产肠毒素脆弱类杆菌聚合酶链反应检测

背景:产肠毒素脆弱类杆菌(ETBF)能导致家畜、儿童和成人腹泻,已引起广泛关注,但国内尚未见相关报道。目的:通过ETBF聚合酶链反应(PCR)检测,调查不同人群粪便标本中ETBF的阳性率。方法:建立ETBFPCR检测,评估其敏感性和特异性,并检测正常对照组和腹泻组的ETBF阳性率。结果:PCR的敏感性为1×104CFU/ml,常见肠道正常菌群和致病菌均为阴性。正常对照组(475例)和腹泻组(498例)的ETBF阳性率分别为3.6%和6.4%(P=0.042)。其中成人对照组和成人腹泻组的阳性率分别为2.3%和5.7%(P=0.047),儿童对照组和儿童腹泻组的阳性率分别为5.1%和7.3%(P=0.33)。结论:ETBFPCR检测具有较好的敏感性和特异性,ETBF可能为腹泻的致病因素之一。     

非同位素标记脆弱类杆菌DNA探针的研究

为探索应用分子生物学技术检测厌氧菌的方法，选择脆弱类村抽提其染色体ＤＮＡ，用生物素和地高辛配基标记制备ＤＮＡ探针，检测厌氧和需氧菌９４株，临床标本４５份，并和＾３２Ｐ标记探针及培养生化鉴定比较，该探针可检出临床上常见的类杆菌属细菌与其它细菌无交叉反应，结果：地高辛配基标记探针的灵敏度（１０ｐｇＤＮＡ和１０＾３ＣＦＵ／ｍｌ）接近＾３２Ｐ标记探针的灵敏度（１ｐｇＤＮＡ和１０＾３ＣＦＵ／ｍｌ）。地高辛配     

A commensal symbiotic factor derived from Bacteroides fragilis promotes human CD39+Foxp3+ T cells and Treg function

Polysaccharide A (PSA) derived from the human commensal Bacteroides fragilis is a symbiosis factor that stimulates immunologic development within mammalian hosts. PSA rebalances skewed systemic T helper responses and promotes T regulatory cells (T_regs). However, PSA-mediated induction of Foxp3 in humans has not been reported. In mice, PSA-generated Foxp3⁺ T_regs dampen Th17 activity thereby facilitating bacterial intestinal colonization while the increased presence and function of these regulatory cells may guard against pathological organ-specific inflammation in hosts. We herein demonstrate that PSA induces expression of Foxp3 along with CD39 among naïve CD4 T cells in vitro while promoting IL-10 secretion. PSA-activated dendritic cells are essential for the mediation of this regulatory response. When cultured with isolated Foxp3⁺ T_regs, PSA enriched Foxp3 expression, enhanced the frequency of CD39⁺HLA-DR⁺ cells, and increased suppressive function as measured by decreased TNFα expression by LPS-stimulated monocytes. Our findings are the first to demonstrate in vitro induction of human CD4⁺Foxp3⁺ T cells and enhanced suppressive function of circulating Foxp3⁺ T_regs by a human commensal bacterial symbiotic factor. Use of PSA for the treatment of human autoimmune diseases, in particular multiple sclerosis and inflammatory bowel disease, may represent a new paradigm in the approach to treating autoimmune disease.     

