Novel Complement C5 Small-interfering RNA Lipid Nanoparticle Prolongs Graft Survival in a Hypersensitized Rat Kidney Transplant Model.

BACKGROUND: Prophylaxis of antibody-mediated rejection (AMR) caused by donor-specific antibodies remains challenging. Given the critical roles of complement activity in antibody-mediated graft injury, we developed a lipid nanoparticle (LNP) formulation of small-interfering RNA against complement C5 (C5 siRNA-LNP) and investigated whether C5 siRNA-LNP could downregulate the complement activity and act as an effective treatment for AMR. METHODS: Lewis recipient rats were sensitized by skin grafting from Brown Norway donor rats. Kidney transplantation was performed at 4 wk post-skin grafting.C5 siRNA- or control siRNA-LNP was administered intravenously, and the weekly injections were continued until the study's conclusion. Cyclosporin (CsA) and/or deoxyspergualin (DSG) were used as adjunctive immunosuppressants. Complement activity was evaluated using hemolysis assays. The deposition of C5b9 in the grafts was evaluated using immunohistochemical analysis on day 7 posttransplantation. RESULTS: C5 siRNA-LNP completely suppressed C5 expression and complement activity (hemolytic activity ≤ 20%) 7 d postadministration. C5 siRNA-LNP in combination with CsA and DSG (median survival time: 56.0 d) prolonged graft survival compared with control siRNA-LNP in combination with CsA and DSG (median survival time: 21.0 d; P = 0.0012; log-rank test). Immunohistochemical analysis of the grafts revealed that downregulation of C5 expression was associated with a reduction in C5b9-positive area ( P = 0.0141, Steel-Dwass test). CONCLUSIONS: C5 siRNA-LNP combined with immunosuppressants CsA and DSG downregulated C5 activity and significantly prolonged graft survival compared with control siRNA-LNP with CsA and DSG. Downregulation of C5 expression using C5 siRNA-LNP may be an effective therapeutic approach for AMR.

Role of Fractalkine-CX3CR1 Axis in Acute Rejection of Mouse Heart Allografts Subjected to Ischemia Reperfusion Injury.

Transplantation outcomes are affected by the increase in rejection associated with ischemia reperfusion injury (IRI). Fractalkine (FKN), a chemokine for recruitment of CX3CR1+ leukocytes, contributes to the pathogenesis of various inflammatory diseases. Herein, we evaluated the importance of the FKN-CX3CR1 axis during IRI-related rejections using a mouse heterotopic heart transplantation model. FKN expression and graft survival was compared between wild-type C57BL/6 recipients transplanted with BALB/c hearts preserved for 8 (WT-IRI) and 0.5 h (WT-control) at 4°C. Graft survival of WT-IRI was shorter than that of WT-control. FKN was expressed on the vascular endothelium in WT-IRI allografts, but minimally in WT-control. The role of the FKN-CX3CR1 axis in IRI-related rejection was directly investigated using the transplant model with CX3CR1-deficient recipients (CX3CR1 KO-IRI) or treatment with anti-mouse FKN monoclonal antibodies. Graft survival of CX3CR1 KO-IRI was longer than that of WT-IRI; antibody treatment prolonged graft survival. The contribution of CX3CR1+ monocytes to IRI-related rejection was evaluated by adoptive transfer to CX3CR1 KO-IRI. Adoptive transfer of CX3CR1+ monocytes attenuated the effect of prolonged graft survival in CX3CR1 KO-IRI. Overall, the FKN-CX3CR1 axis plays a major role during IRI-related rejection; its blockade has the potential to improve the outcomes of deceased donor transplantation.

Triazolopyrimidine derivative NK026680 and donor-specific transfusion induces CD4+CD25+Foxp3+ T cells and ameliorates allograft rejection in an antigen-specific manner.

