL-2 for binding to IL-2R on M07E cells, whereas no competition of GM-CSF binding was observed with IL-2 even at a 400-fold molar excess. These results suggest that IL-2R may be functionally expressed in some cases of acute myeloblastic leukemia cells and raise the possibility that IL-2 may have some effects on human myelopoiesis.

Carbohydrate analysis of immunoglobulin G myeloma proteins by lectin and high performance liquid chromatography: role of glycosyltransferases in the structures.

The carbohydrate structures and the enzymatic basis for glycosylation of IgG by bone marrow plasma cells were determined in 7 patients with monoclonal gammopathy of undetermined significance and 22 patients with IgG MM. Lectin-binding analysis showed that in all cases of monoclonal gammopathy of undetermined significance and normal controls the IgG heavy chains bound to Ricinus communis agglutinin more strongly than to concanavalin A. In contrast, the IgG in 11 of the 17 advanced cases of MM (stages II and III) studied reacted to concanavalin A more strongly. Structural analysis showed that the reduced R. communis agglutinin binding capacity of these MM IgGs was due to hypogalactosylation of IgG. The galactosyltransferase and N-acetylglucosaminyltransferase III activities of the bone marrow myeloma cells from 5 MM cases were found to have a low enzyme activity ratio of galactosyltransferase to N-acetylglucosaminyltransferase III which reflects the hypogalactosylation. This indicates that the difference in the carbohydrate moieties observed in myeloma proteins is due to variations in the activities of the two glycosyltransferases.

Surface immunoglobulin-mediated signal transduction involves rapid phosphorylation and activation of the protooncogene product Raf-1 in human B-cells.

The protooncogene product, Raf-1, is a serine/threonine kinase and has been implicated as an intermediate in signal transduction mechanisms. We examined neoplastic and normal B cells for phosphorylation and activation of Raf-1 protein in response to anti-immunoglobulin antibody (anti-Ig). Anti-Ig induced rapid phosphorylation of Raf-1 protein in both neoplastic B-cells of hairy cell leukemia and normal tonsillar B-cells which proliferated well in response to anti-Ig. The increase in phosphorylation was due primarily to an increase in phosphoserine. The immune complex kinase assay using Histone V-S as an exogenous substrate also showed an increase in Raf-1-associated kinase activity. An inhibitor of protein kinase C, H7, inhibited the proliferation as well as the Raf-1 phosphorylation in response to the proliferative signal of anti-Ig. Further, downregulation of protein kinase C by the treatment with 12-phorbol 13-myristic acid significantly abrogated the induction of Raf-1 phosphorylation. These results suggest that, in human B-cells, Raf-1 protein may be involved in the signal transduction pathway mediated by surface immunoglobulin, and that it may be, at least partially, phosphorylated by activated PKC.

Lovastatin inhibits gene expression of type-I scavenger receptor in THP-1 human macrophages.

Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, inhibits the synthesis of mevalonic acid and is widely used as an anti-atherosclerotic drug. The macrophage scavenger receptor (SCR), a trimeric membrane glycoprotein, is postulated to play a key role in atheroma macrophage foam cell formation. HMG-CoA reductase is involved in the control of the synthesis of glycoproteins and farnesylated proteins, including ras proteins, which are involved in the transcriptional regulation of SCR gene expression. Accordingly, we examined whether lovastatin alters the gene expression of SCRs in THP-1 cell derived human macrophages. Lovastatin (5-15 microM) caused a significant dose-related reduction in steady state levels of type-I SCR mRNA in phorbol 12-myristate 13-acetate (PMA)-treated THP-1 cells. The addition of exogenous mevalonate (1 mM) completely restored the lovastatin-induced decrease of type-I SCR mRNA levels. While the addition of the isoprenoid end-product, isopentenyl adenine (50 microM), had little effect on the type-I SCR mRNA levels in lovastatin treated cells, the addition of isoprenoid farnesol (5 microM) largely restored the lovastatin-induced decrease of type-I SCR mRNA levels. Actinomycin D treatment showed that degradation rates of type-I SCR mRNA did not differ between the THP-1 derived cells with and without lovastatin treatment. Nuclear run-on assays showed that lovastatin markedly decreased the transcription of SCR gene in the cells. These results suggest that lovastatin inhibits the transcription of type-I SCR gene by affecting mevalonate metabolism, possibly through the farnesyl-pyrophosphate related end-product(s) in the THP-1-derived macrophages.

cAMP-induced changes of intracellular free Mg2+ levels in human erythrocytes.

