基于RIP1/RIP3/NLRP3信号通路探讨祛瘀生新方治疗溃疡性结肠炎小鼠的作用机制

目的:研究祛瘀生新方对溃疡性结肠炎(UC)小鼠的治疗效果以及对RIP1/RIP3/NLRP3信号通路的调节作用.方法:将36只小鼠随机分为实验组(祛瘀生新组)、模型组、阳性对照组(美莎拉嗪组)、空白组、中药拆方1组(生新组)、中药拆方2组(祛瘀组),6只/组,采用3.5％葡聚糖硫酸钠盐(DSS)溶液自由饮水法制备UC小...     

Melatonin potentiates running wheel‐induced neurogenesis in the dentate gyrus of adult C3H/HeN mice hippocampus

This study assessed the role of melatonin in modulating running wheel(RW)‐induced hippocampal neurogenesis in adult C3H/HeN mice. Chronic melatonin (0.02 mg/mL, oral for 12 days) treatment did not affect cell proliferation or cell survival determined by the number of BrdU‐positive cells in dentate gyrus of mice with access to fixed wheel (FW). RW activity significantly increased cell proliferation [RW (n = 8) versus FW (n = 6): dorsal, 199 ± 18 versus 125 ± 12, P < 0.01; ventral, 211 ± 15 versus 123 ± 13, P < 0.01] and newborn cell survival [RW (n = 7) versus FW (n = 8): dorsal, 45 ± 8.5 versus 15 ± 1.8, P < 0.01; ventral, 48 ± 8.1 versus 15 ± 1.4)] in the dorsal and ventral dentate gyrus. Oral melatonin treatment further potentiated RW activity‐induced cell survival in both areas of the dentate gyrus [melatonin (n = 10) versus vehicle (n = 7): dorsal, 63 ± 5.4 versus 45 ± 8.5 P < 0.05; ventral, 75 ± 7.9 versus 48 ± 8.1, P < 0.01] and neurogenesis [melatonin (n = 8) versus vehicle (n = 8): dorsal, 46 ± 3.4, versus 34 ± 4.5, P < 0.05; ventral, 41 ± 3.4 versus 25 ± 2.4, P < 0.01]. We conclude that melatonin potentiates RW‐induced hippocampal neurogenesis by enhancing neuronal survival suggesting that the combination of physical exercise and melatonin may be an effective treatment for diseases affecting the hippocampus neurogenesis.     

Regulation of the growth and differentiation of Trypanosoma (Trypanozoon) brucei brucei in resistant (C57B1/6) and susceptible (C3H/He) mice

While Trypanosoma brucei brucei GUTat 3 were equally infective for C3H/He and for C57Bl/6 mice at doses ranging from 5 to 5 x 10(3) organisms and had similar prepatent periods in both strains of mice, infected C57Bl/6 mice displayed lower parasitaemia, shorter times to parasite wave remission and survived for a longer time than infected C3H/He mice. Parasite growth and differentiation rates and host immune responses were similar for the first 5 days in both strains of mice after infection with 10(3) T.b.brucei GUTat 3 but, thereafter, parasite differentiation proceeded more rapidly and specific antibodies reached higher titres in C57Bl/6 than in C3H/He mice. In contrast, parasite growth and differentiation rates were similar in irradiated mice of both strains. Furthermore, following inoculation of intact mice with irradiated T.b.brucei GUTat 3, C3H/He mice actually mounted higher titred antibody responses than C57Bl/6 mice showing that they were not intrinsically defective in their capacity to respond to GUTat 3 antigens. Parasite differentiation occurred at the same rate in irradiated (650r) C57Bl/6 mice and in irradiated C57Bl/6 mice reconstituted with syngeneic spleen cells although T.b.brucei GUTat 3 specific antibody was detected in the latter mice prior to peak parasitaemia. Furthermore, it was shown directly in C57Bl/6 mice that there was no selective destruction of slender form T.b.brucei GUTat 3 parasites during the phase of accumulation of stumpy form parasites. These studies indicate that the more rapid differentiation of T.b.brucei GUTat 3 parasites in infected C57Bl/6 mice as compared to infected C3H/He mice was unlikely to be directly related to the more efficient antibody response in the infected C57Bl/6 mice. The observations suggest that there might be an association between host mechanisms which regulate differentiation of T.b.brucei parasites and those which regulate antibody responses.     