Cell-cell contacts prevent anoikis in primary human colonic epithelial cells

BACKGROUND & AIMS: Colonic epithelial cells (CECs) receive important survival signals from the extracellular matrix and undergo detachment-induced apoptosis (anoikis) as soon as they lose their cell-matrix anchorage. In contrast to the established role of cell-matrix contact, the role of cell-cell contacts as a physiologic survival factor for CECs is less clear. METHODS: Intact CEC crypts gently centrifuged to form a cell aggregate in which cell-cell contacts were maintained. Induction of apoptosis was assessed by Western Blot analysis, colorimetric assays, DNA electrophoresis, 4',6-diamidino-2-phenylindole staining, and flow cytometry. Activation of survival pathways was analyzed by Western blot. The role of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (Erk)1/2, epidermal growth factor receptor, phosphatidylinositol 3-kinase (PI3-K), and Src signaling was investigated using specific inhibitors. RESULTS: Despite a complete loss of cell-matrix adhesion after CEC isolation, activation of caspases was blocked and anoikis was prevented when cell-cell contacts were preserved. CECs with preserved cell-cell contacts exhibited a rapid dephosphorylation of focal adhesion kinase. Aggregated CECs had stable levels of active beta-catenin and phosphorylated Akt, Erk1/2, and epidermal growth factor receptor, but CECs undergoing anoikis rapidly degraded beta-catenin and dephosphorylated Akt. Inhibition of Src- and PI3-K-dependent signaling reversed the antiapoptotic effect of cell-cell contact preservation, while inhibition of the MEK pathway had no effect. CONCLUSIONS: Integrity of cell-cell contacts compensates for the loss of cell-matrix contact-mediated survival signals in CECs and prevents apoptosis. Cell-cell contact-triggered CEC survival involves antiapoptotic signaling through beta-catenin-, Src-, and PI3-K/Akt- but not through MEK- and focal adhesion kinase-dependent pathways.     

大鼠结肠上皮细胞分离及培养方法的建立

目的:建立大鼠结肠上皮细胞的原代培养方法,为结肠上皮形态及功能研究提供理想的细胞模型.方法:采用6-15日龄乳鼠,取结肠剪碎、反复清洗后,弃上清,加10 mL、0.1%Ⅰ型胶原酶和透明质酸酶联合配制的消化液,37℃消化25 min,分离上皮细胞团;吹打消化液,取上清,加入培养基重悬反复离心3次后,细胞沉淀接种于含100 mL/L胎牛血清的完全培养基中,37℃、50 mL/L CO2细胞培养箱中培养.采用差速贴壁法及相差消化法去除成纤维细胞,细胞贴壁长满瓶80%-90%后用2.5 g/L的胰酶消化液消化并传代.结果:成功分离结肠上皮细胞团且活力较高.24 h后可见部分细胞团贴壁,贴壁细胞为多角形,4-8 d逐渐融合成片,呈现明显的"铺路石"样.细胞经传代处理后成纤维细胞明显减少.免疫荧光染色及透射电镜观察鉴定为上皮细胞.冻存处理复苏后细胞状态较佳.结论:通过该法可建立较稳定的大鼠结肠上皮细胞原代培养体系,为结肠上皮生理、病理及药物作用机制提供了体外研究平台.     

Metabolic activation of polycyclic aromatic hydrocarbons by human colonic mucosa and Bacteroides fragilis

Abstract The Ames Salmonella mutagenicity assay has been used to assess the metabolic activation of the following polycyclic aromatic hydrocarbons by human colonic microsomes (S9) and a cell-free extract of Bacteroides fragilis (Bf): 2-aminoanthracene, 1-naphthylamine, 2-naphthylamine, 2-aminofluorene, 2-acetylaminofluorene, anthracene, benzo(a)pyrene, 3-methylcholanthrene, 7, 12-dimethylbenzanthracene, acridine, 9-aminoacridine and 3-methylindole.
2-Aminoanthracene and 2-aminofluorene were the only compounds activated. In both cases, activation was dose-dependent and 2-aminofluorene exhibited synergistic activation by S9+Bf, as has previously been demonstrated with 2-aminoanthracene.
The organospecific aliphatic colonic carcinogen, 1, 2-dimethylhydrazine was not activated in this system. Metabolic activation by S9+Bf is thus restricted to aromatic compounds with three rings and an amino group in position 2.
These findings are consistent with enzymic substrate specificity, and are compatible with an enzymic basis for activation of polycyclic aromatic hydrocarbons by B. fragilis , present in large concentrations in the colonic lumen, and colonic microsomes.     