To examine the role of cAMP in the regulation of intracellular free magnesium concentration ([Mg2+]i), we measured [Mg2+]i in human erythrocytes by 31P-NMR spectroscopy. (-)-Isoproterenol, forskolin, Bt2cAMP and 8-bromo-cAMP decreased [Mg2+]i in human erythrocytes. Bt2cAMP did not increase the efflux rate of Mg2+ from erythrocytes. HA1004, a potent inhibitor of cAMP-dependent kinases, markedly increased the [Mg2+]i in a Mg(2+)-free buffer solution. Addition of 8-bromo-cGMP or 12-O-tetradecanoylphorbol 13-acetate (TPA) did not affect the [Mg2+]i. These results suggest that beta-adrenergic stimulation and cAMP play an important role in the regulation of [Mg2+]i in human erythrocytes.

Angiotensin-converting enzyme inhibitor increases angiotensin type 1A receptor gene expression in aortic smooth muscle cells of spontaneously hypertensive rats.

To examine the regulation of angiotensin receptors in vascular smooth muscle cells, we studied the effects of antihypertensive drugs on angiotensin type 1A (AT1A) receptor gene expression in aortic smooth muscle cells (ASMCs) from spontaneously hypertensive rats (SHRs) using both ribonuclease protection assay and reverse-transcription polymerase chain reaction. An increase in AT1A receptor gene expression in ASMCs of SHRs was induced by treatment with an angiotensin converting enzyme inhibitor (enalapril) for 2 weeks and 4 weeks, but not by other types of antihypertensive drugs such as alpha-blocker (doxazosin), alpha, beta-blocker (arotinolol), Ca antagonist (nicardipine) or vascular smooth muscle relaxant (hydralazine). Since all antihypertensive drugs lowered the blood pressure of the rats almost equally, our results suggest that AT1A receptor gene expression in ASMCs of SHRs may be regulated by the vascular renin-angiotensin system.

Tomato fructokinases exhibit differential expression and substrate regulation

Two divergent genes encoding fructokinase, Frk1 and Frk2, have been previously shown to be expressed in tomato (Lycopersicon esculentum L.) and have now been further characterized with regard to their spatial expression and the enzymic properties of the encoded proteins. Frk1 and Frk2 mRNA levels were coordinately induced by exogenous sugar, indicating that both belong to the growing class of sugar-regulated genes. However, in situ hybridization indicated that Frk1 and Frk2 were expressed in a spatially distinct manner, with Frk2 mRNA primarily localized in cells of the fruit pericarp, which store starch, and Frk1 mRNA distributed ubiquitously in pericarp tissue. To evaluate the biochemical characteristics of the products of the Frk1 and Frk2 genes, each cDNA was expressed in a mutant yeast (Saccharomyces cerevisiae) line defective in hexose phosphorylation and unable to grow on glucose or fructose (Fru). Both Frk1 and Frk2 proteins expressed in yeast conferred the ability to grow on Fru and exhibited fructokinase activity in vitro. Although both Frk1 and Frk2 both utilized Fru as a substrate, only Frk2 activity was inhibited at high Fru concentrations. These results indicate that Frk2 can be distinguished from Frk1 by its sensitivity to substrate inhibition and by its temporal and spatial pattern of expression, which suggests that it plays a primary role in plant cells specialized for starch storage.

Amylase-producing plasmacytoma cell lines, AD3 and FR4, with der(14)t(8;14) and dic(8)t(1;8) established from ascites.