Plasmacytoid dendritic cells as a possible key player to initiate alopecia areata in the C3H/HeJ mouse

BackgroundAlopecia areata (AA) is a tissue-specific autoimmune disease, and interferon (IFN)-γ has been regarded as the key cytokine in the pathogenesis of AA. The clinical observation that AA can occur after viral infection or IFN-α administration implies that IFN-α-producing plasmacytoid dendritic cells (pDCs) may be involved in the AA pathogenesis.MethodsWe generated AA in C3H/HeJ mice by intradermal injection of T cells derived from lymph nodes of AA-bearing syngeneic mice and stimulated IL-2, IL-7, and IL-15. Distribution of IFN-γ producing pDCs were immunohistochemically analyzed. Realtime PCR were also demonstrated to detect the expression of IFN-γ mRNA. Hair follicles were cultured with IFN-α in order to calculate the hair elongation. Imiquimod was employed to induce catagen stage. PDCs were injected into C3H/HeJ mice to initiate AA.ResultsIn this mouse, IFN-α-producing pDCs densely infiltrated around HFs in not only AA lesional but also vicinity of AA lesion. Importantly, intradermal injection of pDCs induced AA lesions. Finally, IFN-α inhibited hair elongation of murine vibrissae and upregulated MHC class I and CXCL10 levels in vitro.ConclusionsThese findings suggest that IFN-α-producing pDCs initiate AA by inducing apoptosis and increasing Th1/Tc1 chemokine production such as CXCL10, that accumulates Th1/Tc1 cells and result in autoimmune reactions against hair follicles.     

BALB/C小鼠核小体H2B组蛋白Th细胞表位的鉴定

目的 鉴定正常BALB／C小鼠核小体。H2B组蛋白Th细胞表位。方法 根据小鼠H2B确切氨基酸组成序列，利用计算机软件预测可能的Th细胞表位，体外固相合成，然后分别刺激BALB/C小鼠淋巴细胞，根据其中的Th细胞增生程度和白细胞介素(IL)-2分泌水平，判断实验肽是否为具有免疫活性的表位。结果 共预测获得4条可能的核小体H2B组蛋白Th细胞表位，即H2B2-15、H2B14-28、H2B61-76和H2B99-113。其中H2B14-28能够明显刺激BALB／C小鼠Th淋巴细胞增生和分泌IL-2，且有明显剂量依赖关系。结论 H2B14-28可能为BALB/c小鼠核小体。H2B组蛋白Th细胞免疫优势表位之一。     

Infectivity and pathogenicity of canine H3N8 influenza A virus in horses

Please cite this paper as: Yamanaka et al. (2010) Infectivity and pathogenicity of canine H3N8 influenza A virus in horses. Influenza and Other Respiratory Viruses 4(6), 345–351. Background  Equine H3N8 influenza A viruses (EIVs) cause respiratory disease in horses and circulate among horses worldwide. In 2004, an outbreak of canine H3N8 influenza A virus (CIV) occurred among dogs in Florida and has spread among dogs in the United States (US). Genetic analyses revealed that this CIV is closely related to the recent EIVs. Although CIV‐infected dogs could be the source of H3N8 influenza A virus for horses, it remains unclear whether the CIV circulating in the United States still maintains its infectivity and/or pathogenicity in horses. To address this, we investigated the infectivity and pathogenicity of CIV in horses and the receptor binding specificity of CIV. Materials and methods  Three horses were inoculated with A/canine/Colorado/30604/2006 (CO06, H3N8). Clinical signs and nasal swabs were recorded or collected every day. We also evaluated the virus binding to α2‐3‐linked 5‐N‐acetylneuraminic acid (NeuAcα2‐3Gal) and 5‐N‐glycolylneuraminic acid (NeuGcα2‐3Gal) receptor analogues. Results  Although all the three horses inoculated with CO06 seroconverted, they showed only mild clinical signs and two of them showed no virus shedding. CO06 had reduced binding to NeuGcα2‐3Gal. Discussion  Our results demonstrated that CO06 had reduced proliferation ability and pathogenicity in horses. As the recognition of NeuGcα2‐3Gal by EIV is known to be essential for binding to the equine respiratory system, the decreased binding of CO06 to NeuGcα2‐3Gal may be one of the important factors that reduces the proliferation ability and pathogenicity of CO06 in horses.     

