Helicobacter pylori Chronic Infection Selects for Effective Colonizers of Metaplastic Glands.

Chronic gastric infection with Helicobacter pylori can lead to progressive tissue changes that culminate in cancer, but how H. pylori adapts to the changing tissue environment during disease development is not fully understood. In a transgenic mouse gastric metaplasia model, we found that strains from unrelated individuals differed in their ability to infect the stomach, to colonize metaplastic glands, and to alter the expression of the metaplasia-associated protein TFF3. H. pylori isolates from different stages of disease from a single individual had differential ability to colonize healthy and metaplastic gastric glands. Exposure to the metaplastic environment selected for high gastric colonization by one of these strains. Complete genome sequencing revealed a unique alteration in the frequency of a variant allele of the putative adhesin sabB, arising from a recombination event with the related sialic acid binding adhesin (SabA) gene. Mutation of sabB in multiple H. pylori strain backgrounds strongly reduced adherence to both normal and metaplastic gastric tissue, and highly attenuated stomach colonization in mice. Thus, the changing gastric environment during disease development promotes bacterial adhesin gene variation associated with enhanced gastric colonization. IMPORTANCE Chronic infection with Helicobacter pylori is the primary risk factor for developing stomach cancer. As disease progresses H. pylori must adapt to a changing host tissue environment that includes induction of new cell fates in the cells that line the stomach. We tested representative H. pylori isolates collected from the same patient during early and later stages of disease in a mouse model where we can rapidly induce disease-associated tissue changes. Only the later-stage H. pylori strains could robustly colonize the diseased stomach environment. We also found that the ability to colonize the diseased stomach was associated with genetic variation in a putative cell surface adhesin gene called sabB. Additional experiments revealed that SabB promotes binding to stomach tissue and is critical for stomach colonization by the late-stage strains. Thus, H. pylori diversifies its genome during disease progression and these genomic changes highlight critical factors for bacterial persistence.

Decontamination of breast pump milk collection kits and related items at home and in hospital: guidance from a Joint Working Group of the Healthcare Infection Society and Infection Prevention Society.

INTRODUCTION: A variety of methods are in use for decontaminating breast pump milk collection kits and related items associated with infant feeding. This paper aims to provide best practice guidance for decontamination of this equipment at home and in hospital. It has been compiled by a Joint Working Group of the Healthcare Infection Society and the Infection Prevention Society. METHODS: The guidance has been informed by a search of the literature in Medline, the British Nursing Index, the Cumulative Index to Nursing and Allied Health Literature, Midwifery and Infant Care, and the results of two surveys of UK neonatal units in 2002/3 and 2006, and of members of the Infection Prevention Society in 2014. Since limited good quality evidence was available from these sources, much of the guidance represents good practice based on the consensus view of the Working Group. CONCLUSION: This guidance provides practical recommendations to support the safe decontamination of breast pump milk collection kits for healthcare professionals to use and communicate to other groups such as parents and carers.

Decontamination of breast pump milk collection kits and related items at home and in hospital: guidance from a Joint Working Group of the Healthcare Infection Society & Infection Prevention Society.

INTRODUCTION: A variety of methods are in use for decontaminating breast pump milk collection kits and related items associated with infant feeding. This paper aims to provide best practice guidance for decontamination of this equipment at home and in hospital. It has been compiled by a joint Working Group of the Healthcare Infection Society and the Infection Prevention Society. METHODS: The guidance has been informed by a search of the literature in Medline, the British Nursing Index, the Cumulative Index to Nursing & Allied Health Literature, Midwifery & Infant Care and the results of two surveys of UK neonatal units in 2002/3 and 2006, and of members of the Infection Prevention Society in 2014. Since limited good quality evidence was available from these sources much of the guidance represents good practice based on the consensus view of the Working Group. KEY RECOMMENDATIONS: Breast pump milk collection kits should not be reused by different mothers unless they have been sterilized in a Sterile Services Department between these different users.When used by the same mother, a detergent wash followed by thorough rinsing and drying after each use gives acceptable decontamination for most circumstances, as long as it is performed correctly.Additional decontamination precautions to washing, rinsing and drying may be used if indicated by local risk assessments and on advice from the departmental clinicians and Infection Prevention and Control Teams. The microbiological quality of the rinse water is an important consideration, particularly for infants on neonatal units.If bottle brushes or breast/nipple shields are used, they should be for use by one mother only. Decontamination should be by the processes used for breast pump milk collection kits.Dummies (soothers, pacifiers or comforters) needed for non-nutritive sucking by infants on neonatal units, should be for single infant use. Manufacturers should provide these dummies ready-to-use and individually packaged. They must be discarded at least every 24 hours or immediately if soiled with anything other than the baby's saliva. No attempt should be made to decontaminate the dummies, either before or during use. CONCLUSION: This guidance provides practical recommendations to support the safe decontamination of breast pump milk collection kits for healthcare professionals to use and communicate to other groups such as parents and carers.