We have previously demonstrated the unique properties of a new triazolopyrimidine derivative, NK026680, which exerts immunosuppressive effects in rat heart transplant model and confers tolerogeneic properties on ex vivo-conditioned dendritic cells in mice. We herein demonstrate that NK026680 promotes the expansion of regulatory T cells (Tregs) with potent immunoregulatory effects when used in combination with donor-specific transfusion (DST). BALB/c (H-2d) heart graft were transplanted into C57BL/6 (H-2b) mice following intravenous injection of donor splenocytes (DST) and oral administration of NK026680. The NK026680 plus DST treatment markedly prolonged the survival time of the donor-graft, but not that of the 3rd party-graft (C3H; H-2k). Treg cells in the recipient spleen on day 0 expanded when stimulated with donor-antigens in vivo and in vitro. After heart transplantation, Treg cells accumulated into the graft and increased in the spleen. NK026680 plus DST also decreased activated CD8+ T cells in the spleen and inhibited infiltration of CD8+ T cells into the graft. Depletion of CD25+ cells inhibited the graft prolonging effect of the NK026680 plus DST treatment. NK026680 administration together with DST induces potent immunoregulatory effects in an antigen-specific manner, likely due to the in vivo generation of donor-specific Tregs.

Involvement of non­triple helical type VI collagen α1 chain, NTH α1(VI), in the proliferation of cancer cells.

It has been reported that a polypeptide encoded by collagen type VI alpha 1 chain (COL6A1), one of the three α chains of type VI collagen, is strongly associated with the migration and invasion of highly metastatic human pancreatic cancer BxPC­M8 cells and excessive proliferation of LNCaP cells. We previously reported that non­triple helical type VI collagen α1 chain, NTH α1(VI), a non­triple helical polypeptide encoded by COL6A1, is not derived from type VI collagen and exists in cancer cell­conditioned media. Therefore, NTH α1(VI) may be involved in cancer cell migration, invasion, and proliferation. The active entity that promotes cellular behaviors in cancer remains unclear. Thus, we predicted that NTH α1(VI) has cancer­promoting activity, such as the ability to induce cell proliferation. This study was conducted to examine whether NTH α1(VI) and/or its derived peptides are involved in cancer cell proliferation. Highly metastatic human pancreatic S2­VP10 cells were used to explore the potential of COL6A1 knockdown in reducing cell proliferation. Moreover, S2­VP10 conditioned medium was assessed after molecular size­fractionation to determine whether the inhibitory effect of COL6A1 knockdown could be rescued by the medium. We showed that S2­VP10­conditioned medium contained COL6A1 polypeptide, but not COL6A2, suggesting that COL6A1 in the conditioned medium of S2­VP10 cells reflects the presence of NTH α1(VI). COL6A1 knockdown repressed S2­VP10 cell proliferation and this repression was rescued using the conditioned medium of S2­VP10 cells. The fraction of conditioned medium containing peptides smaller than 10 kDa rescued the inhibitory effect; however, the fraction containing polypeptides larger than 10 kDa, including NTH α1(VI), did not show rescue activity, indicating that NTH α1(VI) fragmentation is necessary for enhanced cancer cell proliferation. In conclusion, fragmentation of NTH α1(VI) into peptides <10 kDa is required for its cancer cell proliferation­promoting activity.

Combination therapy of an iNKT cell ligand and CD40-CD154 blockade establishes islet allograft acceptance in nonmyeloablative bone marrow transplant recipients.

AIMS: Islet transplantation is an effective therapeutic option for type 1 diabetes. Although maintenance immunosuppression therapy is required to prevent allogeneic rejection and recurrence of autoimmunity, long-term allograft survival has not yet been achieved partly because of its adverse effects. The induction of donor-specific immunotolerance is a promising approach for long-term allograft survival without maintenance immunosuppression therapy. We previously reported that combination therapy using a liposomal ligand for invariant natural killer T cells, RGI-2001, and anti-CD154 antibody established mixed hematopoietic chimerism for the induction of donor-specific immunotolerance. This study investigated whether the protocol could promote islet allograft acceptance in experimental diabetes. METHODS: Streptozotocin-induced diabetic BALB/c mice were transplanted with bone marrow cells from C57BL/6 donors and received combination therapy of RGI-2001 and anti-CD154 antibody after 3-Gy total body irradiation. 3 Weeks after bone marrow transplantation, islets isolated from C57BL/6 donors were transplanted under the kidney capsule. RESULTS: Mixed chimerism was established in diabetic mice receiving the tolerance induction protocol. After islet transplantation, blood glucose levels improved and normoglycemia persisted for over 100 days. Hyperglycemia recurred after islet grafts were removed. Histopathological examinations showed insulin-positive staining and absence of cellular infiltration in the islet grafts. T cells of recipients showed donor-specific hyporesponsiveness, and anti-donor antibodies were not detected. CONCLUSIONS: The tolerance induction protocol with combination therapy of RGI-2001 and anti-CD154 antibody promoted islet allograft acceptance in a mouse diabetic model. This protocol may be clinically applied to islet transplantation for type 1 diabetes mellitus.