Lipopolysaccharide (LPS)-induced cell death of C3H mouse peritoneal macrophages in the presence of cycloheximide: different susceptibilities of C3H/HeN and C3H/HeJ mice macrophages

Lipopolysaccharide (LPS) induced cytotoxicity toward mouse peritoneal macrophages from C3H/HeN mice but not C3H/HeJ mice in vitro in the presence of cycloheximide (CHX). More than 1 ng/ml LPS induced a significant time-dependent release of a cytoplasmic enzyme, lactate dehydrogenase (LDH), while even 1000 ng/ml LPS failed to induce it in LPS-non-responsive C3H/HeJ mouse macrophages. Although similar LPS-induced cytotoxicity was observed in a murine macrophage-like cell line, J774.1, but not in an LPS-resistant mutant of J774.1, the LPS1916 cell line, these results suggest that the induction of this cytotoxicity is linked to the LPS-sensitivity of mouse macrophages. A recombinant TNF-alpha (rTNF-alpha) at 100 ng/ml augmented LDH release from both C3H/HeN and C3H/HeJ macrophages treated with LPS and CHX, while rTNF-alpha alone or in combination with LPS or CHX failed to induce LDH release. These results suggest that this cytotoxicity might be partially regulated by high concentrations of exogenous TNF-alpha in both C3H/HeN and C3H/HeJ macrophages, implying a possibility of paracrine regulation of TNF-alpha in mice toward LPS-treated macrophages under impaired protein synthesis.     

NLRP3炎症小体在甲型流感病毒和MRSA致小鼠肺炎早期的作用差异

目的 探讨NLRP3炎症小体在甲型流感病毒H1N1(甲流病毒H1N1)及耐甲氧西林金黄色葡萄球菌(MRSA)所致肺炎早期的作用。方法 分别以甲流病毒H1N1/FM1株与MRSA滴鼻感染C57BL/6小鼠各5只,引起甲流病毒肺炎与MRSA肺炎,感染24 h后,观察肺组织病理,荧光定量PCR检测各组小鼠肺组织NLRP3蛋白、IL-1β的mRNA表达强度,蛋白免疫印迹法检测小鼠肺组织NLRP3蛋白相对表达量,ELISA检测小鼠血清IL-1β的浓度。结果 NLRP3蛋白的mRNA表达水平及蛋白表达水平各组间比较差异有统计意义(F_mRNA=38.782和F_蛋白=1 325.64,P均为0.000),IL-1β的mRNA表达水平及血清浓度各组间比较差异有统计学意义(F_mRNA=1 253.97和F_血清=41.974,P均为0.000)。H1N1组 NLRP3蛋白的mRNA表达水平及蛋白表达水平均高于空白对照组(P均<0.05),IL-1β的mRNA表达水平及血清浓度均高于空白对照组(P均<0.01)。MRSA组NLRP3蛋白的mRNA表达水平高于空白对照组(P<0.05),蛋白表达水平与空白对照组比较差异无统计学意义(P>0.05),MRSA组IL-1β的mRNA表达及血清浓度均高于空白对照组(P均<0.01)。H1N1组 NLRP3蛋白的mRNA表达水平及蛋白表达水平均高于MRSA组(P均<0.05),但MRSA组IL-1β的mRNA表达水平及血清浓度均明显高于H1N1组(P均<0.01),与肺组织病理变化相一致。结论 在甲流病毒性肺炎及MRSA肺炎早期,NLRP3炎症小体介导甲流病毒引起的炎症反应,但并没有在MRSA引起的剧烈炎症反应中起重要作用。     

Immune responses to human factor IX in haemophilia B mice of different genetic backgrounds are distinct and modified by TLR4

