Kan Saiga

NK026680, a novel compound suppressive of dendritic cell function, ameliorates mortality in acute lethal graft-versus-host reaction in mice

A role for dendritic cells (DCs) has been emphasized in the onset of acute graft-versus-host disease (GVHD). We have made efforts to develop a new strategy for suppression of DC functions with a chemical compound in the treatment of acute GVHD. We here describe the immunological characterization of the new chemical compound NK026680. It was found that NK026680 significantly suppressed (1) expression of CD83, CD86, and major histocompatibility complex (MHC) class I and II antigens on human monocyte-derived DCs, (2) excretion of interleukin-12p40 on activation of monocyte-derived DCs, (3) allogeneic responses of human and mouse T cells and (4) mortality in mice with acute GVHD evoked across MHC class I or II. The beneficial effect of NK026680 administered orally was without any recognizable adverse effects. Early intervention in acute GVHD was required for this effect, indicating that an early event in acute GVHD is a critical target of NK026680. We propose the use of NK026680 as a prophylactic for acute GVHD.

Autoimmune-prone (NZW × BXSB)F1 (W/BF1) mice escape severe thrombocytopenia after treatment with deoxyspergualin, an immunosuppressant

Summary. Male (NZW × BXSB)F1 mice spontaneously develop a disease which closely resembles human systemic autoimmune disease, involving idiopathic thrombocytopenic purpura and glomerulonephritis. We investigated whether autoimmune thrombocytopenia in the mice responded to deoxyspergualin, as immunosuppressant. Deoxyspergualin completely prevented the development of thrombocytopenia and suppressed the increase in circulating autoantibodies against platelets. This agent also ameliorated lupus nephritis. These findings suggest that deoxyspergualin may be effective in the prevention of idiopathic thrombocytopenic purpura.

Mast cell hyperplasia in the skin of Dsg4-deficient hypotrichosis mice, which are long-living mutants of lupus-prone mice

Desmosomal cadherins are essential cell adhesion molecules expressed in the epidermis. We identified a mutation of a cadherin superfamily member, namely, desmoglein 4 (Dsg4), in early onset of death (EOD)hage mice with hypotrichosis. The mutation was induced by the insertion of an early transposon II-β into intron 8 of Dsg4. Mast cell hyperplasia was observed in the skin of EODhage mice. The abnormally expanded population of lpr T cells, i.e., CD4−CD8−B220+Thy1.2+ αβT cells, in the splenocytes of EOD mice was reduced in EODhage mice. Therefore, it was suspected that the long-living mutant EODhage mice were selected from lupus-prone EOD mice because of their immunological immaturity. These findings clearly indicate that Dsg4 is an important molecule for the formation of hair follicles and hypothesize that unorganized hyperplastic hair follicles in anagen due to the Dsg4 mutation provide niches for mast cell precursors in the skin.

A repeat biopsy study in spontaneous crescentic glomerulonephritis mice.

Background: Spontaneous crescentic glomerulonephritis (SCG)/Kj mice are a candidate for suitable animal model of human pauci-immune crescentic glomerulonephritis (GN). In the present study, we used renal biopsy technique and analyzed time sequence correlations among crescent formation and glomerular neutrophil infiltration in SCG/Kj mice. Methods: We observed the progress of GN in SCG/Kj mice according to the urinary abnormalities, and performed the serial renal biopsies. The kinetics of histopathology and glomerular neutrophil influx corresponding disease stage were examined by enzyme-histochemistry and immunohistochemistry. Results: We divided natural course of GN into three periods in view of urinalysis: a proteinuria-negative/hematuria-negative (P− H−) period, followed by a proteinuria-positive/hematuria-negative (P+ H−) period, and finally a proteinuria-positive/hematuria-positive (P+ H+) period. Endocapillary proliferation phase existed in P+ H− period, whereas crescent formation occurred and extended in P+ H+ period. In P+ H− period, prominent glomerular neutrophil infiltration was observed, while these numbers decreased with the progression of crescent formation. Conclusion: These observations suggest that there is a good correlation between urinalysis and histopathological events of SCG/Kj mice, and that endocapillary proliferation, which contains neutrophil infiltration, may contribute to the subsequent crescent formation in these mice.

Preparation and partial characterization of monoclonal antibodies specific for the nascent non-triple helical form of the type IV collagen alpha 1 chain

This report describes the preparation and partial characterization of monoclonal antibodies that are reactive specifically with the nascently produced non-triple helical form of the type IV collagen α1 chain, designated as NTH α1(IV). These antibodies were nonreactive with the α1 chain of the type IV collagen in the triple-helical conformation. Three antibodies, #141, #179 and #370, with different epitopes in NTH α1(IV) were found to be reactive with the nascent polypeptide secreted from human normal cells and a human carcinoma cell line. The antibodies with different epitopes may provide a key method for elucidating the physiological function and tissue distribution of NTH α1(IV), which is distinct from the chain derived from triple-helical type IV collagen.

Long-lasting immunosuppressive effects of tacrolimus-loaded micelle NK61060 in preclinical arthritis and colitis models

AIM Tacrolimus (TAC) is an important drug for inflammatory diseases. However, TAC has several limitations, such as variable trough concentrations among individuals and a high medication frequency. In this study, we created NK61060, a novel micellar TAC formulation, to circumvent these disadvantages. MATERIALS & METHODS Immunosuppressive activity of NK61060 was determined in the collagen-induced arthritis rat model, mannan-induced arthritis mouse model and dextran sodium sulfate-induced colitis mouse model. The pharmacokinetics and toxicology of NK61060 were evaluated in those models. RESULTS In arthritis and colitis models, NK61060 exhibited superior immunosuppressive activity compared with that of TAC. Pharmacokinetic and toxicological analyses indicated that NK61060 had a wider safety margin and could be administered at a reduced medication frequency. CONCLUSION NK61060 mitigates the trough concentration variability and the medication frequency and it may be a safer and more effective option for use in clinical settings. Further studies are needed to determine its clinical usefulness.