Different cytokine response of primary colonic epithelial cells to commensal bacteria

AIM: To determine if primary murine colonic epithelial cells (CEC) respond to commensal bacteria and discriminate between different types of bacteria. METHODS: A novel CEC: bacteria co-culture system was used to compare the ability of the colonic commensal bacteria, Bacteroides ovatus, E coli(SLF) and Lactobacillus rhamnosus (LGG) to modulate production of different cytokines (n = 15) by primary CEC. Antibody staining and flow cytometry were used to investigate Toll-like receptor (TLR) expression by CEC directly ex vivo and TLR responsiveness was determined by examining the ability of TLR ligands to influence CEC cytokine production. RESULTS: Primary CEC constitutively expressed functional TLR2 and TLR4. Cultured in complete medium alone, CEC secreted IL-6, MCP-1 and IP-10 the levels of which were significantly increased upon addition of the TLR ligands peptidoglycan (PGN) and lipopolysaccharide (LPS). Exposure to the commensal bacteria induced or up-regulated different patterns of cytokine production and secretion.E coli induced production of MIP-1α/β and p defensin3 whereas B. ovatus and L. rhamnosus exclusively induced MCP-1 and MIP-2α expression, respectively. TNFa, RANTES and MEC were induced or up-regulated in response to some but not all of the bacteria whereas ENA78 and IP-10 were up-regulated in response to all bacteria. Evidence of bacterial interference and suppression of cytokine production was obtained from mixed bacterial: CEC co-cultures. Probiotic LGG suppressed E coli- and B. ovatus-induced cytokine mRNA accumulation and protein secretion. CONCLUSION: These observations demonstrate the ability of primary CEC to respond to and discriminate between different strains of commensal bacteria and identify a mechanism by which probiotic bacteria (LGG) may exert anti-inflammatory effects in vivo.     

Extended role for insertion sequence elements in the antibiotic resistance of Bacteroides

The Bacteroides species are important micro-organisms, both in the normal physiology of the intestines and as frequent opportunistic anaerobic pathogens, with a deeply-rooted phylogenetic origin endowing them with some interesting biological features. Their prevalence in anaerobic clinical specimens is around 60%-80%, and they display the most numerous and highest rates of antibiotic resistance among all pathogenic anaerobes. In these antibiotic resistance mechanisms there is a noteworthy role for the insertion sequence (IS) elements, which are usually regarded as representatives of ‘selfish’ genes; the IS elements of Bacteroides are usually capable of up-regulating the antibiotic resistance genes. These include the cepA (penicillin and cephalosporin), cfxA (cephamycin), cfiA (carbapenem), nim (metronidazole) and ermF (clindamycin) resistance genes. This is achieved by outward-oriented promoter sequences on the ISs. Although some representatives are well characterized, e.g., the resistance gene-IS element pairs in certain resistant strains, open questions remain in this field concerning a better understanding of the molecular biology of the antibiotic resistance mechanisms of Bacteroides, which will have clinical implications.     

Interferon gamma downregulates IL-8 production in primary human colonic epithelial cells without induction of apoptosis

BACKGROUND: In acute or chronic inflammatory bowel disease (IBD) interferon gamma (IFNgamma) is still considered to be an important pro-inflammatory mediator. In the present study we investigated the impact of IFNgamma on interleukin-8 (IL-8) production as a read-out for cell activation in intestinal epithelial cell (IEC) lines and primary human colonic epithelial cells (CEC). METHODS: Primary cultures of human CEC were established from the mucosa of patients without inflammatory disease. CEC, HT-29 or Caco-2 cells were incubated with either IFNgamma, tumor necrosis factor (TNF)alpha or IL-10. IL-8 and IL-1Ra secretion was determined by ELISA. Competicon PCR was used for quantification of IL-8mRNA. Apoptosis was quantified by propidium iodine incorporation and fluorescence activated cell sorting (FACS) analysis. RESULTS: In contrast to HT-29 cells in primary human CEC 100 U/ml IFNgamma inhibited IL-8 secretion significantly to 70+/-15% of unstimulated primary CEC (p<0.005) more effectively than IL-10 (87+/-21% versus unstimulated cells, n.s.). In HT-29 cells, IL-8 secretion was induced to 405+/-101% of unstimulated cells. In Caco-2 cells, IFNgamma had no significant effect on IL-8 secretion. The effect in HT-29 and CEC was concentration dependent. In primary CEC, 200 U/ml IFNgamma further reduced IL-8 secretion to 48+/-18% of unstimulated CEC (p<0.05). Whereas IL-8 mRNA was strongly upregulated in HT-29 cells, no upregulation or even a downregulation was found in CEC. Pre-incubation with 100 U/ml IFNgamma did not increase the susceptibility to apoptosis mediated by anti-Fas antibody (CH-11) in primary CEC, whereas HT-29 cells showed increased rates of apoptosis after priming with IFNgamma. CONCLUSION: In contrast to HT-29, IFNgamma downregulated IL-8 secretion and did not induce IL-8 mRNA expression in primary human CEC. This effect was not due to induction of apoptosis.     

