Oseltamivir-resistant 2009-2010 pandemic influenza A (H1N1) in an immunocompromised patient.

Although neuraminidase inhibitors are active against most 2009-2010 pandemic influenza A (H1N1) swine-origin strains, sporadic cases of oseltamivir resistance have been described. Since April 2009, 54 cases of oseltamivir-resistant H1N1 swine-origin have been reported in the USA (http://www.cdc.gov/flu/weekly/; accessed 1 February 2010). Approximately 1.4% of tested isolates are oseltamivir resistant. We report a patient with an underlying hematological malignancy who was hospitalized with influenza A (H1N1) swine-origin and whose strain developed oseltamivir resistance during therapy.

tPA-mediated generation of plasmin is catalyzed by the proteoglycan NG2.

Paralysis resulting from spinal cord injury is devastating and persistent. One major reason for the inability of the body to heal this type of injury ensues from the local increase of glial cells leading to the formation of a glial scar, and the upregulation of chondroitin sulfate proteoglycans (CSPGs) at the site of injury through which axons are unable to regenerate. Experimental approaches to overcome this problem have accordingly focused on reducing the inhibitory properties of CSPGs, for example by using chondroitinase to remove the sugar chains and reduce the CSPGs to their core protein constituents, although this step alone does not provide dramatic benefits as a monotherapy. Using in vitro and in vivo approaches, we describe here a potentially synergistic therapeutic opportunity based on tissue plasminogen activator (tPA), an extracellular protease that converts plasminogen (plg) into the active protease plasmin. We show that tPA and plg both bind to the CSPG protein NG2, which functions as a scaffold to accelerate the tPA-driven conversion of plg to plasmin. The binding occurs via the tPA and plg kringle domains to domain 2 of the NG2 CSPG core protein, and is enhanced in some settings after chondroitinase-mediated removal of the NG2 proteoglycan side chains. Once generated, plasmin then degrades NG2, both in an in vitro setting using recombinant protein, and in vivo models of spinal cord injury. Our finding that the tPA and plg binding is in some instances more efficient after exposure of the NG2 proteoglycan to chondroitinase treatment suggests that a combined therapeutic approach employing both chondroitinase and the tPA/plasmin proteolytic system could be of significant benefit in promoting axonal regeneration through glial scars after spinal cord injury.

The trefoil protein TFF1 is bound to MUC5AC in human gastric mucosa.

The trefoil protein TFF1 is expressed principally in the superficial cells of the gastric mucosa. It is a small protein and forms homo- and hetero-dimers via a disulphide bond through Cys58 which is located three amino acids from the C terminus. TFF1 is co-expressed with the secreted mucin MUC5AC in superficial cells of the gastric mucosa suggesting that it could be involved in the packaging or function of gastric mucus. We have previously shown that TFF1 co-sediments with mucin glycoproteins on caesium chloride gradients. To extend this observation we have now used gel filtration under physiological conditions, immunoprecipitation and Western transfer analysis to characterise the interaction of TFF1 with gastric mucin glycoproteins. We show that TFF1 co-elutes with MUC5AC but not MUC6 on gel filtration and that immunoprecipitation and Western transfer analysis confirms that TFF1 interacts with MUC5AC. We also demonstrate that the TFF1 dimer is the predominant molecular form bound to MUC5AC. Salt and chelators of divalent cations such as EDTA and EGTA disrupted the TFF1- MUC5AC interaction and increased the degradation of MUC5AC, whereas calcium increased the amount of TFF1 bound to MUC5AC. These data support the contention that TFF1 is pivotal in the packaging and function of human gastric mucosa.

Development of a two-site ELISA assay for the dimeric form of human TFF1.

TFF1 is one of three human trefoil proteins expressed principally in the gastrointestinal tract in normal tissues. TFF1 protects the gastric mucosa against damage as a result of its ability to facilitate reconstitution of damaged gastric mucosa and its involvement in the secretion and structure of gastric mucus. The most biologically active molecular form in cell culture and animal models tested is a dimer formed by a disulfide bond between two cysteine residues close to the C terminus of the protein. We have therefore developed an assay for this form of TFF1 which should facilitate its measurement in biological samples.

