Cholinergic anti-inflammatory pathway ameliorates murine experimental Th2-type colitis by suppressing the migration of plasmacytoid dendritic cells.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease. Several studies have demonstrated that α7 nicotinic acetylcholine receptors (α7nAChRs) exert anti-inflammatory effects on immune cells and nicotine suppress UC onset and relapse. Plasmacytoid dendritic cells (pDCs) reportedly accumulate in the colon of UC patients. Therefore, we investigated the pathophysiological roles of α7nAChRs on pDCs in the pathology of UC using oxazolone (OXZ)-induced Th2-type colitis with BALB/c mice. 2-deoxy-D-glucose, a central vagal stimulant suppressed OXZ colitis, and nicotine also ameliorated OXZ colitis with suppressing Th2 cytokines, which was reversed by α7nAChR antagonist methyllycaconitine. Additionally, α7nAChRs were expressed on pDCs, which were located very close to cholinergic nerve fibers in the colon of OXZ mice. Furthermore, nicotine suppressed CCL21-induced bone marrow-derived pDC migration due to Rac 1 inactivation, which was reversed by methyllycaconitine, a JAK2 inhibitor AG490 or caspase-3 inhibitor AZ-10417808. CCL21 was mainly expressed in the isolated lymphoid follicles (ILFs) of the colon during OXZ colitis. The therapeutic effect of cholinergic pathway on OXZ colitis probably through α7nAChRs on pDCs were attributed to the suppression of pDC migration toward the ILFs. Therefore, the activation of α7nAChRs has innovative therapeutic potential for the treatment of UC.

The traditional herbal medicine saireito exerts its inhibitory effect on murine oxazolone-induced colitis via the induction of Th1-polarized immune responses in the mucosal immune system of the colon.

BACKGROUND: Ulcerative colitis is an intractable inflammatory colonic disease, and its etiology remains unclear. Saireito, a traditional herbal medicine, is widely used for treating ulcerative colitis in Japan. We analyzed the immunological characteristics of an oxazolone (OXZ)-induced colitis (OC) model and examined the effects of sareito on this model. METHODS: OXZ was injected into the colon of BALB/c mice. Saireito was orally administered once a day for 3 consecutive days. Colitis was assessed by scoring the symptoms and macroscopic findings. The transcription patterns in the middle colon and spleen were analyzed with global transcriptome analysis and real-time polymerase chain reaction (PCR). RESULTS: The above-mentioned scores were increased in the OC mice. The transcription levels of Th2 cytokines were significantly upregulated in the spleen and middle colon of the OC mice, whereas those of the Th1 cytokine interferon (IFN)-gamma decreased in the spleen and increased in the middle colon. Saireito significantly ameliorated OC. In the middle colon of the saireito-treated mice, enhanced expression of Th2 cytokine mRNAs was markedly downregulated, while that of IFN-gamma mRNA was further upregulated. In contrast, in the spleen, saireito had no effect on the transcription of either type of cytokine. After global transcriptome analysis, real-time PCR analysis revealed that saireito greatly downregulated the enhanced expression of the suppressor of cytokine signaling (SOCS)-3 mRNA in the middle colon of OC mice. CONCLUSIONS: Saireito exhibits inhibitory effects on OC by the induction of Th1-polarized immune responses in the mucosal immune system of the colon.

Therapeutic effect of kakkonto in a mouse model of food allergy with gastrointestinal symptoms.

BACKGROUND: The number of patients with food allergy has increased dramatically over the last several decades. However, there is no effective drug for food allergies. In the present study, we evaluated the effects of kakkonto, a traditional Japanese herbal medicine, in a mouse model of food allergy with gastrointestinal symptoms. METHODS: BALB/c mice were systemically sensitized twice with ovalbumin (OVA) and then were repeatedly given OVA by oral intubation (OVA mice). Kakkonto was administered orally before the OVA challenges. RESULTS: The OVA mice developed allergic diarrhea (91.8 +/- 3.8% after 6 OVA challenges), and myeloperoxidase (MPO) activity was dramatically elevated in the colons of the OVA mice. Kakkonto significantly suppressed the occurrence of allergic diarrhea and MPO activity in the OVA mice. Furthermore, the number of mucosal mast cells was greatly increased in the proximal colons of the OVA mice, and this was also suppressed by kakkonto. Interestingly, mRNA expression of helper T cell type 1 (Th1) cytokines (IFN-gamma) and Th2 cytokines (IL-4, IL-5 and IL-10) were significantly upregulated in the proximal colons of the OVA mice, an effect which was also reduced by kakkonto. Transcriptome analysis detected increased mRNA expression of suppressor of cytokine signaling-3 in the proximal colons of OVA mice, which was decreased by kakkonto administration. CONCLUSION: Kakkonto has immunosuppressive effects and interferes with the infiltration of mucosal mast cells in the colons of mice with induced food allergy, leading to improvement of allergic symptoms. Kakkonto has potential as a therapeutic drug for treatment of allergic symptoms induced by the disruption of intestinal mucosal immunity.