We established two human plasma cell lines, FR4 and AD3, from ascitic fluid in a patient with IgA k plasmacytoma (PC). Aberrant amylase production was found in this patient. Both AD3 and FR4 were free of Epstein-Barr virus, and both produced Ig A k in vitro. They produced amylase of the salivary type in vitro. This was confirmed by the demonstration of amylase mRNA comigrating with salivary gland mRNA. These cell lines commonly had unusual chromosomal abnormalities der(14)t(8;14) and dic(8)t(1;8). AD3 had additional chromosomal abnormalities compared with FR4. This suggests that AD3 is a subline of FR4. The oncogene c-myc is rearranged in most case of Burkitt's lymphoma with t(8;14). However, neither rearrangement nor amplification of the c-myc allele was detected in our PC lines. These lines expressed c-myc of 2.4 kb. There were no structural changes in the amylase genes of AD3 and FR4 detectable with Southern blotting analysis. As these lines were authentic PC lines, they would be useful for the future study of the relationship between the mechanism of oncogenesis and the rare tumor aberration, amylase production.

Activating mutations of the c-kit proto-oncogene in a human mast cell leukemia cell line.

The c-kit proto-oncogene encodes a receptor tyrosine kinase that is known to play a crucial role in mast cell growth and differentiation. In a human mast cell leukemia cell line (HMC-1), KitR was found to be constitutively phosphorylated on tyrosine, activated and associated with phosphatidylinositol 3-kinase (P13K) in the absence of autocrine production of SCF. Sequencing of c-kit cDNA revealed that c-kit genes of HMC-1 cells were composed of a normal, wild-type allele and a mutant allele with two point mutations in codon 560 and codon 816, resulting in intracellular amino acid substitutions of Gly-560 for Val and Val-816 for Asp, respectively. Murine c-kit mutants encoding Gly-559 and/or Val-814, corresponding to human Gly-560 and/or Val-816, were constructed by site-directed mutagenesis and expressed in cells of a human embryonic kidney cell line (293T). In the transfected cells, KitR (Gly-559 + Val-814) and KitR (Val-814) were strikingly phosphorylated on tyrosine and activated in the absence of SCF, whereas tyrosine phosphorylation and activation of KitR (Gly-559) or wild-type KitR was modest or little, respectively. These results suggest that constitutive activation of KitR in HMC-1 results from the activating mutations of c-kit gene, and raise the possibility that the activating mutations, particularly at codon 814 of murine c-kit or at codon 816 of human c-kit, may participate in oncogenesis of mast cells.

Correlation between steroid myopathy and serum lactic dehydrogenase in systemic lupus erythematosus.

Examination of 27 patients with systemic lupus erythematosus (SLE) before treatment showed an elevation of the serum level of lactic dehydrogenase (LDH) in 15 patients. In these patients, the LDH level fell to normal in response to corticosteroid therapy. In six of 27 patients, steroid myopathy with elevation of the LDH level developed during corticosteroid therapy. At the same time, there was no or only a slight increase in the creatine phosphokinase level, while the SGOT and aldolase levels remained normal. The elevated LDH levels gradually returned to normal as the corticosteroid dosages were reduced and the myopathic symptoms disappeared. We suggest that the measurement of LDH levels is useful for diagnosis and the subsequent treatment of patients with steroid myopathy in SLE.

Serum lactate dehydrogenase and its isozymes in lupus nephritis.

The relationship between the renal pathologic activity of systemic lupus erythematosus (SLE) and serum lactate dehydrogenase (LDH) was examined in 28 patients with active SLE involving only the kidney. Serum levels of total LDH, LDH1, and LDH2 were significantly higher in the patients with diffuse proliferative lupus nephritis (World Health Organization class IV) than in those with milder renal disease (classes I through III and V). Total LDH levels showed good correlations with the activity index and the total pathologic score of the renal pathologic scoring system, and with the glomerular hypercellularity and overall deposits. The elevated level of LDH was mainly due to elevated levels of its isozymes LDH1 and LDH2. These results suggest that the elevation of serum LDH levels in patients with SLE reflects the renal pathologic changes due to lupus nephritis.