Identification of a common epitope in the sequences of COL4A1 and COL6A1 recognized 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.

Impact of activated invariant natural killer T cells on the expansion of regulatory T cell precursors in murine thymocytes in vitro.

Tolerance induction is a goal of clinical transplantation to prevent graft rejection without the lifelong use of immunosuppressive drugs. In a series of mouse studies, we previously reported that the establishment of mixed chimerism by treatment with a ligand for invariant natural killer T (iNKT) cells with CD40 signal blockade makes it possible to prevent allograft rejection without immunosuppressants, and this approach fails in thymectomized recipient mice. In this study, we showed that iNKT cells in murine thymocyte cultures are indispensable for the expansion of CD4+CD25+Foxp3+ regulatory T (Treg) cells as well as CD4+CD25+Foxp3- cells, which contained precursor Tregs (preTregs). After the culture of BALB/c mouse-derived thymocytes in the presence of α-galactosylceramide (α-GalCer), a representative ligand for iNKT cells, the ratio of CD4+CD25+Foxp3- preTregs to total CD4+CD8- T cells was much higher than that of CD4+CD25+Foxp3+ Treg cells, regardless of anti-CD40 L mAb treatment. The proliferation of CD4+CD25+Foxp3- cells, but not Treg cells, was significantly augmented, and the stability of Treg cells was not affected by α-GalCer. The expansion of thymocyte-derived Tregs was not inhibited by cytokine neutralization. However, in vitro thymus-derived CD4+CD25+Foxp3- cells expressed Foxp3 after IL-2 stimulation in a dose-dependent manner. These results collectively suggest that in vitro thymus-derived Treg cell expansion by α-GalCer treatment was caused by the proliferation of CD4+CD25+Foxp3- preTregs but not existing Treg cells.

Evaluation of the impact of conventional immunosuppressant on the establishment of murine transplantation tolerance - an experimental study.

Regulatory T cells (Tregs) play a significant role in immune tolerance. Since Treg function deeply depends on Interleukin-2 signaling, calcineurin inhibitors could affect their suppressive potentials, whereas mammalian target of rapamycin (mTOR) inhibitors may have less impact, as mTOR signaling is not fundamental to Treg proliferation. We previously reported a novel mixed hematopoietic chimerism induction regimen that promotes Treg proliferation by stimulating invariant natural killer T cells under CD40 blockade. Here, we use a mouse model to show the impact of tacrolimus (TAC) or everolimus (EVL) on the establishment of chimerism and Treg proliferation in the regimen. In the immunosuppressive drug-dosing phase, peripheral blood chimerism was comparably enhanced by both TAC and EVL. After dosing was discontinued, TAC-treated mice showed gradual graft rejection, whereas EVL-treated mice sustained long-term robust chimerism. Tregs of TAC-treated mice showed lower expression of both Ki67 and cytotoxic T lymphocyte antigen-4 (CTLA-4), and lower suppressive activity in vitro than those of EVL-treated mice, indicating that TAC negatively impacted the regimen by interfering with Treg proliferation and activation. Our results suggest that the usage of calcineurin inhibitors should be avoided if utilizing the regimen to induce Tregs in vivo for the establishment of mixed hematopoietic chimerism.

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.

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.

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.

Immunomodulation with eicosapentaenoic acid supports the treatment of autoimmune small-vessel vasculitis.