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.

Pleomorphic carcinoma of the lung associated with loss of heterozygosity of p53 gene.

We report a case with pleomorphic carcinoma of the lung in a 70-year-old man. Pleomorphic carcinoma is characterized by a heterogenous composition that includes epithelial and mesechymal malignancies. In the present case, the tumor was composed of a mixture of unequivocal squamous cell carcinoma and spindle cell components resembling sarcomatous overgrowth. The spindle component did not include a heterologous mesenchymal element characterized by overt differentiation for bone, cartilage, neuron or muscle tissue. To evaluate a state of differentiation of the spindle cell component, we immunohistochemically examined expression of the antigens including vimentin, cytokeratin, sarcomeric actin, alpha-smooth muscle actin, S-100 protein, CD34, Factor VIII, and CD68. The results showed sole expression of vimentin in the spindle cell component, suggesting an immature state of the mesenchymal lineage. Furthermore, the spindle cell component of this case was genetically characterized by loss of heterozygosity (LOH) at a codon 234 of exon 7 of the p53 gene. This mutation causes an amino-acid replacement (Tyr to Cys), which was previously proven to attenuate p53 function. The present case may suggest a relation between somatic alteration of the p53 gene and histogenesis of pleomorphic carcinoma.

Detection of right to left shunting through a patent foramen ovale in Japanese patients with ischemic stroke by transesophageal echocardiography using a standardized Valsalva maneuver.

We attempted to clarify the usefulness of transesophageal echocardiography performed using a standardized Valsalva maneuver to detect the presence of a patent foramen ovale in Japanese patients with ischemic stroke. Four hundred ninety six patients with ischemic stroke who were admitted to the Yokohama City Brain and Stroke Center between September 1999 and February 2002 were enrolled for the study. All the enrolled patients underwent transesophageal echocardiography with contrast injection and color Doppler imaging. During the procedure, a standardized Valsalva maneuver was performed to induce right to left shunting through a patent foramen ovale. Other related structural abnormalities, such as atrial septal aneurysm and the Chiari network anomaly, were also detected by the test. Transesophageal echocardiography without the Valsalva maneuver revealed a functional right to left communication in only 8.2% of the ischemic stroke patients, whereas the procedure conducted using a standardized Valsalva maneuver to provoke shunting revealed a patent foramen ovale in 15.3% of the patients. The presence of an atrial septal aneurysm or the Chiari network anomaly was not sensitive or specific enough to predict the presence of a patent foramen ovale as diagnosed by transesophageal echocardiography using the standardized Valsalva maneuver. Our results suggest that the standardized Valsalva maneuver is a safe and useful technique to detect the presence of a patent foramen ovale, which is potentially known to be associated with paradoxical embolism.

[Successful treatment with drainage of hematoma and chemotherapy in a case of Burkitt leukemia presenting with subdural hematoma].

A 53-year-old male was admitted because of pancytopenia and chronic subdural hematoma. Bone marrow was hypercellular with 97% blasts, which expressed CD10, CD19, CD20, and immunoglobulin mu and gamma chains on the cell surface and had chromosomal abnormalities including t(8 ; 22)(q24 ; q11). The patient was diagnosed as having Burkitt leukemia. Because hemiplegia and disturbance of consciousness developed rapidly, the patient was treated with an emergency drainage operation followed by Hyper-CVAD therapy and achieved a sustained complete remission. Dural infiltration of leukemic cells as well as thrombocytopenia was implicated in the pathogenesis of the subdural hematoma, which contained numerous blasts.

[An abdominal follicular dendritic cell tumor in Castleman's disease].

