A quinazoline-based bromodomain inhibitor, CN210, ameliorates indomethacin-induced ileitis in mice by inhibiting inflammatory cytokine expression.

Inhibitors of bromodomain and extra-terminal motif (BET) proteins are emerging epigenetic therapeutics that suppress gene expressions that drive cancer and inflammation. The present study examined anti-inflammatory effects of a quinazoline-based BET inhibitor, CN210, in a murine ileitis model. CN210 was given orally 30 min before and 24 h after a subcutaneous administration of indomethacin. Macroscopic and histological evidences of ileitis, mucosal myeloperoxidase (MPO) activity and cytokine expressions were evaluated 48 h after the indomethacin administration. To further characterize the anti-inflammatory pathways modulated by CN210, its effects on RAW264 cells treated with lipopolysaccharide (LPS) were investigated. Competitive ligand binding and docking studies of CN210 to CREB-binding protein (CBP) and p300 were also performed. Oral administration of CN210 significantly reduced the severity of ileitis, normalized both proinflammatory MPO activity and concomitant cytokine expressions induced by indomethacin administration. Furthermore, CN210 attenuated the expression of cytokines and reversed the activation of nuclear factor κB (NF-κB) and mitogen-activated protein kinases (MAPK) induced by LPS. Competitive ligand binding assays showed that CN210 bound to the bromodomains of two paralogous histone acetyltransferases, CBP and p300, in addition to the bromodomains of BET proteins. Docking studies of CN210 to the bromodomains of CBP and p300 showed a similarity to the binding mode of SGC-CBP30, a specific CBP/p300 inhibitor. CN210 ameliorates indomethacin-induced ileitis by inhibiting the expression of inflammatory cytokines through the attenuation of NF-κB and MAPK pathways. CN210 thus represents a new mode of therapy for non-steroidal anti-inflammatory drug-induced ileitis and inflammatory bowel disease.

Successful treatment of lipoprotein glomerulopathy in a daughter and a mother using niceritrol.

We report two patients, a daughter and a mother, with lipoprotein glomerulopathy (LPG) who were successfully treated with niceritrol. Both patients carried a mutation in the apolipoprotein E (apoE) gene known as ApoE Tokyo/Maebashi. The daughter was found to have proteinuria at the age of 4 years. Four years later, she was diagnosed as having LPG based on a renal biopsy. She was treated with several medications including pravastatin, ethyl icosapentate, enalapril, warfarin and cyclophosphamide, all of which failed to reduce her proteinuria. At the age of 17 years, she exhibited an increase in proteinuria and a decline in renal function, despite ongoing treatment with pravastatin and enalapril. After switching from pravastatin to niceritrol, a marked reduction in the proteinuria and an improvement in renal function were observed. Her mother was found to have proteinuria at the age of 57 years and was diagnosed as having LPG based on a renal biopsy. She was also treated with niceritrol, resulting in an improvement in her proteinuria and renal function. These cases suggest that niceritrol might be a useful therapeutic option for LPG.

Improved management of intradialytic hypotension (IDH) using vitamin E-bonded polysulfone membrane dialyzer.

BACKGROUND: Intradialytic hypotension (IDH) is a common clinical trait in hemodialysis (HD) which is caused by poor biocompatibility of the dialyzer membrane. Aiming to improve IDH, vitamin E-bonded polysulfone dialyzer (VPS-H) was evaluated in a pilot study. METHODS: Eight IDH patients on standard HD were switched from their conventional high-flux dialyzers to VPS-H, and intradialytic blood pressure (BP) was monitored regularly for 10 months. RESULTS: The results showed that hypotension of systolic BP (SBP), diastolic BP (DBP) and pulse pressure (PP) during the session were improved after changing the dialyzer. Notably, almost all the values recorded from 120 minutes into the session until the end of the treatment in the period between the second and tenth month after treatment were significantly different from the corresponding baseline values. Moreover, after 8 to 10 months, the SBP prior to a dialysis session was significantly reduced compared with baseline values. On the other hand, the pulse rate showed no difference throughout the study period. CONCLUSIONS: This study provides early evidence of the beneficial role that vitamin E-bonded dialyzers may have in preventing IDH. Larger controlled trials are needed to confirm this original finding.

Pure red cell aplasia accompanied by autoimmune hemolytic anemia in a patient with type A viral hepatitis.

A rare case of acute hepatitis A associated with autoimmune hemolytic anemia (AIHA) and pure red cell aplasia (PRCA) is reported. A 55-year-old woman consulted a doctor because of common cold-like symptoms and she was referred to our hospital in January 2007. Laboratory findings showed a marked elevation of serum transaminase and total bilirubin levels (AST 9,605 IU/l, ALT 5,546 IU/l and T-bil 4.14 mg/dl), and prolonged prothrombin time, findings which suggested the risk of progression to fulminant hepatitis, and she was treated with plasmapheresis and hemodialysis filtration on the first and second hospital days. She was diagnosed with severe acute hepatitis A based on the elevation of serum IgM anti-hepatitis A virus. On the 20th hospital day, her hemoglobin level began to decrease in spite of improving transaminase levels without any signs of gastrointestinal bleeding. Bilirubin and LDH elevation, haptoglobin decline and a positive direct Coombs test were detected and these findings indicated AIHA complication; however, the reticulocyte count decreased and bone marrow showed marked erythroid hypoplasia so the co-existence of PRCA was diagnosed. After oral prednisolone administration (1 mg/kg/day), her hemolytic anemia rapidly improved.

