A clinical study of patients with pemphigus vulgaris and pemphigus foliaceous: an 11-year retrospective study (1996-2006).

Only a few reports have been published of detailed clinical studies of pemphigus in Japan. The aim of this study was to determine the clinical characteristics of patients with pemphigus vulgaris (PV) and pemphigus foliaceous (PF), who were newly diagnosed in the dermatology department of Kurume University Hospital, Japan, over the past 11 years. The primary site of involvement was the oral mucosa in 21 patients (75%) with PV. At the initial visit, most of the patients with PV had moderate to severe disease. With regard to management, systemic corticosteroids were the mainstay of treatment for patients with PV, and plasmapheresis was the most frequently used adjuvant therapy. Dapsone was the mainstay of treatment for the patients with PF. The patients were investigated for any association with an underlying malignancy; in patients with PV, lung, stomach and uterine cancers (one patient each) were seen.

Immunosuppressive properties of CD95L-transduced "killer" hybrids created by fusing donor- and recipient-derived dendritic cells.

Allogeneic immune responses, which are initiated by dendritic cells (DCs) of both donor and host origins, remain a major obstacle in organ transplantation. Presentation of intact major histocompatibility complex (MHC) molecules by allogeneic DCs and allogeneic peptides by syngeneic DCs leads to complex allogeneic immune responses. This study reports a novel strategy designed to suppress both pathways. A stable DC line XS106 (A/J mouse origin) was transfected with CD95L cDNA and fused with splenic DCs purified from allogeneic BALB/c mice. The resulting "killer" DC-DC hybrids: (1) expressed CD95L and MHC class I and class II molecules of both A/J and BALB/c origins, while maintaining otherwise characteristic surface phenotypes of mature DCs; (2) inhibited MHC class I- and class II-restricted mixed leukocyte reactions between the parental strains by triggering apoptosis of alloreactive T cells; and (3) abolished delayed-type hypersensitivity responses of A/J (and BALB/c) mice to BALB/c-associated (and A/J-associated) alloantigens when injected intravenously into A/J (and BALB/c) mice. The onset of graft-versus-host disease in (BALB/c x A/J) F1 hosts receiving A/J-derived hematopoietic cell transplantation was suppressed significantly (P <.001) by killer DC-DC hybrid treatment. These results form both technical and conceptual frameworks for clinical applications of CD95L-transduced killer hybrids created between donor DCs and recipient DCs in the prevention of allogeneic immune responses following organ transplantation.

p38 Kinase is a negative regulator of angiotensin II signal transduction in vascular smooth muscle cells: effects on Na+/H+ exchange and ERK1/2.

Activation of the Na+/H+ exchanger isoform-1 (NHE-1) by angiotensin II is an early signal transduction event that may regulate vascular smooth muscle cell (VSMC) growth and migration. Many signal transduction events stimulated by angiotensin II are mediated by the mitogen-activated protein (MAP) kinases. To define their roles in angiotensin II-mediated NHE-1 activity, VSMCs were treated with angiotensin II and the activities of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinases 1 and 2 (ERK1/2) were measured. Angiotensin II rapidly (peak, 5 minutes) activated p38 and ERK1/2, whereas JNK was activated more slowly (peak, 30 minutes). Because angiotensin II stimulated Na+/H+ exchange within 5 minutes, the effects of p38 and ERK1/2 antagonists on Na+/H+ exchange were studied. The MEK-1 inhibitor PD98059 decreased ERK1/2 activity and Na+/H+ exchange stimulated by angiotensin II. In contrast, the specific p38 antagonist SKF-86002 increased Na+/H+ exchange. Two mechanisms were identified that may mediate the effects of p38 and SKF-86002 on angiotensin II-stimulated Na+/H+ exchange. First, angiotensin II activation of ERK1/2 was increased 1. 5- to 2.5-fold (depending on assay technique) in the presence of SKF-86002, demonstrating that p38 negatively regulates ERK1/2. Second, the ability of angiotensin II-stimulated MAP kinases to phosphorylate a glutathione S-transferase fusion protein containing amino acids 625 to 747 of NHE-1 in vitro was analyzed. The relative activities of endogenous immunoprecipitated p38, ERK1/2, and JNK were 1.0, 2.0, and 0.05 versus control, respectively suggesting that p38 and ERK1/2, but not JNK, may phosphorylate NHE-1 in VSMC. These data indicate important roles for p38 and ERK1/2 in angiotensin II-mediated regulation of the Na+/H+ exchanger in VSMC.