Synergistic effects of systemic trefoil factor family 1 (TFF1) peptide and epidermal growth factor in a rat model of colitis.

Novel therapies for the treatment of colitis are required. We therefore examined the potential value of the trefoil factor family 1 (TFF1) peptide and epidermal growth factor (EGF) alone and in combination. Effects of TFF1- Cys58 +/- EGF on an in vitro HT29 cell wounding model of restitution showed synergistic activity when used in combination. In addition, animals had colitis induced by adding 4% dextran sulphate sodium (DSS) to the drinking water for 7 days and they also received twice daily subcutaneous injections of test peptides. Treatment with TFF1-Cys58 alone (100 microg/kg) reduced histological colitis score by 22%, but the TFF1-Ser58 variant was ineffective. In a second study, TFF1-Cys58 reduced histological colitis score by 15%, EGF (600 microg/kg) by 26%, and an additive response (42% reduction) was demonstrated when used together (P < 0.01 versus either peptide given alone). Similar results were found using tissue myeloperoxidase (MPO) activity as a marker of inflammation. Where clinical risk/benefit seems justified, these initial studies suggest that combination therapy of systemic EGF and TFF peptides may prove useful for treatment of colitis in patients with disease extending beyond the reach of topical (enema) therapy.

High expression of the trefoil protein TFF1 in interval breast cancers.

Breast cancer screening is important for the early detection of breast cancer. Tumors that become symptomatic in the screening interval are known as interval cancers but the reasons for their rapid progression are unknown. Estrogen receptor expression is lower in interval cancers suggesting that they may have reduced hormonal responsiveness. To investigate this hypothesis we have measured the expression of the estrogen receptor and three estrogen-responsive genes (cathepsin D, progesterone receptor, and TFF1) in screen-detected and interval breast cancers. The expression of the protease cathepsin D was not associated with estrogen receptor in either group of tumor. Progesterone receptor expression was highly correlated with that of the estrogen receptor in both groups of tumors but it was not expressed at significantly different levels in the two groups of tumors. Expression of TFF1, a cellular motogen, was correlated with estrogen receptor in screen-detected but not interval cancers and was expressed at markedly higher levels in interval breast tumors, the group that expresses lower levels of estrogen receptor. Interval cancers are characterized by high levels of expression of TFF1 and/or Ki67 suggesting that cell migration and cell division play important roles in the rapid progression of interval cancers. The observation that TFF1 expression in interval cancers tends to be estrogen-independent and that interval cancers have reduced estrogen receptor expression suggests they may have a reduced response to hormone therapy.

Activation of cellular invasion by trefoil peptides and src is mediated by cyclooxygenase- and thromboxane A2 receptor-dependent signaling pathways.

We have investigated the possible functional relationships between cellular invasion pathways induced by trefoil factors (TFFs), src, and the cyclooxygenases COX-1 and COX-2. Pharmacological inhibitors of the Rho small GTPase (C3 exoenzyme), phospholipase C (U-73122), cyclooxygenases (SC-560, NS-398), and the thromboxane A2 receptor (TXA2-R) antagonist SQ-295 completely abolished invasion induced by intestinal trefoil factor, pS2, and src in kidney and colonic epithelial cells MDCKts.src and PCmsrc. In contrast, invasion was induced by the TXA2-R mimetic U-46619, constitutively activated forms of the heterotrimeric G-proteins Galphaq (AGalphaq), Galpha12, Galpha13 (AGalpha12/13), which are signaling elements downstream of TXA2-R. Ectopic overexpression of pS2 cDNA and protein in MDCKts.src-pS2 cells and human colorectal cancer cells HCT8/S11-pS2 initiate distinct invasion signals that are Rho independent and COX and TXA2-R dependent. We detected a marked induction of COX-2 protein and accumulation of the stable PGH2/TXA2 metabolite TXB2 in the conditioned medium from cells transformed by src. This led to activation of the TXA2-R-dependent invasion pathway, which is monitored via a Rho- and Galpha12/Galpha13-independent mechanism using the Galphaq/PKC signaling cascade. These findings identify a new intracrine/paracrine loop that can be monitored by TFFs and src in inflammatory diseases and progression of colorectal cancers.