Clinical implications of idiopathic multicentric castleman disease among Japanese: a report of 28 cases.

To clarify the clinicopathologic findings of idiopathic multicentric Castleman disease among Japanese, 28 cases were studied. Two variants were delineated by the clinicopathologic findings (1) idiopathic plasmacytic lymphadenopathy with polyclonal hyperimmunoglobulinemia (n = 18) and (2) nonidiopathic plasmacytic lymphadenopathy type (n= 10). Clinicopathologically, idiopathic plasmacytic lymphadenopathy was defined by the prominent polyclonal hyperimmunoglobulinemia, normal germinal centers, and sheet-like infiltration of plasma cells in the interfollicular area of the lymph node. Histologically, the nonidiopathic plasmacytic lymphadenopathy type was characterized by hyaline-vascular germinal centers of the lymph node lesion. In comparison with idiopathic plasmacytic lymphadenopathy, patients with nonidiopathic plasmacytic lymphadenopathy showed infrequent prominent polyclonal hyperimmunoglobulinemia and frequent association with autoimmune disease. However, there was no difference in the overall 5-year survival between the 2 subtypes. Compared with idiopathic multicentric Castleman disease in Western countries, the chronic course of the disease of idiopathic multicentric Castleman disease in Japan appears to be related to negativity for human herpesvirus 8 infection.

The c-Cbl/CD2AP complex regulates VEGF-induced endocytosis and degradation of Flt-1 (VEGFR-1).

Vascular endothelial growth factor (VEGF) and its receptors are key regulators of angiogenesis and are potential targets in cancer therapy. Here we report the down-regulation of activated VEGF receptor (VEGFR)-1/Flt-1 by endocytosis and proteolytic degradation. VEGF stimulation induced a ternary complex of Flt-1, c-Cbl, and CD2AP. Substitution of tyrosine 1333 in Flt-1 with phenylalanine (Y1333F) impaired its binding to c-Cbl. In a transient expression system, VEGF stimulated colocalization of Flt-1, CD2AP, and c-Cbl in endocytic vesicles. This colocalization was significantly impaired by an inhibitor of VEGFR kinase SU5416, the Y1333F mutation in Flt-1, or by a dominant negative form of CD2AP. In Flt-1-overexpressing NIH3T3 cells, expression of the wild-type CD2AP enhanced VEGF-stimulated internalization as well as ubiquitination of Flt-1 whereas that of a mutated form of either CD2AP or c-Cbl failed to do so. These results suggest that the c-Cbl/CD2AP complex binds to activated Flt-1 and plays a crucial role in its endocytosis and subsequent degradation.

Clinical characteristics of asymptomatic hepatitis B virus carriers with YMDD mutant not treated with lamivudine.

We previously developed a method of detecting drug-resistant mutation of hepatitis B virus (tyrosine (Y)-methionine (M)-aspartic acid (D)-aspartic acid (D) (YMDD) mutation) caused by the antiviral agent lamivudine. Using this method, we also reported that YMDD mutation is present in asymptomatic carriers that had not been administered antiviral agents. Thus, we investigated the clinical characteristics of 18 asymptomatic carriers of hepatitis B virus by various biochemical and virological examinations, and compared the results between five subjects with YMDD mutation and 13 subjects without mutation. Although there was no significant difference in the results of various examinations between the two groups, the quantity of virus was generally small, and S-antigen disappeared in 2 patients in whom YMDD mutation was detected. These results suggest that there is no particular cause for the appearance of mutant viruses, but that they occur spontaneously as they gain fitness and, with a subsequent decrease in the absolute quantity of viruses, become relatively easy to detect.

Atypical lymphoplasmacytic and immunoblastic proliferation of autoimmune disease : clinicopathologic and immunohistochemical study of 9 cases.