A 90-kD Na(+)-H+ exchanger kinase has increased activity in spontaneously hypertensive rat vascular smooth muscle cells.

Increased activity of the Na(+)-H+ exchanger (NHE-1 isoform) has been observed in cells and tissues from hypertensive humans and animals, including the spontaneously hypertensive rat (SHR). No mutation in NHE-1 DNA sequence or alteration in NHE-1 mRNA and protein expression has been demonstrated in hypertension, indicating that alterations in proteins that regulate NHE-1 activity are responsible for increased activity. The recent finding that NHE-1 phosphorylation in SHR vascular smooth muscle cells (VSMCs) was greater than in Wistar-Kyoto rat (WKY) VSMCs suggested that NHE-1 kinases may represent an abnormal regulatory pathway present in hypertension. To define NHE-1 kinases altered in the hypertensive phenotype. We measured NHE-1 kinase activity by an in-gel-kinase assay using a recombinant glutathione S-transferase NHE-1 fusion protein as a substrate. At least 7 NHE-1 kinases (42 to 90 kD) were present in VSMCs. We studied a 90-kD kinase because it was the major NHE-1 kinase and exhibited differences between SHR and WKY. Comparison of 90-kD kinase activity revealed that SHR VSMCs had increased activity in growth-arrested cells and in cells stimulated by angiotensin II (100 nmol/L for 5 minutes). Activation of the 90-kD kinase by angiotensin II was Ca2+ dependent, PKC independent, and partially dependent on the mitogen-activated protein kinase pathway. These findings indicate that increased activity of a 90-kD NHE-1 kinase is a characteristic of SHR VSMCs in culture and suggest that alterations in the 90-kD NHE-1 kinase and/or proteins that regulate its activity may be a pathogenic component in hypertension in the SHR.

Differentiating agent, hexamethylene bisacetamide, inhibits neointimal formation after vascular injury.

BACKGROUND: Hexamethylene bisacetamide (HMBA), a potent differentiating agent of transformed cell lines, has been reported to affect phenotypic modulation and suppress proliferation of vascular smooth muscle cells (VSMCs). The effects of HMBA on the growth of VSMCs were studied in the rat carotid injury model. METHODS: HMBA was added to culture medium of VSMCs explanted from aorta of Sprague Dawley rats and the cell number was counted after 5 days of incubation. The carotid artery of Sprague Dawley rats was denuded with a balloon embolectomy catheter. The rats were treated with continuous infusion of HMBA (1 g/day) or saline for 7 days after injury. RESULTS: HMBA inhibited VSMC proliferation in a dose-dependent manner (at a HMBA concentration of 1-5 mM). Plasma concentration of HMBA during continuous infusion was in the range 0.8-1.0 mM. Immunohistochemistry of proliferating cell nuclear antigen showed that HMBA inhibited VSMC proliferation after vascular injury. Cell number in the intima and neointimal thickening were significantly reduced in HMBA-treated rats at 14 days after injury. CONCLUSIONS: Systemic administration of HMBA inhibited VSMC proliferation in the rat carotid injury model.

Oxidized LDL stimulates mitogen-activated protein kinases in smooth muscle cells and macrophages.