A 57-year-old man was referred to our hospital because of elevated ALP. CT and MRI scans together with abdominal angiography showed multiple masses in his abdomen and portal vein obstruction. A diagnostic laparoscopic examination revealed a tumor of 3 cm x 3 cm near the portal vein and para-aortic lymphadenopathy. Histopathological examination of the tumor showed abnormal follicles with poorly formed germinal centers, scattered large spindle cells with proliferation of small lymphocytes, and hypervascular interfollicular tissue. The spindle cells were positive for follicular dendritic cell markers CD21, CD35, and epithelial membrane antigen. The diagnosis was made of a follicular dendritic cell (FDC) tumor in Castleman's disease (CD) of the hyaline-vascular type. Although the portal vein was obstructed by the FDC tumor, blood flow to the liver was retained by collateral vein. The patient did not show any response to four courses of CHOP therapy and died of obstructive jaundice, biliary tract infection and sepsis. So far, 17 cases of FDC tumor complicating CD have been reported, with a poor prognosis in all cases.

Enhancement effect of p-menthane-3,8-diol on in vitro permeation of antipyrine and indomethacin through Yucatan micropig skin.

The enhancing effect of p-Menthane-3,8-diol (MDO) on skin permeation of antipyrine (ANP) and indomethacin (IM) through Yucatan micropig skin in vitro was compared with 1-menthol. p-Menthane-3,8-diol is a metabolite of 1-menthol and has little odor. It is easy to combine the vehicle because of lower lipophilicity than 1-menthol. All formulations contained 40% (v/v) ethanol. The permeation of ANP increased with MDO about three times that without enhancer by increasing ANP concentration in the skin. However, the MDO effect was about a quarter that of 1-menthol. The permeation of IM with MDO was about 15 times that with no enhancer and it was almost the same as that with 1-menthol. The lag time of permeation was not significantly changed by MDO, which was not so in the case of 1-menthol. Skin concentration of IM increased about 11 times and six times with MDO and 1-menthol, respectively. MDO and 1-menthol partitioned to the skin relatively high concentrations, 5.9 and 2.5 mg/ cm3, respectively. The solubility of IM in the skin was improved by MDO, and consequently, the permeation of IM was enhanced.

Possible contribution of acetylamrinone and its enhancing effects on platelet aggregation under shear stress conditions in the onset of thrombocytopenia in patients treated with amrinone.

BACKGROUND: Thrombocytopenia is recognized as one of the most common complications when the patients with severe heart failure are treated with cardiotropic phosphodiesterase (PDE)-3 inhibitors. To understand the mechanism of the onset of this complication, we focused on the effects of various PDE-3 inhibitors and its stable metabolite of acetylamrinone on platelet aggregation occurring under physiological shear stress conditions. METHOD: Blood specimens were obtained from eight apparently healthy adult donors. Platelet-rich plasma was separated after anticoagulation by citrate. The effects of PDE-3 inhibitors of amrinone and olprinone, as well as the stable metabolite of the former of acetylamrinone, on platelet aggregation induced by its exposure to a shear rate of 1200 and 10,800 s(-1) were determined by optically modified cone-plate viscometer. RESULTS: Both olprinone and amrinone inhibited platelet aggregation at 10,800 s(-1) in a dose-dependent manner with the IC(50) value of 14 +/- 1 and 61 +/- 8 microM (mean +/- S.D.), respectively, while amrinone significantly inhibited platelet aggregation at 1200 s(-1) only at highest concentration tested (100 microM). Contrary to the effects shown with PDE-3 inhibitors, acetylamrinone did not inhibit platelet aggregation at all. Moreover, it even enhanced the aggregation at 1200 s(-1) when used with 5 microM. CONCLUSIONS: Our results demonstrate possible contribution of the enhancing effects of acetylamrinone on platelet aggregation occurring under blood flow conditions, which reduced the platelet count when occurring in real circulation, to the higher incidence of thrombocytopenia in patients treated with amrinone.

Internalization of antibodies by endothelial cells via fibronectin implicating a novel mechanism in lupus nephritis.