Dramatic diurnal variation in the concentration of the human trefoil peptide TFF2 in gastric juice.

BACKGROUND: TFF2, a member of the trefoil factor family of proteins, is a glycosylated protein of 106 amino acids. It is secreted by gastric antral and pyloric glands and by Brunner's glands of the duodenum. TFF2 is found in high concentrations around sites of ulceration. It stimulates cell motility and is probably the principal cytoprotective trefoil peptide in the stomach. AIMS: To determine if production of TFF2 follows a circadian rhythm and to measure changes in secretion of TFF2 in response to food intake and during sleep. SUBJECTS: Young healthy adults were recruited. They were asymptomatic and were not receiving medication. The 24 hour regimen was designed to allow normal stimulation of gastric secretion in response to food intake and sleep. Gastric juice was collected two hourly via a nasogastric tube. METHODS: Glycosylated and non-glycosylated TFF2 proteins were measured by quantitative western transfer analysis. The results were analysed statistically using SPSS software. RESULTS: There was a dramatic diurnal variation in the concentration of TFF2. The mean concentration was lowest in the early evening (0.29 microg/ml), increased gradually during the evening, and then sharply during the night to reach 7.9 microg/ml. The ratio of glycosylated to non-glycosylated TFF2 varied and was higher during the night than in the afternoon. pH, total protein, and pepsin concentrations in gastric juice did not vary significantly over 24 hours. CONCLUSION: The data suggest that diurnal variations in TFF2 secretion occur independently of pepsin and gastric acid secretion. The concentration of glycosylated TFF2 in the gastric lumen falls in response to food intake. TFF2 secretion increases during inactivity and sleep. These results suggest that secretion of TFF2 in the stomach is highest during the night and that the cytoprotective effects of TFF2 on the gastric mucosa occur mainly during sleep.

The solution structure of the disulphide-linked homodimer of the human trefoil protein TFF1.

The trefoil factor family protein, TFF1, forms a homodimer, via a disulphide linkage, that has greater activity in wound healing assays than the monomer. Having previously determined a high-resolution solution structure of a monomeric analogue of TFF1, we now investigate the structure of the homodimer formed by the native sequence. The two putative receptor/ligand recognition domains are found to be well separated, at opposite ends of a flexible linker. This contrasts sharply with the known fixed and compact arrangement of the two trefoil domains of the closely related TFF2, and has significant implications for the mechanism of action and functional specificity of the TFF of proteins.

Induction of scattering and cellular invasion by trefoil peptides in src- and RhoA-transformed kidney and colonic epithelial cells.

Trefoil factors (TFFs) are protease-resistant peptides that promote epithelial cell migration and mucosal restitution during inflammatory conditions and wound healing in the gastrointestinal tract. To date, the molecular mechanism of TFFs action and their possible role in tumor progression are unclear. In the present study, we observed that premalignant human colonic PC/AA/C1 and canine kidney MDCK epithelial cells are not competent to invade collagen gels in response to exogenously added TFFs (pS2, spasmolytic polypeptide, and intestinal trefoil factor). In contrast, activated src and RhoA exert permissive induction of invasion by the TFFs that produce similar parallel dose-response curves in src-transformed MDCKts.src and PCmsrc cells (EC50=20-40 nM). Cell scattering is also induced by TFFs in MDCKts.src cells. Stable expression of the pS2 cDNA promotes constitutive invasiveness in MDCKts.src-pS2 cells and human colonic HCT8/S11-pS2 cells established from a sporadic tumor. Furthermore, we found that TFF-mediated cellular invasion is dependent of several signaling pathways implicated in cell transformation and survival, including phosphoinositide PI3'-kinase, phospholipase C, protein kinase C, and the rapamycin target TOR. Constitutive and intense expression of pS2 was revealed by Western blot analyses and immunohistochemistry in human colorectal tumors and their adjacent control mucosa during the neoplastic progression, from the adenoma to the liver metastases. Our studies indicated that TFFs can be involved in cell scattering and tumor invasion via autocrine loops and may serve as potential targets in the control of colon cancer progression.

