The role of urease in the acid stress response and fimbriae expression in Klebsiella pneumoniae CG43

Background/purposeTwo urease operons were identified in Klebsiella pneumoniae CG43, ure-1 and ure-2. This study investigates whether a differential regulation of the expression of ure-1 and ure-2 exists and how urease activity influences the acid stress response and expression of type 1 and type 3 fimbriae.MethodsThe ureA1 and ureA2 gene specific deletion mutants were constructed. Promoter activity was assessed using a LacZ reporter system. The sensitivity to acid stress was determined by assessing the survival after pH 2.5 treatment. The influence on type 1 and type 3 fimbriae expression was assessed using western blotting and mannose-sensitive yeast agglutination and biofilm formation assay, respectively.ResultsBacterial growth analysis in mM9-U or modified Stuart broth revealed that ure-1 was the principal urease system, and ure-2 had a negative effect on ure-1 activity. Deletion of the fur or nac gene had no apparent effect on the activity of P_ure1, P_ure2-1, and P_ure2-2. The P_ure2-2 activity was enhanced by deletion of the hns gene. ureA1 deletion increased acid stress sensitivity, whereas the deleting effect of ureA2 was notable without hns. Deletion of ureA1 or ureA2 significantly induced the expression of type 1 fimbriae but decreased MrkA production and biofilm formation.Conclusionure-1 is the primary expression system in K. pneumoniae CG43, while ure-2 is active in the absence of hns. Impairment of urease activity increases the sensitivity to acid stress, and the accumulation of urea induces the expression of type 1 fimbriae but represses type 3 fimbriae expression.     

Expression of type 1 and P fimbriae in situ and localisation of a uropathogenic Escherichia coli strain in the murine bladder and kidney

Adhesion is an important aspect of bacterial colonisation and induction of human disease. Escherichia coli which infects and causes disease of the urinary tract expresses several adherence factors including type 1 and P fimbriae. Their expression has been implicated in the virulence of E. coli strains infecting the urinary tract, however, the evidence for the expression of these fimbriae in situ has been implied rather than proven. Here we describe in situ detection of E. coli and of fimbrial expression in urinary tract tissue. Kidneys and bladders were isolated from mice infected with the uropathogenic isolate E. coli AD110. The tissue was sectioned and subjected to DNA-rRNA hybridization and indirect immunofluorescent staining with antibodies against type 1 and P fimbriae. Sections of both kidney and bladder stained positive for bacterial cells using a Cy3-labelled E. coli-specific rRNA probe. The same cells in these sections also stained positive for type 1 or P fimbriae using fluorescein-labelled antibodies. Tissue taken from several different time points (2, 6, and 24 hours post infection) showed the presence of bacterial cells which stained positive for fimbrial expression. Bacteria in kidney and bladder sections were observed either as individual cells associated with the mucosa or as members of microcolonies.     

Reactivation of tuberculosis is associated with a shift from type 1 to type 2 cytokines

The pattern of cytokines produced by T cells from mice with latent tuberculosis and during reactivation of tuberculosis was determined. A type 1 cytokine pattern was observed in T cells isolated from the lung of mice with latent disease. Reactivation of mycobacterial growth, by activation of the hypothalamic-pituitary-adrenal (HPA) axis, resulted in a shift from a type 1 to a type 2 cytokine pattern in both CD4 and CD8 T cells. Classification of the T cells based on their differential expression of CD45 and CD44 showed that the phenotypically different populations of CD4 and CD8 cells exhibited a type 1 cytokine pattern at latency and that reactivation of latent tuberculosis was associated with a shift in cytokines produced by these populations to a type 2 cytokine response. Control of mycobacterial growth resulted in a return to the type 1 cytokine pattern found during latent disease.     

Treatment responsiveness in CIDP patients with diabetes is associated with unique electrophysiological characteristics,and not with common criteria for CIDP

Objectives: Characterize treatment responsiveness in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) patients with diabetes mellitus (DM). Methods: We performed a retrospective chart review of CIDP subjects assessed between 1997 and 2013 and compared treatment response rates in those with and without DM, using different sets of criteria. Results: 99 CIDP patients were included, 34 CIDP+DM and 65 CIDP?DM patients, both having similar treatment response rates. CIDP patients fulfilling European Federation of Neurological Societies/Peripheral Nerve Society (EFNS/PNS) criteria had higher treatment response rates. Responders fulfilled a higher number of American Academy of Neurology (AAN) and EFNS/PNS criteria and had a higher number of demyelinating features in the total cohort and in CIDP?DM but not in CIDP+DM patients. CIDP+DM responders, however, had unique electrophysiologic characteristics. Conclusion: Fulfilling EFNS/PNS and AAN criteria, and higher number of demyelinating features, are associated with higher treatment response rates in CIDP?DM but not in CIDP+DM patients, implying the need for adjusting current criteria to predict treatment response rates in CIDP?DM patients.     