BACKGROUND: One of the crucial events in lupus nephritis is the glomerular deposition of immunoglobulins (Igs), of which pathogenic properties have been proposed mostly to be either type IIor type III allergic reactions. Some of IgG3-producing hybridoma clones established from an MRL/MpTn-gld/gld (MRL/gld) lupus mouse generate wire loop-like lesions in glomeruli resembling lupus nephritis when injected into SCID mice. These clones are useful for analyzing the mechanisms of glomerular deposition of antibodies in lupus nephritis at the monoclonal level. METHODS: Glomerular lesions of SCID mice injected with the hybridoma clones, 17H8a or 1G3 as control were analyzed by light and electron microscopy. Interaction of the antibodies with human glomerular endothelial cells (HGECs) and human umbilical vein endothelial cells (HUVECs) in vitro was studied by fluorescence microscopy, electron microscopy, and flow cytometry. RESULTS: Both antibodies did not show any antigen specificity for mouse glomeruli. The glomerular lesions generated by 17H8a, but not by 1G3, contained electron-dense deposits not only in subendothelial regions but also in the cytoplasm of endothelial cells, suggesting internalization of the 17H8a antibodies by endothelial cells. In cell culture studies, internalization of only 17H8a antibodies by HGECs and HUVECs was observed, but the antibodies did not have antigen specificity for both types of endothelial cells. The internalization by HUVECs was mediated by actin polymerization, and it was inhibited by RGDS (Arg-Gly-Asp-Ser) tetrapeptide, antihuman fibronectin and antihuman integrin beta1 monoclonal antibodies. CONCLUSION: The interaction between particular antibodies and endothelial cell surface integrins via fibronectin may be involved in their subsequent internalization by endothelial cells leading to antibody deposition in glomeruli. This may be one of the mechanisms of glomerular injury in lupus nephritis.

Comparison of skin permeation enhancement by 3-l-menthoxypropane-1,2-diol and l-menthol: the permeation of indomethacin and antipyrine through Yucatan micropig skin and changes in infrared spectra and X-ray diffraction patterns of stratum corneum.

3-l-Menthoxypropane-1,2-diol (MPD) is a derivative of l-menthol, which has an enhancement effect on drug permeation through skin. In this study, the effect of MPD on drug permeation through skin was compared with that of l-menthol. MPD or l-menthol at final concentrations of 3% in 40% ethanol was added to the drugs indomethacin or antipyrine and each mix then applied to Yucatan micropig skin in vitro. Drug concentrations in the skin were higher in the presence of either MPD or l-menthol, however, only l-menthol shortened the lag time of permeation. MPD enhanced the skin permeation of the drugs only by increasing the skin concentration of the drugs. In contrast, l-menthol enhanced the skin permeation of the drugs by increasing both the skin concentration and the diffusion rate in skin. The infrared (IR) spectra and X-ray diffraction patterns of stratum corneum after treatment with MPD did not differ from those of intact stratum corneum. A change in the IR spectra of stratum corneum after treatment with l-menthol was observed at the CH band, and the peaks representative of the lipid structure in the X-ray diffraction patterns decreased in intensity. These results suggest that l-menthol, but not MPD, disrupts the intercellular lipid structure of stratum corneum. Thus, MPD is expected to be a moderate skin permeation enhancer.

Shear-induced von Willebrand factor-mediated platelet surface translocation of the CD40 ligand.

BACKGROUND: Platelets, which can adhere to damaged vascular surfaces and release bioactive substances upon activation, may play important roles in regulating local inflammatory responses. We focused on the surface translocation of CD40 ligand (CD40L) molecules when the platelets are exposed to a high shear stress. METHOD: Blood specimens were obtained from eight apparently healthy adult donors. The number of CD40L molecules appearing on the surface of platelets after exposure of platelet-rich plasma to a shear rate of 10,800 s(-1) was determined by quantitative flow cytometry. RESULTS: The number of anti-CD40L IgG molecules bound per platelet increased from 15+/-80/platelet before to 355+/-122/platelet after exposure of the platelets to a shear rate of 10,800 s(-1) (p<0.01), but not after their exposure to the relatively low shear rate of 1200 s(-1). This shear-induced platelet surface translocation of CD40L, mediated by the von Willebrand factor (VWF)-GP Ibalpha interaction, was enhanced in the presence of a low concentration of epinephrine (100 nM), which by itself, however, could not cause platelet activation. Our results demonstrate that fluid force induces the appearance of CD40L on the surface of platelets, and also that this phenomenon is enhanced in the presence of a low concentration of epinephrine, corresponding to that released by sympathetic stimulation.