Comparison of the vector potential of different mosquito species for the transmission of heartworm, Dirofilaria immitis, in rural and urban areas in and surrounding Stillwater, Oklahoma, U.S.A.

Dirofilaria immitis Leidy (Spirurida: Onchocercidae), or heartworm, is a mosquito-borne nematode that causes a fatal disease in carnivores. Although infection is preventable through prophylactic drugs, compliance and the spectre of resistance suggest vector control is a viable alternative. There were two main objectives in this study: (a) to evaluate the relationships between landscape and social factors and the number and species of heartworm-positive mosquitoes, with a specific focus on the importance of the invasive Asian tiger mosquito, Aedes albopictus (Skuse) (Stegomyia albopicta) (Diptera: Culicidae), and (b) to test the hypothesis that dog heartworm is more prevalent in suburban than in rural areas. To achieve these objectives, mosquitoes were collected from May to November 2010 at 16 rural and 16 urban locations in Payne County, Oklahoma, U.S.A. using three trapping methods that utilized, respectively, resting boxes, carbon dioxide traps and BG Sentinel traps. Urban areas showed greater numbers of Ae. albopictus and a higher overall likelihood of infection with D. immitis. Because many species of mosquito are responsible for heartworm transmission, current prophylactic treatment remains the best method of controlling this parasite.

Real-time studies of hypertension using non-mydriatic fundus photography and computer-assisted intravital microscopy.

Hypertension is asymptomatic until late stages of pathogenesis, rendering an effective means of detection for early diagnosis essential. The current method of diagnosing hypertension requires two or more sphygmomanometric readings over two or more office visits, which potentially hinders early detection. Though retinopathy is an indicator of vascular damage, it generally presents in later stages of hypertension. Previous and related studies have suggested that the microvasculature in the bulbar conjunctiva may be a sensitive site to assess vasculopathy. Conjunctival microangiopathy was assessed using CAIM and reported on a severity index (SI). Images of the retinal fundus were taken via non-mydriatic fundus photography and graded using the Scheie scale in the same subjects to compare with CAIM. Conjunctival microangiopathy was significantly elevated in hypertensive subjects (SI = 5.35 ± 1.04, n = 20) compared to control subjects (SI = 1.75 ± 1.39, n = 8; p ≤ 0.05), and correlated with time since disease diagnosis (R² = 0.33). Hypertensive subjects with Grade 1 retinopathy displayed increased conjunctival microangiopathy (SI = 5.85 ± 0.90, n = 13) compared to those without retinopathy (SI = 4.43 ± 0.53, n = 7; p ≤ 0.05). These data indicate a possible pre-retinopathy time window during which conjunctival microangiopathy may indicate the risk of organ damage, supporting the hypothesis that the conjunctival microcirculation may serve as a platform for early detection and monitoring disease progression.

Positive futures: a qualitative study on the needs of adolescents on antiretroviral therapy in South Africa.

With the increasing availability of highly active antiretroviral therapy, vertically infected children have a better chance of surviving into adolescence and adulthood. Additionally, sexual transmission of human immunodeficiency virus (HIV) remains a problem, and incidence and prevalence among youth remain high. Therefore, HIV-infected adolescents are becoming a more prominent sub-group in the HIV/AIDS epidemic. Experience from the developed world indicates that providing effective care and treatment for adolescents poses unique challenges. This study aimed to identify the experiences and needs of adolescents growing up in care or on treatment for HIV in Cape Town, South Africa. Four focus groups interviews were conducted with a total of 26 young people attending an adolescent infectious diseases clinic at a tertiary hospital. Questions explored participant's perceptions on their present and future lives, and their self-identified needs. Focus groups revealed that adolescents viewed their illness negatively, but that social issues such as violence and poverty were also concerns. Despite these stressors, most respondents remained positive about the present and future, and wanted support for achieving their goals. As increasing numbers of HIV-infected children enter adolescence, healthcare providers and communities must find ways to support these young people to transition into adulthood.

Cytokeratin 18 in plasma of patients with gastrointestinal adenocarcinoma as a biomarker of tumour response.