It has been proposed that oxidized LDL is more atherogenic than native LDL. However, the mechanisms by which native LDL and oxidized LDL alter function of cells in the vessel wall remain undefined. A signal transduction pathway that mediates many changes in cell function is the mitogen-activated protein (MAP) kinase cascade. We therefore examined the effect of native LDL and oxidized LDL on MAP kinase activity in cultured vascular smooth muscle cells (VSMC), endothelial cells, and macrophages by using an in-gel-kinase assay and anti-phosphotyrosine MAP kinase antibodies. Native LDL and LDL oxidized by the addition of Cu2+ (Cu(2+)-oxidized LDL) stimulated MAP kinase in a time- and dose-dependent manner in baboon and rat VSMC but not in bovine endothelial cells. Cu(2+)-oxidized LDL stimulated MAP kinase in human monocyte-derived macrophages, but the effect was much greater in cells cultured for 7 days compared with 1 day, suggesting dynamic regulation of the cellular response to oxidized LDL. In rat VSMC, the maximal MAP kinase response to Cu(2+)-oxidized LDL was significantly greater than the response to native LDL. Cu(2+)-oxidized LDL was more potent, with half-maximal activation at 15 micrograms/mL versus 30 micrograms/mL for native LDL. Stimulation of MAP kinase appeared to involve protein kinase C, since phorbol ester pretreatment for 24 hours blocked MAP kinase activation. Oxidation of LDL by other methods showed that activation of MAP kinase was not well correlated with lipid peroxides or aldehydes, suggesting that other components present in oxidized LDL were responsible. The active moiety appeared to be lipid based on extraction of oxidized LDL with organic solvents. These data indicate that LDL stimulates MAP kinase in VSMC, oxidation of LDL potentiates the effect, a lipid moiety is involved, and Cu(2+)-oxidized LDL activation of MAP kinase is cell-type specific. These findings suggest a role for MAP kinase in the pathways by which oxidized LDL contributes to altered cellular function associated with atherogenesis.

Big mitogen-activated protein kinase 1 (BMK1) is a redox-sensitive kinase.

Mitogen-activated protein (MAP) kinases are a multigene family activated by many extracellular stimuli. There are three groups of MAP kinases based on their dual phosphorylation motifs, TEY, TPY, and TGY, which are termed extracellular signal-regulated protein kinases (ERK1/2), c-Jun N-terminal kinases, and p38, respectively. A new MAP kinase family member termed Big MAP kinase 1 (BMK1) or ERK5 was recently cloned. BMK1 has a TEY sequence similar to ERK1/2 but has unique COOH-terminal and loop-12 domains. To define BMK1 regulation, its activation in cultured rat vascular smooth muscle cells was characterized. Angiotensin II, phorbol ester, platelet-derived growth factor, and tumor necrosis factor-alpha were the strongest stimuli for ERK1/2 but were weak activators of BMK1. In contrast, H2O2 caused concentration-dependent activation of BMK1 but not ERK1/2. Sorbitol activated both BMK1 and ERK1/2. BMK1 activation by H2O2 was calcium-dependent and appeared ubiquitous as shown by stimulation in human skin fibroblasts, human vascular smooth muscle cells, and human umbilical vein endothelial cells. These findings demonstrate that activation of BMK1 is different from ERK1/2 and suggest an important role for BMK1 as a redox-sensitive kinase.

Bullous pemphigoid in a 19-year-old woman. A case with unusual target antigens.

We report a 19-year-old woman with typical clinical histological and immunofluorescence features of bullous pemphigoid. By immunoblotting, the serum was shown to detect antigens at 240 and 138 kDa. Elevated serum IgE levels were present, and there was a marked peripheral blood eosinophilia. The degree of eosinophilia correlated with small changes in the severity of the skin lesions, and the IgE level showed correlation with marked changes in severity. Another unusual feature was an exacerbation of her disease at the time of her first menses after the onset of the bullous pemphigoid.

Purification and characterization of prolyl endopeptidase from rat skin.

An enzyme with the specificity of a prolyl endopeptidase was purified approximately 329-fold from rat skin. The enzyme has a molecular weight of 70,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pH optimum of 5.8 as checked with 7-(Succinyl-Gly-Pro)-4- methylcoumarinamide (Suc-Gly-Pro-MCA) as the substrate. The optimal temperature for the enzyme activity was 40 degrees C. The Km and Vmax values for Suc-Gly-Pro-MCA were 0.7 mM and 68 nmol/min per mg protein, respectively. The enzyme activity was markedly inhibited by diisopropyl fluorophosphate, p-chloromercuribenzoic acid, N-ethylmaleimide, Zn2+ and Cu2+, while it was partially inhibited by phenylmethanesulphonyl fluoride. The purified enzyme was shown to release the N-terminal tetrapeptide, Arg-Pro-Lys-Pro, from substance P producing the C-terminal heptapeptide, Gln-Gln-Phe-Phe-Gly-Met- CONH2. In the skin, this enzyme might be related to the inactivation of substance P.