Insulin receptor substrate-1 expression is regulated by estrogen in the MCF-7 human breast cancer cell line.

Estrogens can stimulate the proliferation of estrogen-responsive breast cancer cells by increasing their proliferative response to insulin-like growth factors. The mechanism underlying the increased proliferation could involve the induction of components of the insulin-like growth factor signal transduction pathway by estrogen. In this study we have examined the regulation of the expression of insulin receptor substrate-1, a major intracellular substrate of the type I insulin-like growth factor receptor tyrosine kinase. Estradiol increased insulin receptor substrate-1 mRNA and protein levels at concentrations consistent with a mechanism involving the estrogen receptor. Insulin receptor substrate-1 was not induced significantly by the antiestrogens tamoxifen and ICI 182,780, but they inhibited the induction of insulin receptor substrate-1 by estradiol. Analysis of tyrosine-phosphorylated insulin receptor substrate-1 showed that the highest levels were found in cells stimulated by estradiol and insulin-like growth factor-I, whereas low levels were found in the absence of estradiol irrespective of whether type I insulin-like growth factor ligands were present. Insulin receptor substrate-2, -3, and -4 were not induced by estradiol. These results suggest that estrogens and antiestrogens may regulate cell proliferation by controlling insulin receptor substrate-1 expression, thereby amplifying or attenuating signaling through the insulin-like growth factor signal transduction pathway.

The human two domain trefoil protein, TFF2, is glycosylated in vivo in the stomach.

BACKGROUND: TFF2, a member of the trefoil factor family (TFF) of peptides, is a secreted protein of 106 amino acids that is expressed in mucous neck cells of the fundus and glands at the base of the antrum in normal human stomach. TFF2 is also detected at high concentrations around sites of ulceration. It is protective against mucosal damaging agents and stimulates cell motility. AIMS: To measure the expression of TFF2 in normal human stomach and its secretion into gastric juice. METHODS: TFF2 cDNA was amplified by reverse transcription polymerase chain reaction from gastric mucosa and sequenced. Gastric juice or cytosol, prepared from gastric mucosa, was obtained from individuals with macroscopically normal stomachs. TFF2 concentrations were measured by quantitative western transfer analysis. RESULTS: Sequencing of TFF2 cDNA revealed a single amino acid change from the published sequence. Significant amounts of 12 kDa TFF2 were detected in human gastric juice. Larger quantities of a protein of higher apparent molecular mass were also detected. This was shown to be N-glycosylated TFF2 using the endoglycosidase, peptide-N-Gycosidase F. The majority of TFF2 in normal gastric mucosa was also glycosylated. CONCLUSIONS: Human TFF2 is glycosylated via an N-linkage, presumably on Asn(15) which forms part of the single consensus site for N-glycosylation in human TFF2. The glycosylation may be of functional significance. Future studies of human TFF2 should use antibodies raised against the correct amino acid sequence. Biological studies should be performed with recombinant protein of the correct sequence, and the biological consequences of glycosylation investigated.

The human trefoil peptide, TFF1, is present in different molecular forms that are intimately associated with mucus in normal stomach.

BACKGROUND: TFF1 is a 6.5 kDa secreted protein that is expressed predominantly in normal gastric mucosa. It is coexpressed with mucins and it can form dimers via a free carboxy terminal cysteine residue. AIMS: To investigate the molecular forms of TFF1 that are present in normal human stomach and the association of the different molecular forms with mucus. SUBJECTS: All subjects had macroscopically normal stomachs at gastroscopy. None had a significant past medical history. METHODS: TFF1 was detected in normal gastric mucosa and adherent mucus by western transfer analysis after electrophoresis on reducing and non-reducing polyacrylamide gels. In some instances, proteins were fractionated by caesium chloride density gradient centrifugation prior to detection of TFF1. The location of TFF1 in gastric mucosa with an intact adherent mucus layer was assessed by immunohistochemistry. RESULTS: Three different molecular forms of TFF1 were detected: TFF1 monomer, TFF1 dimer, and a TFF1 complex with an apparent molecular mass of about 25 kDa. TFF1 was present at higher concentrations than realised previously. The TFF1 complex was present in the adherent mucus gel layer but while its interaction with mucin was destabilised by caesium chloride, the interaction between mucin and the TFF1 dimer was resistant to caesium chloride. CONCLUSIONS: Most of TFF1 in normal human gastric mucosa is present in a complex that is stabilised by a disulphide bond. TFF1 is intimately associated with mucus. The high concentration, colocalisation, and binding of TFF1 to gastric mucus strongly implicate TFF1 in gastric mucus function.