BACKGROUND: Plasma biomarkers may be particularly useful as a predictor or early marker of clinical response to treatment in addition to radiological imaging. Cytokeratin 18 (CK18) is an epithelial-specific cytokeratin that undergoes cleavage by caspases during apoptosis. Measurement of caspase-cleaved (CK18-Asp396) or total cytokeratin 18 (CK18) from epithelial-derived tumours could be a simple, non-invasive way to monitor or predict responses to treatment. METHODS: Soluble plasma CK18-Asp396 and CK18 were measured by ELISA from 73 patients with advanced gastrointestinal adenocarcinomas before treatment and during chemotherapy, as well as 100 healthy volunteers. RESULTS: Both CK18-Asp396 and total CK18 plasma levels were significantly higher in patients compared with the healthy volunteers (P=0.015, P<0.001). The total CK18 baseline plasma levels before treatment were significantly higher (P=0.009) in patients who develop progressive disease than those who achieve partial response or stable disease and this correlation was confirmed in an independent validation set. The peak plasma levels of CK18 occurring in any cycle following treatment were also found to be associated with tumour response, but peak levels of CK18-Asp396 did not reach significance (P=0.01, and P=0.07, respectively). CONCLUSION: Plasma levels CK18 are a potential marker of tumour response in patients with advanced gastrointestinal malignancy.

Cell transformation by the E5/E8 protein of bovine papillomavirus type 4. p27(Kip1), Elevated through increased protein synthesis is sequestered by cyclin D1-CDK4 complexes.

The E5/E8 hydrophobic protein of BPV-4 is, at only 42 residues, the smallest transforming protein identified to date. Transformation of NIH-3T3 cells by E5/E8 correlates with up-regulation of both cyclin A-associated kinase activity and, unusually, p27(Kip1) (p27) but does not rely on changes in cyclin E or cyclin E-CDK2 activity. Here we have examined how p27 is prevented from functioning efficiently as a CDK2 inhibitor, and we investigated the mechanisms used to achieve elevated p27 expression in E5/E8 cells. Our results show that normal subcellular targeting of p27 is not subverted in E5/E8 cells, and p27 retains its ability to inhibit both cyclin E-CDK2 and cyclin A-CDK activities upon release from heat-labile complexes. E5/E8 cells also have elevated levels of cyclins D1 and D3, and high levels of nuclear p27 are tolerated because the inhibitor is sequestered within an elevated pool of cyclin D1-CDK4 complexes, a significant portion of which retain kinase activity. In agreement with this, pRB is constitutively hyperphosphorylated in E5/E8 cells in vivo. The increased steady-state level of p27 is achieved largely through an increased rate of protein synthesis and does not rely on changes in p27 mRNA levels or protein half-life. This is the first report of enhanced p27 synthesis as the main mechanism for increasing protein levels in continuously cycling cells. Our results are consistent with a model in which E5/E8 promotes a coordinated elevation of cyclin D1-CDK4 and p27, as well as cyclin A-associated kinase activity, which act in concert to allow continued proliferation in the absence of mitogens.

Quercetin, E7 and p53 in papillomavirus oncogenic cell transformation.

Bovine papillomavirus type 4 (BPV-4) infects the upper alimentary canal of cattle causing benign papillomas which can progress to squamous carcinomas in cattle grazing on bracken fern (BF). We have previously shown that quercetin, a well characterized and potent mutagen found in BF, causes cell cycle arrest of primary bovine cells (PalF), but that a single exposure to quercetin can cause full oncogenic transformation of PalF cells partially transformed by BPV-4. Here we show that cell cycle arrest correlates with an increase in p53 protein levels and transcriptional activity. However, in cells transformed but non-tumorigenic, p53 protein is elevated and transcriptionally activated in response to quercetin or other DNA damaging stimuli, but the cells bypass quercetin-induced G1 arrest likely due to E7 expression. In transformed tumorigenic cells, p53 is elevated in response to quercetin but its transcriptional activity is inhibited due to mutation, and the cells fail to stop in G1 in the presence of quercetin.

Binding of bovine papillomavirus type 4 E8 to ductin (16K proteolipid), down-regulation of gap junction intercellular communication and full cell transformation are independent events.

The E8 open reading frame of bovine papillomavirus type 4 encodes a small hydrophobic polypeptide that contributes to primary cell transformation by conferring to cells the ability to form foci and to grow in low serum and in suspension. Wild-type E8 binds in vitro to ductin, a component of gap junctions, and this binding is accompanied by a loss of gap junction intercellular communication in transformed bovine fibroblasts. However, through the analysis of a panel of E8 mutants, we show here that binding of E8 to ductin is not sufficient for down-regulation of gap junction communication and that there is no absolute correlation between down-regulation of gap junction communication and the transformed phenotype.