Improvement in long-term prognosis by coronary bypass surgery in patients with 3-vessel coronary disease--a matched case control study.

We compared survival patterns in 61 medically treated and 78 surgically treated patients at a Japanese community hospital. The 2 groups were matched for presence of significant 3 vessel disease, resting ejection fraction of more than 40%, a bypassable left anterior descending artery, sex, and age. All surgical patients received saphenous vein grafts. The patients treated surgically had better 5 and 9 years survival rates than the medically treated patients (93% and 85% vs 74% and 55%, respectively; p < 0.01 by Cox-Mantel analysis). Five and 9 years rates of absence of ischemic events (non-fatal myocardial infarction and primary cardiac death) were also better in the surgical group than the medical group (92% and 87% vs 66% and 52%, respectively; p < 0.001). Of the surgically treated patients, 5 died perioperatively, 3 had late cardiac deaths and 2 had a nonfatal infarction. Among the medically treated patients, 16 had cardiac deaths, and 6 had non-fatal infarctions. Although our study was non-randomized, we have shown an advantage for surgical treatment of patients with 3-vessel coronary disease.

Stimulation of anchorage-independent cell growth by endothelin in NRK 49F cells.

Endothelin (ET) is a vasoconstrictor peptide originally isolated from vascular endothelial cells. Recent studies have revealed that ET has many biological functions including growth factor-like activity. The present study aims to clarify whether ET-1 possesses the ability to stimulate anchorage-independent cellular growth, an indicator of factors with transforming activity. We found that NRK 49F cells possess a large number of high-affinity ET-1 receptors; labeled 125I-ET-1 binding was displaced by unlabeled ET-2 in a similar dose response, but in the case of ET-3, 100-fold more was required. Specific 125I-ET-3 binding was undetectable in NRK 49F cells, indicating that ET receptors in NRK 49F cells are ET-1/ET-2 selective. NRK 49F is a cell line which is most commonly used to assay for anchorage-independent cellular growth. Therefore, we explored whether ETs promote anchorage-independent cellular growth in this cell line. ET-1 and ET-2 stimulated NRK colony formation dose dependently in the presence of 1 nM epidermal growth factor (EGF). In contrast, ET-3 did not have colony-stimulating ability. In the presence of EGF, the maximal effect of ET-1 was approximately 90% of that of transforming growth factor-beta. Moreover, in the presence of maximal stimulating concentrations of EGF and transforming growth factor-beta, ET-1 additionally induced colony formation. These results indicate that ET-1 and -2 possess transforming growth factor-like activity for NRK 49F cells. Since ET-1 and -2 increased intracellular calcium levels, this ion may participate in signal transduction pathways by which ET-1 and -2 promote colony formation.

Surface charge of fractionated guinea pig keratinocytes measured by free-flow cell electrophoresis.

Keratinocytes differentiate from basal cells to spinous, granular, and horny layer cells. It is known that alterations in the surface charge of cell membranes in most cases reflect the processes of differentiation. By using a continuous colloidal silica (Percoll) density gradient, keratinocytes may be separated into three fractions which correspond to their arrangement in vivo. Using a free-flow cell electrophoretic technique, we measured the electrophoretic mobility of guinea pig keratinocytes. Electrophoretic mobility histograms of basal and granular cells showed slow and fast monophasic patterns, respectively. In spinous cells, a biphasic pattern of slow and fast electrophoretic mobility was present. The electrophoretic mobility level of guinea pig keratinocytes was slightly reduced with neuraminidase digestion. Those of human red blood cells and lymphocytes, however, were markedly decreased. These results indicate that membrane charge density is lower in basal cells and higher in granular cells and that the membrane charge density of guinea pig keratinocytes involves not only neuraminic acid residues but also other substance(s). Our results illustrate the alterations of cell membrane charge properties during epidermal cell differentiation.

Endothelin-1 inhibits adipogenic differentiation of 3T3-L1 preadipocytes.