Selective promoter usage of the human estrogen receptor-alpha gene and its regulation by estrogen.

Three promoters have been identified for the human estrogen receptor-alpha gene. The positions of promoters A and B are known whereas that of the recently identified promoter C is not. Cloning and hybridization experiments demonstrated that promoter C is located more than 21 kb upstream of promoter A. The use of the three promoters was examined in estrogen receptor-positive breast cancer cell lines, cell lines derived from other malignancies, and some normal tissues by RT-PCR and transient transfection. All estrogen-responsive breast cancer cell lines used all three promoters, apart from ZR-75 cells, which did not use promoter B in one of two sublines examined. Cell lines derived from other malignancies and other normal tissues that express lower levels of estrogen receptor-alpha showed more selective promoter usage. This suggests that the level of expression of estrogen receptor-alpha is determined by the number of promoters used, rather than the selective use of specific promoters. We also show that promoter C is used more widely than suggested by others. Analysis of a series of estrogen receptor-positive primary breast tumors showed that all three promoters were used in all the tumors. All three promoters were modulated by estrogen in estrogen-responsive breast cancer cell lines: all three promoters were down-regulated by estrogen in MCF-7 cells in which estrogens reduce receptor expression whereas all promoters used were upregulated in T47D, ZR-75, and EFM-19 cells in which estrogens increase receptor expression. This suggests that it is the repertoire of transcription factors present within a cell rather than the selective use of a specific promoter that determines whether estrogen receptor-alpha expression is increased or decreased by estrogen.

Differences in Ki67 and c-erbB2 expression between screen-detected and true interval breast cancers.

Breast cancer screening facilitates the early detection of breast cancer, although a significant number of tumors still arise in the interval between screening. The objective of this study was to measure the expression of five markers of proven prognostic significance in symptomatic breast cancer (estrogen receptor, progesterone receptor, p53, Ki67, and c-erbB2) in screen-detected and interval breast cancers to identify biological markers that may be associated with the emergence of symptomatic breast cancer in the screening interval. The expression of estrogen receptor, progesterone receptor, p53, Ki67, and c-erbB2 was assessed in a series of 51 true interval and 84 screened-detected invasive tumors by immunohistochemistry. Interval cancers tended to be of higher histological grade and were of larger pathological size than screen-detected cancers. Expression of estrogen receptor was 1.7-fold lower (P<0.001), whereas expression of p53 was 2.5-fold (P<0.01), Ki67 2.4-fold (P<0.001), and c-erbB2 3.6-fold higher (P<0.01) in true interval cancers compared with screen-detected invasive cancers. There was no significant difference in progesterone receptor expression. The most important differences identified by multiple logistic regression analysis were in the expression of Ki67 and c-erbB2. The differences in the expression of these markers were more important than clinical features such as pathological grade and size. Using the logistic regression model, 83% of the tumors analyzed in this study could be correctly assigned as interval or screen-detected tumors on the basis of Ki67 and c-erbB2 expression. The importance of high expression of Ki67 in interval cancers compared with screen-detected cancers suggests that tumors may become symptomatic in the screening interval as a result of increased levels of cell proliferation. The inclusion of c-erbB2 in the regression equation suggests that this growth factor receptor may play a significant role in stimulating the rapid growth of interval cancers.

The trefoil peptide TFF1 inhibits the growth of the human gastric adenocarcinoma cell line AGS.