Promoter orientation of the immunomodulatory Bacteroides fragilis capsular polysaccharide A (PSA) is off in individuals with inflammatory bowel disease (IBD)

ABSTRACT

Bacteroides fragilis is a member of the normal microbiota of the lower gastrointestinal tract, but some strains produce the putative tumourigenic B. fragilis toxin (BFT). In addition, B. fragilis can produce multiple capsular polysaccharides that comprise a microcapsule layer, including an immunomodulatory, zwitterionic, polysaccharide A (PSA) capable of stimulating anti-inflammatory interleukin-10 (IL-10) production.

It is known that the PSA promoter can undergo inversion, thereby regulating the expression of PSA. A PCR digestion technique was used to investigate B. fragilis capsular PSA promoter orientation using human samples for the first time. It was found that approximately half of the B. fragilis population in a healthy patient population had PSA orientated in the ‘ON’ position. However, individuals with inflammatory bowel disease (IBD) had a significantly lower percentage of the B. fragilis population with PSA orientated ‘ON’ in comparison with the other patient cohorts studied. Similarly, the putative tumourigenic bft-positive B. fragilis populations were significantly associated with a lower proportion of the PSA promoter orientated ‘ON’.

These results suggest that the proportion of the B. fragilis population with the PSA promoter ‘ON’ may be an indicator of gastrointestinal health.     

Cyr 61基因过表达对人肾小管上皮细胞凋亡的影响

摘　要　目的:观察Cyr61基因过表达对人肾小管上皮细胞 (HKC)凋亡的影响,探讨Cyr61在常染色体显性多囊肾病(ADPKD)发病中的作用及机制。　方法:RT PCR扩增Cyr61基因全长,构建pcDNA3. 1 Cyr61重组质粒,转染并筛选获得稳定转染pcDNA3. 1 Cyr61的HKC细胞。经RT PCR、Northern、Westernblot鉴定转染细胞系基因的整合及表达。对空白HKC、空质粒pcDNA3. 1转染的HKC、Cyr61转染的HKC和囊肿衬里上皮细胞去血清培养 72h后,流式细胞仪检测细胞凋亡指数,Westernblot检测磷酸化Akt和Bad含量。　结果:构建了pcDNA3. 1 Cyr61重组质粒并获得稳定转染该质粒的HKC细胞。空白HKC与质粒pcDNA3. 1转染组之间上述指标无显著差异(P>0 05),Cyr61基因转染组与空白组、pcDNA3. 1转染组相比,凋亡指数显著降低 (P<0. 01),磷酸化Akt和磷酸化Bad含量显著增高 (P<0 .01 ),Cyr61基因转染组与囊肿衬里上皮细胞之间上述指标无显著差异 (P>0. 05);在Cyr61抗体存在下,Cyr61基因转染组的上述指标与空白组无显著差异 (P>0 .05 )。　结论:过表达Cyr61的HKC对去血清诱导的凋亡特性与囊肿衬里上皮细胞相似,过表达的Cyr61可能通过自分泌途径,促进Akt和Bad磷酸化,抑制细胞凋亡,参与ADPKD囊肿形成和发展。     

Bacteroides fragilis type VI secretion systems use novel effector and immunity proteins to antagonize human gut Bacteroidales species