1型自身免疫性胰腺炎的流行病学及临床特点

目的总结自身免疫性胰腺炎(AIP)的流行病特征、临床表现和诊断经验,提高诊治水平。方法回顾性分析了北京协和医院自1998年1月至2016年12月所有确诊为AIP的临床资料。结果共纳入194例1型AIP。该病发病的男女比例为3.51/1,平均发病年龄为(57±15)岁。最常见的症状是黄疸和腹痛等。最常见的胰腺外受累疾病是硬化性胆管炎、涎腺炎和IgG4肾病。36.68%AIP患者合并糖尿病。IgG4、ANA、ESR、C反应蛋白和CA199均有助于疾病诊断和病情判断。病理、pet-CT和激素治疗反应均有助于AIP的诊断。结论作为一种系统性疾病的胰腺表现,1型AIP有特殊的临床特点和实验室检查特点。AIP的诊断可应用多种检测手段和方法。     

2型糖尿病患者免疫功能变化与血糖和胰岛素水平相关性的研究

探讨2型糖尿病患者免疫功能变化及其与血糖和胰岛素的相关性.检测34例2型糖尿病(T2DM)患者在空腹和口服75克葡萄糖后120min时的红细胞C3b受体花环(RBC-C3bRR)、红细胞免疫复合物花环(RBC-ICR)、T淋巴细胞亚群(CD3、CD4、CD8、CD4/CD8)、可溶性白介素-2受体(sIL-2R)、免疫球蛋白(IgG、IgA、IgM)、血糖和胰岛素.对照组30名.2型糖尿病患者与对照组相比,RBC-C3bRR、CD3、CD4、CD4/CD8和IgG、IgA均明显降低(P＜0.01,P＜0.05),而RBC-ICR、sIL-2R和CD8明显增高(P＜0.01,P＜0.05),服葡萄糖后120min时较空腹时更明显.RBC-C3bRR与血糖和胰岛素呈负相关(r1=-0.354,r2=-0.335,P＜0.05);RBC-ICR与血糖和胰岛素呈正相关(r3=0.368,r4=0.342,P＜0.05);CD3、CD4、CD4/CD8、IgA与血糖呈负相关(r5=-0.302,r6=-0.378,r7=-0.413,P＜0.01;r8=-0.332, P＜0.01);CD8和sIL-2R与血糖呈正相关(r9=0.214,P＜0.05;r10=0.437,P＜0.01).T2DM患者存在多项免疫指标变化,其免疫功能低下与血糖和胰岛素升高有相关性.     

Reduced Food Intake is the Major Contributor to the Protective Effect of Rimonabant on Islet in Established Obesity-Associated Type 2 Diabetes

Purpose

Although the presence of cannabinoid type 1 (CB1) receptor in islets has been reported, the major contributor to the protective effect of rimonabant on islet morphology is unknown. We determined whether the protective effect of rimonabant on pancreatic islet morphology is valid in established diabetes and also whether any effect was independent of decreased food intake.

Materials and Methods

After diabetes was confirmed, Otsuka Long-Evans Tokushima Fatty rats, aged 32 weeks, were treated with rimonabant (30 mg/kg/d, rimonabant group) for 6 weeks. Metabolic profiles and islet morphology of rats treated with rimonabant were compared with those of controls without treatment (control group), a pair-fed control group, and rats treated with rosiglitazone (4 mg/kg/d, rosiglitazone group).

Results

Compared to the control group, rats treated with rimonabant exhibited reduced glycated albumin levels (p<0.001), islet fibrosis (p<0.01), and improved glucose tolerance (p<0.05), with no differences from the pair-fed control group. The retroperitoneal adipose tissue mass was lower in the rimonabant group than those of the pair-fed control and rosiglitazone groups (p<0.05). Rimonabant, pair-fed control, and rosiglitazone groups showed decreased insulin resistance and increased adiponectin, with no differences between the rimonabant and pair-fed control groups.

Conclusion

Rimonabant had a protective effect on islet morphology in vivo even in established diabetes. However, the protective effect was also reproduced by pair-feeding. Thus, the results of this study did not support the significance of islet CB1 receptors in islet protection with rimonabant in established obesity-associated type 2 diabetes.     