The effect of endothelin (ET)-1 on the adipogenic differentiation of 3T3-L1 preadipocytes was examined. Cellular morphology and lipoprotein lipase activity were used as differentiation markers. ET-1 inhibited the hormone-induced adipogenic differentiation of 3T3-L1 preadipocytes morphologically and biochemically in a dose-dependent manner. These findings promote ET-1 as a potent inhibitor of adipogenic differentiation, playing an important role in cellular differentiation of preadipocytes and making it a significant regulator of lipid metabolism.

Subepicardial microischemia formation induced by epicardial application of endothelin-1.

The effects of endothelin-1 (ET-1), applied topically on the epicardium, on the coronary microcirculation of the beating canine heart were investigated in situ. ET-1 (1, 10, 100, and 1,000 pmol) induced a dose-dependent elevation of the ST segment in the epicardial ECG (n = 5). After application of ET-1 (100 pmol), the beating hearts were rapidly cross-sectioned and freeze-clamped in 120 ms using a specially developed device (n = 6). By NADH fluorescence photography of the cross-sectioned frozen sample, an increased fluorescent area in the subepicardium was clearly observable. The fluorescent dye injected from the left atrium was negative in the NADH fluorescent area. It can be concluded that ET-1, when administered extravascularly, induces severe vasoconstriction and myocardial ischemia in the microcirculation of the beating canine heart.

Intracoronary endothelin-1 increases coronary retrograde pressure by constricting arterioles.

STUDY OBJECTIVE: The aim was to determine the site of coronary vasoconstriction induced by endothelin, by investigating the response in terms of retrograde pressure and reactive hyperaemia. EXPERIMENTAL MATERIAL: Twelve anaesthetised mongrel dogs, 12-14 kg, were used for the studies. DESIGN: The left anterior descending coronary artery was cannulated and perfused with blood through an extracorporeal bypass. The effects of intracoronary endothelin-1 (1-500 pmol) on coronary blood flow, coronary flow reserve (the peak reactive flow and the repayment after 15 s coronary occlusion), and retrograde coronary pressure during coronary occlusion were studied (n = 7). The retrograde coronary flow was collected from the bypass at each dose (n = 5). MEASUREMENTS AND MAIN RESULTS: At doses of greater than 20 pmol the coronary flow decreased dose dependently and reached almost zero flow at 500 pmol. The coronary flow reserve also decreased; however, the retrograde pressure was raised dose dependently at doses of greater than 10 pmol. At a dose of 500 pmol, the retrograde pressure was increased to 61 mm Hg [82(SEM 12)% of the coronary perfusion pressure]. Retrograde flow remained unchanged throughout the experiment. CONCLUSIONS: The endothelin-1 induced rise in retrograde pressure is in accordance with a dose dependent reduction in coronary flow reserve, and collateral flow was not augmented by endothelin. It is concluded that the effect of endothelin-1 on coronary circulation in situ was mainly due to the constriction of small resistant vessels.

Production of endothelin in human cancer cell lines.

Endothelin (ET)-1 is a vasoconstrictor peptide derived from endothelial cells and now known to be a local regulator of vascular tonus. Recent studies, however, have revealed that ET-1 functions also as growth factor in various cells. By using a specific ET-1 radioimmunoassay, immunoreactive (IR) ET-1, ranging from 4.2 to 150 pM (minimum detectable amount, 4.0 pM), was detected in 13 of 42 human cancer cell lines. The frequencies of IR-ET-1 production and its concentrations were high in mammary, pancreatic, and colon carcinoma cell lines. IR-ET-1 produced by cancer cells possessed the same molecular size as synthetic ET-1 and also had ET-1-like biological activity. Moreover, Northern blot analysis revealed bands corresponding to ET-1 mRNA in cancer cell lines, indicating that IR-ET-1 produced by cancer cells is a product of the ET-1 gene. Since ET-1 in the spent media is present in a sufficient amount to stimulate cellular growth, we sought ET-1 receptors in four pancreatic carcinoma cell lines and human skin fibroblasts. No ET-1 receptors were detected in the pancreatic carcinoma cell lines. However, human skin fibroblasts possessed a large number of ET-1 receptors. This finding raises the possibility that ET-1 produced by cancer cells plays a modulatory role in the growth of stromal cells surrounding cancer cells.