Type VI secretion systems (T6SSs) are multiprotein complexes best studied in Gram-negative pathogens where they have been shown to inhibit or kill prokaryotic or eukaryotic cells and are often important for virulence. We recently showed that T6SS loci are also widespread in symbiotic human gut bacteria of the order Bacteroidales, and that these T6SS loci segregate into three distinct genetic architectures (GA). GA1 and GA2 loci are present on conserved integrative conjugative elements (ICE) and are transferred and shared among diverse human gut Bacteroidales species. GA3 loci are not contained on conserved ICE and are confined to Bacteroides fragilis. Unlike GA1 and GA2 T6SS loci, most GA3 loci do not encode identifiable effector and immunity proteins. Here, we studied GA3 T6SSs and show that they antagonize most human gut Bacteroidales strains analyzed, except for B. fragilis strains with the same T6SS locus. A combination of mutation analyses, trans-protection analyses, and in vitro competition assays, allowed us to identify novel effector and immunity proteins of GA3 loci. These proteins are not orthologous to known proteins, do not contain identified motifs, and most have numerous predicted transmembrane domains. Because the genes encoding effector and immunity proteins are contained in two variable regions of GA3 loci, GA3 T6SSs of the species B. fragilis are likely the source of numerous novel effector and immunity proteins. Importantly, we show that the GA3 T6SS of strain 638R is functional in the mammalian gut and provides a competitive advantage to this organism.Bacteria that live in communities have numerous mechanisms to compete with other strains and species. The ability to acquire nutrients is a major factor dictating the success of a species in a community. In addition, the production of secreted factors, such as bacteriocins, that competitively interfere or antagonize other strains/species, also contributes to a member’s fitness in a community. In the microbe-dense human gut ecosystem, such factors and mechanisms of antagonism by predominant members are just beginning to be described, as are models predicting the relevance of these competitive interactions to the microbial community (1). Bacteroidales is the most abundant order of bacteria in the human colonic microbiota, and also the most temporally stable (2). The fact that numerous gut Bacteroidales species stably cocolonize the human gut at high density raises the question of how these related species and strains interact with each other to promote or limit each other’s growth. We previously showed that coresident Bacteroidales strains intimately interact with each other and exchange large amounts of DNA (3) and also cooperate in the utilization of dietary polysaccharides (4). To date, two types of antagonistic factors/systems have been shown to be produced by human gut Bacteroidales species: secreted antimicrobial proteins (5) and T6SSs (3, 6, 7). However, neither of these antagonistic processes has been analyzed to determine if they provide a competitive advantage in the mammalian intestine.Type VI secretion systems (T6SSs) are contact-dependent antagonistic systems used by some Gram-negative bacteria to intoxicate other bacteria or eukaryotic cells. The T6 apparatus is a multiprotein, cell envelope spanning complex comprised of core Tss proteins. A key component of the machinery is a needle-like structure, similar to the T4 contractile bacteriophage tail, which is assembled in the cytoplasm where it is loaded with toxic effectors (8–10). Contraction of the sheath surrounding the needle apparatus drives expulsion of the needle from the cell, delivering the needle and associated effectors either into the supernatant of in vitro grown bacteria, or across the membrane of prey cells. Identified T6SS effectors include cell wall degrading enzymes (11), proteins that affect cell membranes such as phospholipases (12) and pore-forming toxins (13, 14), proteins that degrade NAD(P)+ (15), and nucleases (16). The effector protein is produced with a cognate immunity protein, typically encoded by the adjacent downstream gene (17), which protects the producing cell from the toxicity of the effector. Although both eukaryotic and bacterial cells are targeted by T6SS effectors (18), most described T6SSs target Gram-negative bacteria.We previously performed a comprehensive analysis of all sequenced human gut Bacteroidales stains and found that more than half contain T6SS loci (7). These T6SSs are similar to the well-described T6SSs of Proteobacteria in that remote orthologs of many Proteobacterial Tss proteins are encoded by Bacteroidales T6SS regions, with the exception of proteins that likely comprise the transmembrane complex, which are distinct. The T6SS loci of human gut Bacteroidales species segregate into three distinct genetic architectures (GA), designated GA1, GA2, and GA3, each with highly identical segments within a GA comprising the core tss genes (7). GA1 and GA2 T6SS loci are present on large ∼80- to 120-kb integrative conjugative elements (ICE) that are extremely similar at the DNA level within a GA. Due to the ability of these T6SS regions to be transferred between strains via ICE, GA1 and GA2 T6SS loci are present in diverse human gut Bacteroidales species. GA3 T6SS loci are confined to Bacteroides fragilis and are not contained on conserved ICE (7).Although T6SS loci of a particular GA are highly identical to each other, each GA has internal regions of variability where the genes differ between strains (7). The variable regions of GA1 and GA2 T6SS loci contain genes encoding the identifiable toxic effector and cognate immunity proteins found in these regions. Unlike the GA1 and GA2 T6SS loci, there are no identifiable genes encoding toxin or immunity proteins in the two variable regions or other areas of GA3 T6SS loci. The present study was designed to answer three fundamental questions regarding GA3 T6SS loci: (i) Because no known effectors/immunity proteins are encoded by these regions, are they involved in bacterial antagonism? And if so, what prey cells do they target? (ii) Do the variable regions contain genes encoding effector and immunity proteins? and (iii) If GA3 T6SSs mediate bacterial antagonism, do they provide a competitive advantage in the mammalian gut?     