Small-vessel vasculitis is a life-threatening autoimmune disease that is frequently associated with anti-neutrophil cytoplasmic antibodies (ANCAs). Conventional immunotherapy including steroids and cyclophosphamide can cause serious adverse events, limiting the efficacy and safety of treatment. Eicosapentaenoic acid (EPA), a key component of fish oil, is an omega-3 polyunsaturated fatty acid widely known to be cardioprotective and beneficial for vascular function. We report two elderly patients with systemic ANCA-associated vasculitis (AAV) in whom the administration of EPA in concert with steroids safely induced and maintained remission, without the use of additioal immunosuppressants. To explore the mechanisms by which EPA enhances the treatment of AAV, we employed SCG/Kj mice as a spontaneous murine model of AAV. Dietary enrichment with EPA significantly delayed the onset of crescentic glomerulonephritis and prolonged the overall survival. EPA-derived anti-inflammatory lipid mediators and their precursors were present in the kidney, plasma, spleen, and lungs in the EPA-treated mice. Furthermore, a decrease in ANCA production and CD4/CD8-double negative T cells, and an increase in Foxp3(+) regulatory T cells in the lymph nodes of the kidney were observed in the EPA-treated mice. These clinical and experimental observations suggest that EPA can safely support and augment conventional therapy for treating autoimmune small-vessel vasculitis.

Direct activation of glomerular endothelial cells by anti-moesin activity of anti-myeloperoxidase antibody.

BACKGROUND: Glomerular neutrophil infiltration has been thought to be a key pathological event in the development of myeloperoxidase (MPO)-specific anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis involving glomerulonephritis. Accordingly, we sought to explore the molecules responsible for glomerular neutrophil accumulation. METHODS: Glomerular neutrophil infiltration and renal chemokine expression in mice treated with anti-MPO IgG were evaluated. Chemokine expression in vitro induced by anti-MPO IgG was measured in the primary mouse glomerular endothelial cells (mGEC). The target molecule reacted with anti-MPO IgG on the mGEC was determined by peptide mass fingerprint analysis. RESULTS: A significant glomerular neutrophil infiltration was observed in the mice administered with anti-MPO IgG. The expressions of CXC chemokines, keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2 (MIP-2), were significantly increased in the renal cortex, indicating that these chemokines contribute to the neutrophil infiltration. Based on the previous findings of upregulation of adhesion molecule expression in mGEC treated with anti-MPO IgG, we examined whether mGEC secrete these chemokines in response to anti-MPO IgG. Indeed, anti-MPO IgG induced secretion of KC and MIP-2, leading to neutrophil chemotaxis in vitro. Furthermore, complete depletion of MPO in mGEC and serum using MPO-deficient mice showed an upregulation of intercellular adhesion molecule-1, indicating cross-reactive molecule(s) were existing on mGEC. We identified the molecule as moesin by a proteomic approach. CONCLUSIONS: The endothelial CXC chemokines, KC and MIP-2, contribute to infiltration of neutrophils in MPO-ANCA-associated vasculitis involving glomerulonephritis. The activation of glomerular endothelial cells by anti-MPO IgG appeared to directly involve a signaling through moesin.

Reduction of MPO-ANCA epitopes in SCG/Kj mice by 15-deoxyspergualin treatment restricted by IgG2b associated with crescentic glomerulonephritis.

OBJECTIVES: The MPO-specific antineutrophil cytoplasmic antibodies (MPO-ANCA) are associated with renal failure. Epitopes of MPO-ANCA and immunoglobulin G (IgG) subclass and cytokine levels in the recovery phase were analysed by the administration of 15-deoxyspergualin (DSG) to SCG/Kj mice, which show spontaneous crescentic GN (CrGN). METHODS: We treated SCG/Kj mice by using DSG and MPO deletion mutants to investigate epitopes of MPO-ANCA associated with renal failure in SCG/Kj mice. After DSG treatment for 30 days, we observed histological changes in a crescentic formation and infiltration of neutrophils and lymphocytes into kidney, cytokines/chemokines and MPO-ANCA epitopes by deletion mutants. RESULTS: MPO-ANCA were reduced by the administration of DSG, and epitopes of MPO-ANCA, mainly H-6, decreased. Moreover, the IgG2b subclass of the H-6 epitope of MPO-ANCA was greatly decreased by DSG treatment. These observations correlated with a decrease in renal failure and proteinuria, infiltration of neutrophils and lymphocytes into glomeruli, and crescent formation. The CD4/CD8 ratio of splenocytes ranged from 1.68 (0.24) in the non-treated group to 0.90 (0.12) at 100 microg/mouse/day in the DSG-treated group. In addition, elevated levels of IL-12p40, IL-10 and IL-13 in the active phase of CrGN clearly decreased with DSG treatment but not with TNF-alpha. In contrast, the IL-1alpha level increased, and IL-17 and IL-12p70 slightly increased with DSG treatment. CONCLUSION: These results strongly suggest that DSG treatment of SCG/Kj mice leads to the reduction of risk antibodies in IgG2b and normalization of B-cell clones accompanied by recovery of the cytokine and chemokine balance.