Our laboratory develops protocols to prevent or reverse ongoing anti‐hFIX IgG inhibitors in haemophilia B mice with a F9 gene deletion on BALB/c and C3H/HeJ backgrounds. C3H/HeJ F9^?/Y mice develop high titre anti‐hFIX IgG1 inhibitors and anaphylaxis, whereas most BALB/c F9^?/Y mice have mild anti‐hFIX IgG1 inhibitors and no anaphylaxis. Our aim was to determine if hFIX‐specific B‐ and T‐cell responses in BALB/c and C3H/HeJ F9^?/Y mice trigger the difference in anti‐hFIX immune responses. BALB/c and C3H/HeJ F9^?/Y mice were challenged weekly with recombinant hFIX protein. Humoral immune responses were determined by IgG1 and IgG2a anti‐hFIX ELISA, Bethesda assay for inhibitors and B‐cell ELISpot on bone marrow and spleen cells. T‐cell studies measured the T_H1 (IFN‐γ) and T_H2 (IL‐4) cytokine responses in splenocytes at the mRNA and protein level in response to hFIX protein. Antibody responses were also measured in C3H/HeJ/OuJ F9^?/Y mice with restored toll‐like receptor 4 (TLR4) function. BALB/c F9^?/Y mice have a T_H2 skewed response and a reduction in anti‐hFIX secreting plasma cells in the bone marrow. Independent antigen challenge revealed both strains generated equivalent IgG1 antibody titres to an intravenously delivered antigen. C3H/HeJ F9^?/Y mice have a mixed T_H1 and T_H2 response (mainly T_H2). Importantly, TLR4 signalling has a modulatory role in the C3H background on the levels of anti‐hFIX IgG1 and incidence of anaphylaxis. The background strain strongly impacts the immune response to hFIX, which can be significantly impacted by mutations in innate immune sensors.     

Cytokine responses of C3H/HeN mice infested with Ixodes scapularis or Ixodes pacificus nymphs

Lyme borreliosis, caused by Borrelia burgdorferi, is transmitted by Ixodes scapularis in the eastern and midwestern United States and by Ixodes pacificus in the far-Western United States. Studies have shown that infestation with I. scapularis nymphs modulates host cytokine production; however, the influence of I. pacificus infestation on host cytokines remains uninvestigated. This study demonstrated how repeated infestations with pathogen-free I. scapularis or I. pacificus nymphs affects the production of the macrophage cytokines IL-1beta, IL-6 and tumour necrosis factor-alpha and the T lymphocyte cytokines IL-2, IL-4, IL-5, IL-6, IL-10 and IFN-gamma by C3H/HeN mice. Female mice were infested once or twice with pathogen-free I. scapularis or I. pacificus nymphs, with a 14-day tick-free period between exposures. After each infestation, tick biology parameters were assessed and macrophage and T lymphocyte cytokine production measured by antigen capture ELISA. Acquired resistance to tick feeding did not develop after infestation with either tick species. Differences in cytokine production were observed between infested and noninfested mice, and between mice infested with either I. scapularis or I. pacificus nymphs. Infestations polarized cytokine production towards a Th2 cytokine profile, with suppression of pro-inflammatory Th1 cytokines. This pattern of cytokine production is more pronounced for I. pacificus infested mice.     

趋化因子CXCL10(IP-10)/CXCR3在类风湿关节炎发病中的作用及应用前景

类风湿关节炎（RA）是一类以关节炎为主要临床表现的系统性自身免疫性疾病。实验证明,T细胞尤其是CD4~＋T细胞的异常活化及其分泌的细胞因子所形成的网络参与了RA激发和延续。趋化因子在炎症细胞向滑膜组织迁移及活化过程中发挥了关键作用,趋化因子C-X-C配体10/干扰素诱导蛋白-10（CXCL10/IP-10）可与表达在T细胞表面的受体趋化因子C-X-C受体3（CXCR3）结合促进其活化并向CD4~＋Th1细胞方向分化,从而促进炎症反应。研究发现,CXCL10在RA血清及滑膜中表达增高。目前作为RA的一个可能的致病因素,CXCL10/CXCR3在发病机制中的作用越来越受到重视。研究发现,CXCL10抗体及裸DNA疫苗可对RA关节炎有抑制及治疗作用,其可能作为RA治疗新靶点的研究日益增多。     

慢性重型肝炎肝衰竭患者血浆对C 3A细胞增殖和生物转化功能的影响

急性肝衰竭患者血浆对生物反应器中肝细胞的影响研究较多，并证实患者血浆对所接触的细胞有明显毒性作用。C3A细胞株与正常肝细胞功能接近，易于培养，并已成功应用于临床。现采用C3A细胞为对象，研究慢性重型肝炎肝衰竭患者血浆在体外对其影响。     

The effect of phorbol ester treatment on migration of C3H 10T1/2 and BT5C glioma cells: possible application to carcinogenesis

Treatment with the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) and the second-stage promotor 12-O-retinoylphorbol 13-acetate induced down-regulation of protein kinase C (PKC) and enhanced the migration of C3H 10T1/2 cells. In the case of BT5C glioma cells the same negative correlation was observed only after treatment with TPA. The negative control 4-phorbol affected neither PKC activity nor the migratory ability of both cell lines.Abbreviations PKC protein kinase C - TPA 12-O-tetradecanoylphorbol 13-acetate - RPA 12-O-retinoylphorbol     