支架蛋白IQGAP1-细胞黏附和迁移的关键纽带

支架蛋白IQGAP1发现能与许多重要的细胞活动紧密相关,如黏附和迁移。研究发现在损伤修复、组织发展、肿瘤转移等过程中,IQGAP1的表达和功能有明显改变。同时作为Rho GTPase信号的主要效应因子,IQGAP1能传递细胞外信号至黏附素复合体、细胞骨架以及细胞核。通过上述信号通路,IQ-GAP1协助细胞完成一系列重要生理病理活动。     

Structural and Functional Study of the Receptor Binding Site for FimH Adhesin in Uropathogenic Strains of Escherichia coli

We evaluated binding capacity of FimH-FocH hybrid adhesins during their interaction with model 1M and 3M substrates and epithelial cells. Introduction of the Glu89Lys point mutation into the fimH gene induced a new 1M-specific phenotype of adhesin. The role of a new pathoadaptive sign in the population of E. coli is discussed.     

Basement membrane formation and re-distribution of the β1 integrins in a human intestinal co-culture system

We have developed a co-culture system suited for the study of epithelial-mesenchymal interactions in the human fetal small intestine. As the epithelial component of this model, we used the human intestinal cell line Caco-2 that is unique in its property to differentiate in vitro into a mature fetal enterocyte-like cell type. A sheet of human intestinal mesenchymal cells, which we derived from an 18-week-old fetus, was used as mesenchymal element. Expression and distribution of cell-specific markers (cytokeratin 18 and dipeptidyl peptidase IV), major basement membrane components, and β1 integrins were analyzed. In 14-day co-cultures, Caco-2 cells formed a cytokeratin 18-positive epithelial-like sheet covering the vimentin-positive HIM cell layers. As assessed by brush border dipeptidyl peptidase IV expression, co-cultured Caco-2 cells achieved cytodifferentiation as when cultured on plastic. A complete deposition of all known major human fetal intestinal basement membrane components occurred at the Caco-2/HIM interface. Type IV collagen and tenascin were produced from the mesenchymal compartment, whereas laminin and fibronectin were contributed by both cell types. Interestingly, synthesis and deposition of basement membrane heparan sulfate proteoglycan were exclusively observed in co-cultures, suggesting modulation of epithelial expression of this molecule by HIM cells. Finally, we observed that epithelial integrin-β1 chains redistributed at the basal domain of co-cultured Caco-2 cells. Taken to gether, these observations indicate that the Caco-2/HIM co-culture model is a valuable system to study in vitro human basement membrane formation in the context of intestinal epithelial-mesenchymal interactions. © 1993 Wiley-Liss, Inc.     

Therapeutic effects of a fermented soy product on peanut hypersensitivity is associated with modulation of T‐helper type 1 and T‐helper type 2 responses

Background ImmuBalance^? is a koji fungus (Aspergillus oryzae) and lactic acid fermented soybean product. This unique production process is believed to create a food supplement that helps to induce or maintain normal immune response. Objective To assess possible therapeutic effects of ImmuBalance^? on peanut (PN) hypersensitivity using a murine model of peanut allergy (PNA). Methods PN allergic C3H/HeJ mice were fed standard mouse chow containing 0.5% or 1.0% ImmuBalance (ImmuBalance 2X), radiation‐inactivated 1.0% ImmuBalance (I‐ImmuBalance 2X), or regular diet chow (sham) for 4 weeks, beginning 10 weeks after the initial PN sensitization, and then challenged with PN. Anaphylactic symptom scores, plasma histamine, serum PN specific‐IgE levels and splenocyte cytokine profiles were determined. Results While 100% of sham‐treated PNA mice developed anaphylactic reactions with a median score of 3.3 following PN challenge, only 50% of ImmuBalance, 30% of ImmuBalance 2X and 40% of I‐ImmuBalance 2X‐treated mice developed allergic reactions with median scores of 1.0, 0.4 and 0.5 respectively, which were significantly less than that in the sham‐treated mice (P<0.05). Plasma histamine and PN specific‐IgE levels were also significantly less in all treated mice than in sham‐treated mice (P<0.05). Furthermore, IL‐4, IL‐5 and IL‐13 production by PN‐stimulated splenocytes in vitro from ImmuBalance fed mice were markedly reduced compared with sham‐treated mice, whereas IFN‐γ production was moderately increased. TGF‐β and TNF‐α production were similar. Conclusions ImmuBalance protects against PN‐induced anaphylaxis when administered as a food supplement in this model. Protection was associated with down‐regulation of Th2 responses. This supplement may provide a potential novel therapy for PNA.     