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-beta into intron 8 of Dsg4. Mast cell hyperplasia was observed in the skin of EOD( hage ) mice. The abnormally expanded population of lpr T cells, i.e., CD4(-)CD8(-)B220(+)Thy1.2(+) alphabetaT cells, in the splenocytes of EOD mice was reduced in EOD( hage ) mice. Therefore, it was suspected that the long-living mutant EOD( hage ) 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.

Evaluation of the ameliorative effects of immunosuppressants on crescentic glomerulonephritis in SCG/Kj mice.

The therapeutic efficacy of immunosuppressants for treating rapidly progressive glomerulonephritis (RPGN) with crescent formation remains controversial. SCG/Kj mice spontaneously develop RPGN-like symptoms, characteristic of crescentic glomerulonephritis and systemic small vessel vasculitis, associated with the presence of anti-neutrophil cytoplasmic antibodies (ANCA). We evaluated the "ameliorative", not prophylactic, effects of immunosuppressive agents, deoxyspergualin (DSG), cyclophosphamide (CYC) and prednisolone (PDN), on RPGN in these mice. DSG at intraperitoneal doses of 3 and 6 mg/kg, CYC at an oral dose of 12 mg/kg, or PDN at an intraperitoneal dose of 120 mg/kg was administered once a day for 21 days to female mice "at the onset of hematuria". A set of control SCG/Kj mice received only saline injections. DSG and CYC significantly prolonged survival, improved the proteinuria, hematuria and hyperuremia, and decreased the serum level of myeloperoxidase-ANCA. Moreover, DSG significantly suppressed the formation of crescents in glomeruli. PDN failed to affect any of the parameters. DSG might be useful for inducing remission in crescentic glomerulonephritis involved in RPGN.

NK026680, a novel suppressant of dendritic cell function, prevents the development of rapidly progressive glomerulonephritis and perinuclear antineutrophil cytoplasmic antibody in SCG/Kj mice.

OBJECTIVE: NK026680 is a newly identified type of immunosuppressive agent that inhibits dendritic cell (DC) functions and consequently reduces the mortality of mice with experimental acute graft-versus-host disease. This study was undertaken to evaluate NK026680 suppression of DC functions in preventing development of rapidly progressive glomerulonephritis (RPGN) and perinuclear antineutrophil cytoplasmic antibodies (pANCA) in SCG/Kj mice. METHODS: Oral administration of NK026680 to SCG/Kj mice began when mice were 8-10 weeks old, before the onset of disease, and continued for 56 days. The efficacy of NK026680 was evaluated using the mortality of mice, the results of urinalysis, histopathologic evaluation for glomerular injury, and immunofluorescence staining for the detection of immune complex (IC) deposition in glomeruli, and by assessing lymphadenopathy and measuring autoantibody titers. RESULTS: Oral administration of NK026680 at a dosage of 25 mg/kg once daily or 50 mg/kg once daily significantly suppressed 1) spontaneous mortality, 2) proteinuria and hematuria, 3) blood urea nitrogen levels, 4) glomerular damage characterized histopathologically, 5) IC deposition in glomeruli, 6) the development of pANCA and anti-DNA antibodies, and 7) lymphadenopathy. CONCLUSION: The newly identified DC inhibitor, NK026680, prevented the onset of RPGN, autoantibody production, and lymphadenopathy in SCG/Kj mice, suggesting a crucial role for DC function in these autoimmune phenotypes. NK026680 may be a potent immunosuppressive agent for the treatment of ANCA-associated renovascular disorders.