Moxibustion down-regulates colonic epithelial cell apoptosis and repairs tight junctions in rats with Crohn's disease

AIM: To investigate the effects of moxibustion on down-regulation of the colonic epithelial cell apoptosis and repair of the tight junctions in rats with Crohn’s disease (CD). METHODS: Sixty male Sprague-Dawley rats were randomly divided into a normal control (NC) group, a model control (MC) group, an herbs-partitioned moxibustion (HPM) group, a mild-warm moxibustion (MWM) group and a salicylazosulphapyridine (SASP) group, with 12 rats in each group. The CD model rats were treated with trinitrobenzene sulph...     

A commensal symbiotic factor derived from Bacteroides fragilis promotes human CD39+Foxp3+ T cells and Treg function

Polysaccharide A (PSA) derived from the human commensal Bacteroides fragilis is a symbiosis factor that stimulates immunologic development within mammalian hosts. PSA rebalances skewed systemic T helper responses and promotes T regulatory cells (T_regs). However, PSA-mediated induction of Foxp3 in humans has not been reported. In mice, PSA-generated Foxp3⁺ T_regs dampen Th17 activity thereby facilitating bacterial intestinal colonization while the increased presence and function of these regulatory cells may guard against pathological organ-specific inflammation in hosts. We herein demonstrate that PSA induces expression of Foxp3 along with CD39 among naïve CD4 T cells in vitro while promoting IL-10 secretion. PSA-activated dendritic cells are essential for the mediation of this regulatory response. When cultured with isolated Foxp3⁺ T_regs, PSA enriched Foxp3 expression, enhanced the frequency of CD39⁺HLA-DR⁺ cells, and increased suppressive function as measured by decreased TNFα expression by LPS-stimulated monocytes. Our findings are the first to demonstrate in vitro induction of human CD4⁺Foxp3⁺ T cells and enhanced suppressive function of circulating Foxp3⁺ T_regs by a human commensal bacterial symbiotic factor. Use of PSA for the treatment of human autoimmune diseases, in particular multiple sclerosis and inflammatory bowel disease, may represent a new paradigm in the approach to treating autoimmune disease.     