Identification of a common epitope in the sequences of COL4A1 and COL6A1 recognised by monoclonal antibody #141

Identification of a type IV collagen α1 polypeptide in non-triple helical form [NTH α1(IV)], possibly involved in angiogenesis, introduces the further possibility of the existence of non-triple helical forms of other collagen chains. We previously reported that an anti-NTH α1(IV) monoclonal antibody #141 recognizes not only NTH α1(IV) but also a novel non-triple helical collagen polypeptide NTH α1(VI) encoded by COL6A1. In this study, we identified the recognition sequence in order to better understand the properties of antibody #141 and provide clues regarding the biological function of the two non-triple helical molecules. Additionally, we determined the common epitope between COL4A1 and COL6A1 as PXXGXPGLRG, with surface plasmon resonance analyses revealing KD values for the COL4A1 epitope as 5.56±1.81×10-9 M and for the COL6A1 epitope as 7.15±0.44×10-10 M. The specific recognition of NTH α1(IV) and NTH α1(VI) by antibody #141 can be explained by the common epitope sequence. Moreover, epitope localization supports previous finding that NTH α1(IV) and NTH α1(VI) differ in conformation from the α1 chains in triple-helical type IV and type VI collagen. These findings suggest that antibody #141 might be useful for diagnosis of type VI collagen myopathies.

Type VI collagen α1 chain polypeptide in non-triple helical form is an alternative gene product of COL6A1

Expression of type IV collagen α1 chain in non-triple helical form, NTH α1(IV), is observed in cultured human cells, human placenta and rabbit tissues. Biological functions of NTH α1(IV) are most likely to be distinct from type IV collagen, since their biochemical characteristics are quite different. To explore the biological functions of NTH α1(IV), we prepared some anti-NTH α1(IV) antibodies. In the course of characterization of these antibodies, one antibody, #141, bound to a polypeptide of 140 kDa in size in addition to NTH α1(IV). In this study, we show evidence that the 140 kDa polypeptide is a novel non-triple helical polypeptide of type VI collagen α1 chain encoded by COL6A1, or NTH α1(VI). Expression of NTH α1(VI) is observed in supernatants of several human cancer cell lines, suggesting that the NTH α1(VI) might be involved in tumourigenesis. Reactivity with lectins indicates that sugar chains of NTH α1(VI) are different from those of the α1(VI) chain in triple helical form of type VI collagen, suggesting a synthetic mechanism and a mode of action of NTH α1(VI) is different from type VI collagen.

Abstract 557: Single-chain type IV collagen (SCCOL-IV), an alternative gene product of COL4A1 as a therapeutic target in tumor tissue expansion as well as a diagnostic marker

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Type IV collagen (COL-IV) is a major component of basement membrane (BM), comprising a polygonal meshwork structure underlying the basal side of polarized epithelia in normal tissues. Modulation of BM due to COL-IV degradation by matrix metalloproteinases is often associated with tumor aggravation and metastasis; Degradation products of BM components, including laminin and type IV collagen e.g., are involved in promoting tumor cell motility. Our previous studies showed that cultured human cells, in particular under ascorbate-deficiency, secrete single-chain COL-IV (SCCOL-IV), which is not derived from the denatured polypeptide of COL-IV triple-helical molecule. SCCOL-IV undergoes posttranslational modifications distinct from triple-helical COL-IV; SCCOL-IV has prolyl- and lysyl-hydroxylation to a lower extent than triple-helical COL-IV. SCCOL-IV contains the O-glycan structure of Galβ1-3GalNAc with a positive reaction to Agaricus bisporus agglutinin. The lectin does not recognize the triple-helical COL-IV-derived polypeptide. The alternative form of COL4A1 gene product is detected in vivo by the antibody that recognizes only SCCOL-IV. The polypeptide is isolated from human placenta as well as cultured media. In the present study, we demonstrate that SCCOL-IV is expressed specifically by cultured tumor cells and in tumor tissues. One of the antibodies against SCCOL-IV repressed tumor growth in an in vivo mouse model. The culture cell production of SCCOL-IV was demonstrated by Western blotting of cultured media, with an anti-SCCOL IV antibody, JK132 (Connective Tissue 31:161-168, 1999) for human cancer cell lines; Lu65A and NCI-H460 (pulmonary cancer), HLF (hepatoma), UO31 and A498 (renal cancer), and Panc-1 (pancreatic cancer). SCCOL-IV expression level was as high in tumor tissues of Lu65A-bearing nude mice as in tumor lesions of pulmonary cancer patients. One of the monoclonal antibodies against purified SCCOL-IV, NK46141, which exhibits a higher affinity compared with JK132, has no cross-reactivity with triple-helical COL-IV. Prophylactic interventions with NK46141 suppressed tumor growth in Lu65A-bearing nude mice. NK46141 had no effect, however, on the proliferation of cultured Lu65A cells. In order to obtain a clue for a mechanism how the antibody suppresses in vivo tumor growth, biological properties of SCCOL-IV in tumor interstitium were examined. SCCOL-IV was localized predominantly in vessel lumens in an in vitro vasculogenesis model, suggesting that SCCOL-IV may play a critical role for vasculogenesis in tumor progression. Prevention tumor growth by the antibody might be due to vasculogenesis inhibition. We propose that SCCOL-IV is a useful target in tumor diagnosis and therapy. Anti-SCCOL-IV antibodies including NK46141 could be potential therapeutics for tumor progression involving vasculogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 557. doi:10.1158/1538-7445.AM2011-557