Atypical lymphoplasmacytic immunoblastic proliferation (ALPIB) is a rare lymphoproliferative disorder (LPD) associated with autoimmune disease (AID). To further clarify the clinicopathologic, immunohistological, and genotypic findings of ALPIB in lymph nodes associated with well-documented AIDs, 9 cases are presented. These 9 patients consisted of 4 patients with systemic lupus erythematosus, 3 patients with rheumatoid arthritis, and one case each with Sjögren's syndrome and dermatomyositis. All 9 patients were females aged from 25 to 71 years with a median age of 49 years. Four cases presented with lymphadenopathy as the initial manifestation. In 4 patients, immunosuppressive drugs were administered before the onset of lymph node lesion. However, none of the 9 patients received methotrexate therapy. The present 9 cases were characterized by : (i) prominent lymphoplasmacytic and B-immunoblastic infiltration ; (ii) absence of pronounced arborizing vascular proliferation ; (iii) absence of CD10(+) "clear cells" ; (iv) presence of hyperplastic germinal center in 7 cases ; (v) immunohistochemistry, flow cytometry, and polymerase chain reaction demonstrated a reactive nature of the T- and B-lymphocytes ; and (vi) on in situ hybridization, there were no Epstein-Barr virus -infected lymphoid cells in any of the 9 cases. Overall 5-year survival of our patients was 83%. The combination of clinical, immunophenotypic, and genotypic findings indicated that the present 9 cases can be regarded as having an essentially benign reactive process. Finally, we emphasized that ALPIB should be added to the differential diagnostic problems of atypical LPDs, particularly lymph node lesions of IgG4-related diseases.

Mesangial cells stimulate differentiation of endothelial cells to form capillary-like networks in a three-dimensional culture system.

BACKGROUND: There are conflicting results regarding the role of periendothelial mural cells in angiogenesis. In the current study, we investigated the role of mesangial cells (MCs) in endothelial vascularization by using a three-dimensional co-culture system in basement-membrane reconstruct gel (Matrigel). METHODS: Human umbilical vein endothelial cells (ECs) and human MCs were used. In the contact co-culture system, ECs and MCs were mixed and then plated together onto Matrigel. In the non-contact co-culture system, MCs were cultured within an intercup chamber, which prevented direct physical contact with the ECs on the Matrigel but allowed both cell types to share culture medium. To visualize ECs and MCs, the cells were labelled with two different fluorescent dyes prior to the co-culture. A capillary-like network formation was observed under a fluorescent microscope and confocal microscope, and the length of the network formation was quantified by the image analyzer. RESULTS: ECs barely formed capillary-like networks when cultured alone in growth factor-free medium. However, ECs cultured with MCs in a contact condition remarkably formed capillary-like networks (9.10+/-0.96 vs 0.20+/-0.07 mm/mm2 at 6 h, contact vs ECs alone, P<0.001). Direct contact between ECs and MCs was clearly demonstrated by confocal microscopy. Differentiation into branching capillary-like structures was also observed in the non-contact co-culture system (3.02+/-1.21 mm/mm2 at 6 h, P<0.001 vs ECs alone), but less prominently than in the contact co-culture condition. Vascular endothelial growth factor (VEGF) was secreted from MCs, as determined by enzyme-linked immunosorbent assay and immunofluorescent study. Adding neutralizing antibodies against VEGF into the co-culture system partially inhibited capillary network formation. CONCLUSIONS: Our data indicate that MCs help ECs differentiate toward vascularization, in which the direct cell-cell contact between ECs and MCs plays an important role. VEGF is a mediator in this process.

Nox1 regulates apoptosis and potentially stimulates branching morphogenesis in sinusoidal endothelial cells.

Tubulogenic transformation of a nontubulogenic endothelial cell line NP31 by a constitutively activated form of the Flt-1 kinase (NP31/kinase) was accompanied by an increased expression of Nox1 by sixfold over NP31. Overexpression of Nox1 in NP31 cells (NP31/Nox1) stimulated branching morphogenesis in Matrigel but surprisingly cords lacked a lumen. The branching morphogenesis by NP31/kinase and NP31/Nox1 cells was blocked either by N-acetyl-l-cysteine (NAC) or Tiron. Vascular endothelial growth factor (VEGF)-dependent sinusoidal endothelial cells (SEC) in primary culture showed fivefold increase in Nox1 expression 4 days after VEGF stimulation. Interestingly, VEGF-resistant apoptosis in SEC at day 7 was inhibited by NAC or by anti-Nox1 siRNA. These results suggest that Nox1 regulates apoptosis in SEC and can potentially stimulate branching morphogenesis in SEC-derived NP 31 cells.

Dynamic regulation of gene expression by the Flt-1 kinase and Matrigel in endothelial tubulogenesis.

A nontubulogenic endothelial cell line, NP31, can be transformed by the active form of the Flt-1 kinase (BCR-FLTm1) into Tb3 cells, which show a tubulogenic property only when cultured in Matrigel. By utilizing this strict dependence of NP31 on BCR-FLTm1 and Matrigel for experimental angiogenesis, we performed microarray analyses under several conditions and found 97 genes whose dynamically regulated profiles of gene expression are divided into nine groups, in two major clusters. In one major cluster, gene expression is interdependently regulated by BCR-FLTm1 or Matrigel. The second major cluster contains genes whose expression patterns under BCR-FLTm1 influence are reversed by Matrigel. Based on these gene expression patterns in NP31 driven by BCR-FLTm1 and/or Matrigel, we propose a model in which sequential and alternate stimulation by BCR-FLTm1 and Matrigel induces cooperative regulation of subsets of genes. Microarray analyses of Tb3 under 11 different conditions revealed 5 candidate genes whose gene expression regulation is most closely associated with tubulogenesis.