A single major gene controls most of the difference in susceptibility to streptozotocin-induced diabetes between C57BL/6J and C3H/HeJ mice

Summary To assess genetic factors determining sensitivity to streptozotocin-induced diabetes in inbred strains of mice, a genetic analysis of streptozotocin-sensitive C57BL/6J and streptozotocin-resistant C3H/HeJ mice was performed. One week after a single dose of streptozotocin (200 mg/kg body weight), differences in plasma glucose concentration were marked between male mice of the C57BL/6J and C3H/HeJ strains (p<0.001). To determine the number of genes responsible for the difference, F₁ male progeny of a cross between parental strains were produced, and found to be streptozotocin resistant like C3H/HeJ parents. F₁ mice were, therefore, backcrossed with streptozotocin-sensitive C57BL/6J mice (Backcross: F₁ X C57BL/6J ). The plasma glucoses of backcrossed male mice (n=41) following streptozotocin treatment appeared to segregate into two populations, half like the C57BL/6J parent, and half like the F₁ parent. Statistical analysis of the data revealed that the data fit a model with two distributions better than one with a single distribution, suggesting a single major gene responsible for the difference in streptozotocin susceptibility. This hypothesis was also supported by the observation that streptozotocin sensitivity in 12 recombinant inbred strains of C57BL/6J and C3H/HeJ mice appeared to segregate into two classes. Resistance to streptozotocin induced diabetes in F₁ mice suggested that the expression of this gene is recessive, although X-chromosome linked inheritance could not be excluded.Efforts to map the streptozotocin-sensitivity gene revealed lack of right linkage to several loci including the H-2 locus. If inherited differences in the ability to resist a B-cell toxin play a role in genetic susceptibility to diabetes in man, then mapping the streptozotocin-susceptibility gene in mice may provide a means to evaluate the role of a putative homologous locus in the aetiology of diabetes in man.     

旋毛虫抗C57BL/6小鼠体内Hepa1-6肝癌细胞作用的研究

目的观察旋毛虫对C57BL/6小鼠体内Hepa1-6肝癌细胞生长的抑制作用。方法将C57BL/6小鼠随机分成8组,每组10只。第1和5、2和6、3和7组分别感染未处理旋毛虫、^60Co处理和紫外线处理旋毛虫,4和8组为不感染旋毛虫对照组,1～4组和5～8组分别于接种旋毛虫前7 d和接种后11 d接种Hepa1-6肝癌细胞。荷瘤后25 d后处死小鼠,测量肿瘤体积、重量及脾脏CD3^＋、CD4^＋、CD8^＋淋巴细胞数量。结果未处理旋毛虫组、^60Co处理组和紫外线处理组小鼠肿瘤体积和重量均显著低于对照组（P〈0.05）;脾脏CD3^＋、CD4^＋百分率和CD4^＋/CD8^＋、CD4^＋/CD3^＋的比值显著高于对照组（P〈0.01或P〈0.05）。结论未处理的旋毛虫、经^60Co和紫外线处理的旋毛虫对C57BL/6小鼠体的Hepa1-6肝癌细胞的生长均有抑制作用,未处理旋毛虫的抑瘤效果最好。     

Effects of antisecretory agents on parietal cell structure and H/K-ATPase levels in rabbit gastric mucosain Vivo

The effect of inhibition of acid secretion on parietal cell morphology and the concentration of H,K-ATPase -subunit protein was determined by electron microscopy and western blotting. Omeprazole or famotidine alone or in combination were used. Control animals showed a morphological stimulation index (0=resting, 1.0=fully stimulated) of 0.60; omeprazole treatment (1 mg/kg, twice a day) resulted a stimulation index of 0.63, famotidine injection (20 mg/kg twice a day) an index of 0.11, famotidine infusion (0.2 mg/hr) for five days an index of 0.38, and the combination of omeprazole and famotidine injection twice a day gave an index of 0.02. No change in the frequency of degenerating or damaged parietal cells was observed in any of the groups. In control animals, the number of lysosomes was 0.9/cell, with famotidine 1.8 and with omeprazole 5.6/cell. H/K-ATPase levels fell by about 25% with omeprazole and rose by about 23% with famotidine.This work was supported by USVA-SMI and NIH grants RO1 DK 40165 and 41301.