A mutational analysis of the transforming functions of the E8 protein of bovine papillomavirus type 4.

The E8 protein of BPV-4 contributes to transformation of primary bovine cells (PalFs) by inducing anchorage-independent growth and by down-regulating gap junction intercellular communication, likely due to its binding to 16K ductin. We show here that, in addition, E8 confers on PalF cells the ability to grow in low serum and to escape from contact inhibition (focus formation). E8 also transactivates an exogenous human cyclin A gene promoter, suggesting that overexpression of cyclin A is responsible for the transformed phenotype. Mutant forms of E8 were generated to establish whether the transforming functions of the protein could be segregated. Mutations were introduced both in the hydrophobic domain and in the hydrophilic C-terminal "tail", and chimeras with BPV-1 E5 were constructed. Cells expressing either wild-type E8 or mutant forms were analyzed for their ability to grow in low serum and in suspension and to form foci. Wild-type E8 and its mutants were also analyzed for their ability to transactivate the cyclin A promoter. We show here that the transforming functions of E8 can be segregated and that both the hydrophilic C-terminal tail and the residue at position 17 in the hydrophobic domain are crucial for E8 functions and for the transactivation of the cyclin A promoter. These results support the hypothesis that the different aspects of cellular transformation brought about by E8 might be due to interaction with different cellular targets. They suggest that E8 might function differently from BPV-1 E5 and demonstrate that the separate domains of E5 and E8 are not functionally interchangeable.

BPV-4 E8 transforms NIH3T3 cells, up-regulates cyclin A and cyclin A-associated kinase activity and de-regulates expression of the cdk inhibitor p27Kip1.

The E8 open reading frame of Bovine papillomavirus type 4 (BPV-4) encodes a small (42 amino acid) hydrophobic polypeptide localized to cellular membranes and capable of conferring an anchorage-independent (AI) growth phenotype on primary bovine cells co-transfected with BPV-4 E7 ORF and an activated ras gene. To further study the function of E8 independently of other viral gene products, we have expressed it in the murine fibroblast cell line, NIH3T3. Cells expressing E8 are capable of AI growth and escape growth arrest after serum withdrawal. E8 deregulates cyclin A expression, induces transactivation of the human cyclin A gene promoter and increases endogenous protein levels in cells maintained in short-term suspension culture and in low-serum (LS). Both these culture conditions promote downregulation of cyclin A in control cells. In LS growth conditions E8 permits sustained cyclin A-associated kinase activity but not cyclin E-cdk2 activity. Cyclin A-cdk2 activity and, in part, cyclin A gene expression are regulated by the cdk inhibitor p27Kip1. Expression of this cdk inhibitor is also de-regulated in E8 cells, with high levels being detected under all culture conditions tested. These data suggest that the ability of BPV-4 E8 to transform NIH3T3 cells is associated with upregulation of cyclin A-associated kinase activity and de-regulated expression of the cdk inhibitor p27Kip1 and does not rely on down-regulation of p27Kip1 expression. Analysis of E8 mutants indicate that the hydrophilic 'tail' of the molecule (residues 31-42) is required for cell transformation, as assessed by anchorage-independent growth, while a form of E8 with expression restricted to the Endoplasmic Reticulum/cis-Golgi membranes by addition of a 'KDEL' retention signal revealed that the sub-cellular localization is an important determinant of E8 biological activity.

Expression of the integrin alpha 5 subunit in HT29 colon carcinoma cells suppresses apoptosis triggered by serum deprivation.

It is clear that certain integrins can regulate the growth of tumors, probably by contributing to signal transduction processes. In the present study we have used HT29 human colon carcinoma cells stably transfected with human cDNA for the integrin alpha 5 subunit and studied the effects of alpha 5 expression on the induction of apoptosis. We observe that apoptosis can be triggered in HT29 cells by removal of serum and that this process can be suppressed by the stable expression of full-length integrin alpha 5 subunits. While the mechanism underlying this effect is still unclear, these observations suggest that the alpha 5 beta 1 integrin plays an important role in modulating tumor cell responses to growth factors and nutrients.

Signal transduction by cell adhesion receptors.