Sclerosing haemangioma of the lung is positive for MIB-1 in cell membrane and cytoplasmic staining pattern

AIMS: MIB-1 is a monoclonal antibody raised against the recombinant part of the Ki67 antigen, which is expressed in the nuclei of all the active part of the cell cycle. Recently, cell membrane and cytoplasmic pattern of staining with MIB-1 in hyalinizing trabecular adenoma of the thyroid was reported. Sclerosing haemangioma of the lung is a tumour of uncertain histogenesis with a usually benign course. The tumour is mainly composed of cuboidal cells that line the papillary or tubular structure and pale cells that are found in the stroma. METHODS AND RESULTS: We examined a cell membrane and cytoplasmic immunoreactive pattern for MIB-1 antibody in sclerosing haemangioma. In all of the five cases examined, distinct cell membrane and cytoplasmic MIB-1 positivity mainly in the pale cells was noted. None of the carcinomas of the lung showed this type of staining with MIB-1. CONCLUSION: This result shows that, although the staining may be a result of cross reaction, cell membrane and cytoplasmic MIB-1 positivity is unique in that it could distinguish sclerosing haemangioma from other malignant epithelial tumours of the lung.     

3-Hydroxyglutaric acid is transported via the sodium-dependent dicarboxylate transporter NaDC3

Patients with glutaryl-CoA dehydrogenase (GCDH) deficiency accumulate glutaric acid (GA) and 3-hydroxyglutaric acid (3OH-GA) in their blood and urine. To identify the transporter mediating the translocation of 3OH-GA through membranes, kidney tissue of Gcdh-/- mice have been investigated because of its central role in urinary excretion of this metabolite. Using microarray analyses of kidney-expressed genes in Gcdh-/- mice, several differentially expressed genes encoding transporter proteins were identified. Real-time polymerase chain reaction analysis confirmed the upregulation of the sodium-dependent dicarboxylate cotransporter 3 (NaDC3) and the organic cation transporter 2 (OCT2). Expression analysis of NaDC3 in Xenopus laevis oocytes by the two-electrode-voltage-clamp technique demonstrated the sodium-dependent translocation of 3OH-GA with a K (M) value of 0.95 mM. Furthermore, tracer flux measurements in Chinese hamster ovary cells overexpressing OCT2 showed that 3OH-GA inhibited significantly the uptake of methyl-4-phenylpyridinium, whereas 3OH-GA is not transported by OCT2. The data demonstrate for the first time the membrane translocation of 3OH-GA mediated by NaDC3 and the cis-inhibitory effect on OCT2-mediated transport of cations.     

A Dominant Mutation in the Stereocilia‐Expressing Gene TBC1D24 is a Probable Cause for Nonsyndromic Hearing Impairment

Mutations in TBC1D24 have been linked to a variety of epileptic syndromes and recently to syndromic hearing impairment DOORS syndrome and nonsyndromic hearing impairment DFNB86. All TBC1D24 mutations reported so far were inherited in the recessive mode. In a dominant family segregated with late‐onset, progressive, nonsyndromic hearing impairment, linkage analysis revealed a 2.07 Mb candidate region on chromosome 16p13.3 that contains TBC1D24. Whole‐exome sequencing identified a heterozygous p.Ser178Leu variant of TBC1D24 as the only candidate mutation segregating with the hearing loss within the family. In perinatal mouse cochlea, we detected a restricted expression of Tbc1d24 in the stereocilia of the hair cells as well as in the spiral ganglion neurons. Our study suggested that the p.Ser178Leu mutation of TBC1D24 is a probable cause for dominant, nonsyndromic hearing impairment. Identification of TBC1D24 as the stereocilia‐expressing gene may shed new light on its specific function in the inner ear.     

Cytokine production by CD4 and CD8 T cells during the growth of Mycobacterium tuberculosis in mice

Changes in the pattern of cytokines found in CD4 and CD8 T cells during the growth of Mycobacterium tuberculosis that resulted in the establishment of a latent infection were monitored. Subsets of T cells were identified based on their differential expression of CD45 and CD44 which allowed them to be classified as naive, activated or memory. We found that the T cells in the lung produced a predominantly type 1 cytokine response. The appearance of large numbers of Th1 cells coincided with the establishment of latency. In contrast, the predominant response in the mediastinal lymph node and spleen was a Th2-type response.     

Rwdd1, a thymus aging related molecule, is a new member of the intrinsically unstructured protein family

We had previously identified a novel protein termed Rwddl whose expression in thymus is decreased in aged or oxidatively stressed mice. In the present study, we found that Rwddl expressed in both prokaryotic and eukaryotic ceils showed a slower migration rate on SDS-PAGE gel. In addition, Rwddl was more sensitive to proteinase proteolysis. Furthermore, being a highly acidic protein which contains an RWD domain, Rwddl shared a high level of sequence similarity with Gir2, a member of the intrinsically unstructured protein （IUP）. These findings suggest that Rwddl is a novel member of the IUP family.