Cappuccino mutation in an autoimmune-prone strain of mice suggests a role of platelet function in the progression of immune complex crescentic glomerulonephritis.

OBJECTIVE: Crescent formation in the renal glomerulus is a typical manifestation of progressive glomerulopathy associated with fatal renal failure; therefore, its prevention is of clinical importance. Little is known about the pathogenic mechanism for crescent formation. This study was undertaken in an attempt to identify the events that are critical for crescent formation in immune complex crescentic glomerulonephritis (CGN) by analyzing a novel mutant strain of mice. METHODS: A spontaneous mutant strain of mice was isolated from the autoimmune-prone strain EOD, which stably develops fatal CGN. The mutant phenotypes were assessed histopathologically, hematologically, and immunologically. The mutation was searched for with positional cloning using microsatellite markers. RESULTS: Compared with wild-type EOD (WT-EOD) mice, mutant EOD (mut-EOD) mice showed marked improvement in CGN in conjunction with an improvement in spontaneous mortality. In WT-EOD mice, an inverse correlation between blood urea nitrogen concentration and blood platelet count and massive accumulation of platelets in the glomerulus were evident, suggesting that an accumulation of platelets in the glomerulus contributes to the progression of CGN. The mutant platelets showed an abnormal aggregation in response to collagen and thrombin, associated with a bleeding tendency in mut-EOD mice. Genetic analysis revealed a deleterious mutation in the cappuccino gene (cno), which encodes a protein that belongs to a complex called the biogenesis of lysosome-related organelle complex 1 and is profoundly involved in platelet function. Morphologic examination revealed a partial defect in dense body formation in the delta-granule of platelets. CONCLUSION: The present findings suggest that platelet functions have a critical role in crescent formation in autoimmune GN.

NK95806, a newly synthesized microtubule-disrupting agent that suppresses collagen-induced arthritis in mice.

We investigated the anti-arthritic effects of NK95806, a novel inhibitor of microtubule polymerization, on collagen-induced arthritis in mice. The suppressive effect of NK95806 on the induction and development of arthritis was shown as a significant reduction in clinical arthritis scores. Histological analysis of the hind paws confirmed the improvement in clinical severity and showed marked decreases in granulomatous formation and further bone destruction. Further, under the experimental conditions in which methotrexate had little, if any, effect, NK95806 significantly suppressed the development of arthritis. These results suggest that the disruption of microtubules might be a novel target for anti-rheumatic drugs and NK95806 may be a candidate for further development.

Neutrophil contribution to the crescentic glomerulonephritis in SCG/Kj mice.

BACKGROUND: Myeloperoxidase-specific anti-neutrophil cytoplasmic auto-antibody (MPO-ANCA) has been a useful diagnostic marker in systemic vasculitis with crescentic glomerulonephritis (CrGN). It is highly suspected that the antigenic enzyme MPO released from activated neutrophils is involved in these lesions. We evaluated the relationship between neutrophil functions including peripheral neutrophil counts and renal lesions in SCG/Kj mice as a model of ANCA-associated CrGN and vasculitis. METHODS: Peripheral neutrophil counts, the plasma levels of MPO-ANCA and tumour necrosis factor alpha (TNF-alpha) were measured. The capacity of MPO release and superoxide generation were evaluated as neutrophil activity. The renal lesions were estimated by grade of proteinuria, histopathological lesion, such as glomerular neutrophil infiltration and active or chronic renal injury scores with crescent formation. RESULTS: MPO-ANCA and TNF-alpha levels were higher than those of normal mice C57BL/6 even before overt proteinuria; subsequently, peripheral neutrophils increased. In the phase of nephritis with low grade proteinuria, the spontaneous release of MPO from peripheral neutrophils increased, while superoxide generation increased before spontaneous MPO release occurred. In addition, the renal lesion in histological observations was aggravated with ageing and the glomerular neutrophil infiltration was positively correlated with MPO-ANCA levels, as well as with histological indices of nephritis, active renal injury score; in particular, crescent formation was correlated with spontaneous MPO release. In contrast, superoxide generation was negatively correlated with the severity of this lesion during the progression. CONCLUSIONS: These findings indicate that neutrophils are activated and contribute to the development of the active crescentic lesion in SCG/Kj mice.