Over the last few years, it has become clear that cell adhesion receptors function in signal transduction processes leading to the regulation of cell growth and differentiation. Signal transduction by both integrins and CAMs has been shown to involve activation of tyrosine kinases, while CAM signaling in neural cells involves G proteins as well. In the case of integrins, some of the downstream signaling events intersect with the Ras pathway, particularly the activation of MAP kinases. In fibroblasts, integrin mediated anchorage to the substratum regulates cell cycle traverse, while in epithelial cells, loss of anchorage can trigger programmed cell death. In many cell types, but particularly monocytic cells, integrin ligation has a profound impact on gene expression. Preliminary evidence also implicates CAMs and selectins in gene regulation. A consistent theme in signal transduction mediated by adhesion receptors concerns the role of the cytoskeleton. Integrin mediated signaling processes are interrupted by cytoskeletal disassembly. Identification of the APC and neurofibromatosis type 2 tumor suppressors suggest that cytoskeletal complexes also play a key role in signaling by cadherins and CD44, respectively. Thus, signaling by cell adhesion receptors may involve aspects that impinge on previously known signaling pathways including the RTK/Ras pathway and serpentine receptor/G protein pathways. However, novel aspects of signal transduction involving cytoskeletal assemblies may also be critical.

The development of competence in resting B cells. The induction of cyclic AMP and ornithine decarboxylase activity after direct contact between B and T helper cells.

In the present communication, an experimental approach is utilized that facilitates the study of biochemical processes induced in B cells after their interaction with Th cells. In this approach, Th cell clones are stimulated for 18 h upon anti-CD3-coated plates, fixed with paraformaldehyde, and added at a 2 to 3:1 ratio to small, resting B cells (isolated from Percoll gradients). Th cells not stimulated on anti-CD3-coated plates, but fixed with paraformaldehyde, serve as controls for these experiments. The activated, fixed Th cells induce a transient, sixfold increase in B cell levels of cAMP, as well as an increase in B cell expression of ornithine decarboxylase (ODC) activity. This enzyme initiates the synthesis of polyamines and has been shown to be increased as cells enter the growth phase. In addition, previous studies have shown that the cellular levels of ODC activity are controlled by a multi-tiered regulatory cascade. To examine this aspect, polyclonally stimulated B cells were studied. Such cells demonstrated a gradual increase in ODC mRNA levels that peaked between 6 and 15 h and can be partially explained by a three- to fourfold increase in mRNA stability but not by changes in the enzyme affinity for substrate. The increase in ODC mRNA occurs in the absence of protein synthesis, suggesting that the ODC gene is a member of the immediate/early gene family. Finally, the early increase in ODC mRNA was enhanced in cells in which cAMP levels were artificially elevated, suggesting the possibility that the cAMP-dependent signaling pathway participates during the regulation of this gene expression. The significance of these experimental results concerning the process of B cell activation is discussed.

The relationship between form and space perception, constructional abilities, and clumsiness in children.

The Sensory Integration and Praxis Tests (SIPT) (Ayres, 1989) were administered to 21 children with learning disabilities and 18 children without learning disabilities, aged 5 to 8 years. The children with learning disabilities were divided into two groups, clumsy and nonclumsy, on the basis of their scores on the Test of Motor Impairment (Stott, Moyes, & Henderson, 1984). It was hypothesized that the learning-disabled children in the clumsy group would score significantly lower than the learning-disabled children in the nonclumsy group on the six SIPT subtests that measure form and space perception and visual construction and that the nonclumsy learning-disabled children, in turn, would score significantly lower than the non-learning-disabled children. It was further hypothesized that there would be a significant correlation between the degree of clumsiness and the degree of visual-perceptual and constructional deficits. An analysis of the data indicated that both groups of learning-disabled children scored lower than the non-learning-disabled children on four of the six SIPT subtests. The clumsy and nonclumsy children with learning disabilities, however, differed from each other on only two subtests. The degree of clumsiness correlated significantly with three of the six subtests. The results are discussed in terms of variations in perceptual and motor skills related to subtypes of learning disabilities.

Malnutrition in aboriginal children at Yalata, South Australia.

Comparative studies of body weight, height, intracellular water representing cell mass, and age, and plasma concentrations of albumin, vitamins, trace elements and iron stores in Aboriginal children aged 6 to 13.5 years, from two rural Aboriginal settlements and one rural Caucasian school (Hawker) provided evidence of significant deficits in one of the Aboriginal settlements (Yalata). Yalata Aboriginal children had lower body weights and heights for age and lower intracellular water values. Plasma albumin, zinc, iron, alpha-tocopherol, beta-carotene and retinol concentrations were lower relative to the normally grown Aboriginal children at Nepabunna. The latter children did not differ from rural Caucasian children for the parameters studied. The reasons for this poorer growth at Yalata may reside in poor nutrition, or repeated bowel infection in postnatal life leading to malabsorption, or both. Limited observational evidence suggests that Giardiasis has a high prevalence at Yalata, and it has been shown elsewhere that Giardiasis is capable of inducing malabsorption with resulting nutritional deficiencies.