TFF1 is a 60-amino acid peptide produced in normal gastric mucosa which forms dimers spontaneously. Tumours of patients with gastric cancer usually have reduced TFF1 levels and disruption of the TFF1 gene causes animals to develop gastric adenomas and carcinomas. The effect of normal sequence human recombinant TFF1 and an analogue (Cys(58)-->Ser(58)), which is unable to dimerize, on the proliferation and morphology of the human gastric adenocarcinoma cell line AGS was therefore investigated. Proliferation, assessed by total cell number and [methyl-(3)H]thymidine incorporation, was reduced by dimeric TFF1 in a dose-dependent manner. Monomeric TFF1 also reduced proliferation but was less potent than the dimeric form. It is concluded that TFF1 may be an important controller of gastric cell proliferation, that dimerization of TFF1 is important in this effect, and that the reduced levels of TFF1 seen in gastric cancer may be of clinical relevance.

Diversity of human endogenous retrovirus class II-like sequences.

Class II human endogenous retroviruses (HERVs), often referred to as mouse mammary tumour virus (MMTV)-like or HERV-K elements, have similarities to several animal infectious retroviruses. Single clones from each of nine class II HERV groups (NMWV 1 to NMWV 9), isolated from a human breast cancer cell genomic library, were sequenced over a 244 bp stretch of the conserved reverse transcriptase region. These sequences were aligned to related exogenous and endogenous retroviruses and a phylogenetic tree was constructed. Sequences with more than 80% identity were considered as members of one group and we report here that the class II HERV family consists of at least ten groups. Three of the sequenced clones, from groups NMWV 3, 7 and 9, could not be related to any other previously identified elements and constituted their own groups. NMWV 8 had no similarity to any retroviral sequences in the sequenced region and is so far considered to be non-retroviral.

Dimerization of human pS2 (TFF1) plays a key role in its protective/healing effects.

Human pS2 (trefoil factor family 1, TFF1), a 60-amino acid member of the trefoil peptide family, forms dimers via Cys58 and may stimulate gut repair. The effects of dimeric pS2-TFF1 and monomeric pS2-TFF1 (Cys58 replaced by Ser58) were compared in models of wound healing. Rats given dimeric pS2-TFF1 at 25 and 50 micrograms/kg per h had 50 per cent and 70 per cent reduction in gastric damage induced respectively by indomethacin (20 mg/kg subcutaneously) and restraint (P < 0.01). Monomeric pS2-TFF1, at the same doses, was significantly less effective at reducing injury (about half the amount of protection, P < 0.01 vs. same doses of dimeric). The rate of migration of cells at the leading edge of wounded monolayers of the human colonic cell line HT29 was increased by addition of dimeric or monomeric forms of pS2-TFF1 (0.65-325 micrograms/ml). Dimeric pS2-TFF1 had a greater effect than the monomeric form at all doses tested (P < 0.05). Cell migration induced by pS2-TFF1 was blocked by a pS2-TFF1 antibody, but not by a transforming growth factor beta neutralizing antibody. pS2-TFF1 did not influence cell proliferation as assessed by thymidine incorporation. The increased biological effects of dimeric pS2-TFF1 might be due to direct interaction of Cys58 with a putative trefoil receptor or, more likely, dimerization of pS2-TFF1 might stabilize the interaction with its receptor. This may involve a bivalent interaction of residues on the surfaces of the two trefoil domains.

A prolonged outbreak of Shigella sonnei infections in traditionally observant Jewish communities in North America caused by a molecularly distinct bacterial subtype.

During 1994-1996, Shigella sonnei outbreaks occurred in 8 North American traditionally observant Jewish communities. These communities remain relatively separate from neighboring populations while maintaining close contact by travel with coreligionists in other cities. Epidemiologic investigations suggested community-to-community transmission via travel. Outbreak-related and control isolates of S. sonnei from each city were subtyped by pulsed-field gel electrophoresis (PFGE) to confirm an epidemiologic linkage between outbreaks. Forty-three (94%) of 46 outbreak-related isolates had closely related PFGE patterns, constituting a single subtype; 33 (94%) of 35 control isolates demonstrated unrelated PFGE patterns. Several patterns differing by < or = 3 bands were identified within the outbreak subtype; one of these accounted for 65% of outbreak isolates. Hence, a single subtype of S. sonnei caused an international outbreak involving 8 traditionally observant Jewish communities, but not neighboring populations, over a 2-year period, suggesting sustained propagation of the epidemic strain between communities.