CRF通过CRF2受体介导肠上皮细胞中TLR4的表达

目的:研究促肾上腺皮质释放因子(CRF)介导肠上皮细胞中Toll样受体4(toll-like receptor4,TLR4)的表达,并探讨其可能通过的受体途径.方法:常规培养人结肠上皮细胞株HT-29细胞,将HT-29细胞分为正常对照组(不加刺激剂),脂多糖(lipopolysaccharide,LPS)刺激组(LPS20g/L刺激24h),促肾上腺皮质释放因子(corticotrophin-releasing factor,CRF)刺激组(CRF20g/L刺激24h),CRF+LPS刺激组(预先CRF20g/L刺激12h,更换细胞液后再与LPS20g/L刺激12h),CRF+Antalarmin 组(CRF与Antalarmin20g/L共刺激24h),CRF+LPS+Antalarmin组(CRF与Antalarmin20g/L共刺激12h后再以LPS刺激12h),CRF+Astressin2B组(CRF与Astressin2B20g/L共刺激24h),CRF+LPS+Astressin2B组(CRF与Astressin2B20g/L共刺激12h后再以LPS刺激12h).刺激结束后,收取各组HT-29细胞,RT-PCR法和免疫印迹法检测各组上皮细胞中TLR4mRNA和蛋白的表达.ELISA法检测各组细胞上清液中IL-8的表达.结果:CRF可以诱导人结肠上皮细胞株HT-29细胞中TLR4表达导致IL-8分泌增多(P<0.05),C R F1受体拮抗剂不能有效地阻滞C R F对TLR4的诱导(P>0.05,CRF+LPS组vs CRF组),CRF2受体拮抗剂可阻滞CRF对TLR4的诱导(P<0.05,CRF+LPS组vs CRF组).结论:CRF通过CRF2受体通路介导肠上皮细胞中TLR4的表达.     

MMP-7 is involved in the aging of primary human mammary epithelial cells (HMEC)

Primary cultures of human mammary epithelial cells underwent significant morphological and functional changes during the aging process between passage 12 (P12) and passage 16 (P16). Concomitant with a progressive and significant expression of senescence-associated beta-galactosidase as aging marker, the cells restructured their attachment, increased in size and ceased to divide. Young HMEC until P11 demonstrated a nearly 100% expression of distinct adhesion molecules such as CD24, integrin beta1 (CD29) and CD44 similar to the human mammary tumor cell line MCF-7. In parallel with the aging-associated alterations of the cell adhesion, expression of CD24 and CD44 dropped in senescent P16 HMECs. However, levels of CD29 remained unchanged during the aging process. The tumor-associated Muc-1 (CD227), which was expressed to about 100% in the tumorigenic MCF-7 cells, was detectable in 51% of young HMEC in P11 and declined to 37% in aged HMEC in P16. In association with the remodeling of cell shape, expression levels of distinct matrix metalloproteinases including MMP-7 markedly decreased in aging HMEC. In contrast, MMP-1, MMP-2 and MMP-9 remained unchanged indicating a possible functional role of MMP-7 during the HMEC aging process. Indeed, down-modulation of MMP-7 by RNAi revealed a significantly elevated G(2)/M cell cycle arrest and a 2- to 3-fold enhanced senescence-associated beta-galactosidase expression as compared to control siRNA transfectants and control HMEC, respectively. Together, these findings suggested that decreasing MMP-7 expression contributes to accelerated aging of human mammary epithelial cells.     

Regulation of chemokine responses in intestinal epithelial cells by stress and Toxoplasma gondii infection

Infection with Toxoplasma gondii induces chemokine up-regulation in several cell types. Here, we investigated the role of stress products (norepinephrine, NE) on chemokine production in mouse intestinal epithelial cells (IECs). Purified IECs were used to determine the expression levels of chemokines by real-time PCR. There was significantly increased expression in CCL2, CCL3, CCL5, CXCL2, CXCL9 and CXCL10 in IECs following peroral infection with T. gondii (INF) on day eight post-infection (PI) compared to infected mice subjected to cold-water stress (INF+CWS). In vitro studies using the MODE-K cell line showed increased chemokine mRNA and protein expression in infected but not in cells exposed to parasite antigen. Down-regulation of chemokine expression was more pronounced when active infection was used in combination with NE. Chemokine receptor expression was increased in IECs isolated from INF and decreased in the INF+CWS group. In MODE-K cells, there was decreased mRNA expression of chemokine receptors when incubated with β-adrenergic antagonists. Neither, adrenergic antagonists blocked the effect of infection on chemokine receptor expression. Cold-water stress was able to decrease expression of chemokines and their receptors in IECs in vivo and in vitro. Cold-water stress-mediated modulation of innate intestinal responses are beneficial in C57BL/6 mice during T. gondii infection.