Helicobacter pylori infection and the effect on growth in a cohort of children in the US/Mexico border

Helicobacter pylori (H. pylori) is an S-shaped or curved gram-negative bacterium that is mostly found in the human stomach. H. pylori causes gastritis in adults and children, which can lead to gastric ulcers or risk of cancer. Transmission of this bacterial infection remains to be unknown but is mostly acquired during childhood. Little is known about the effect H. pylori has on growth in children. Although some studies have reported that H. pylori is associated with subnormal growth, the association of H. pylori with growth retardation and malnutrition is poorly described. Data from this study comes from The Pasitos Cohort Study which draws its population from three border communities which include Socorro and San Elizario in Texas, as well as Ciudad Juarez, Chihuahua, Mexico. Birth documentation was obtained for 803 infants and 472 entered follow-up. This cohort study allowed us to assess the growth of children from 6 months to the seventh anniversary, and describe the prevalence of underweight, short stature and overweight in the study population. We also tested the hypothesis that children in the Pasitos Cohort Study who were ever infected with H. pylori show an increased risk of growth retardation or malnutrition at 66 months of age. Using the 2000 CDC Growth Reference, we found that the mean BMI of the study population increased as children grew older, while the mean of their height for age decreased slightly. The proportion of children who were classified as of short stature was under 5%, while those considered underweight were less than 10% at selected six-months of age intervals. Using the subset of children who were 66 months of age we found that the risk of underweight was higher among those who ever tested positive for H. pylori infection using the urea breath test; however, due to small numbers of children with 'wasting' this difference was not statistically significant. Moreover, since the six cases of under weight occurred among children of low socio-economic status we could not rule out potential confounding. The risk of developing short stature was not different among those ever infected and those who never tested positive for H. pylori infection. ^

Protease-activated receptor-1 signaling plays a major role in melanoma growth and metastasis

Overexpression of the thrombin receptor (Protease-Activated-Receptor-1), PAR-1, in cell lines and tissue specimens correlates with the metastatic potential of human melanoma. Utilizing lentiviral shRNA to stably silence PAR-1 in metastatic melanoma cell lines results in decreased tumor growth and lung metastasis in vivo. Since the use of viral technology is not ideal for clinical therapies, neutral liposomes (DOPC) were utilized as a delivery vehicle for PAR-1 siRNA. Our data suggest that PAR-1 siRNA-DOPC treatment by systemic delivery significantly decreases tumor growth and lung metastasis in nude mice. Concomitant decreases in angiogenic and invasive factors (IL-8, VEGF, MMP-2) were observed in PAR-1 siRNA-DOPC-treated mice. Utilizing a cDNA microarray platform, several novel PAR-1 downstream target genes were identified, including Connexin 43 (Cx-43) and Maspin. Cx-43, known to be involved in tumor cell diapedesis and attachment to endothelial cells, is decreased after PAR-1 silencing. Furthermore, the Cx-43 promoter activity was significantly inhibited in PAR-1-silenced cells suggesting transcriptional regulation of Cx-43 by PAR-1. ChIP analysis revealed a reduction in SP-1 and AP-1 binding to the Cx-43 promoter. Moreover, melanoma cell attachment to HUVEC was significantly decreased in PAR-1-silenced cells as well as in Cx-43 shRNA transduced cells. As both SP-1 and AP-1 transcription factors act as positive regulators of Cx-43, our data provide a novel mechanism for the regulation of Cx-43 expression by PAR-1. Maspin, a serine protease inhibitor with tumor-suppressor function, was found to be upregulated after PAR-1 silencing. Our results indicate that PAR-1 transcriptionally regulates Maspin, as the promoter activity was significantly increased after PAR-1 silencing. ChIP analysis revealed that silencing PAR-1 increased binding of Ets and c-Jun to the Maspin promoter. As Maspin was recently found to be a tumor-suppressor in melanoma by reducing the invasive capacity of melanoma cells, invasion assays revealed a decrease in invasion after PAR-1 silencing and in cells transduced with a Maspin expression vector. We propose that PAR-1 is key to the progression and metastasis of melanoma in part by regulating the expression of Cx-43 and Maspin. Taken together, we propose that PAR-1 is an attractive target for the treatment of melanoma.^

The survival role and the potential therapeutic targeting of MET in multiple myeloma

Multiple myeloma (MM) is a debilitating and incurable B-cell malignancy. Previous studies have documented that the hepatocyte growth factor (HGF) plays a role in the pathobiology of MM. The receptor tyrosine kinase MET induced signaling initiates when its ligand HGF binds to the MET receptor. However, the direct importance of MET in MM has not been elucidated. The present work used three different but complementary approaches to reduce MET protein levels or its activity to demonstrate the importance of MET in MM. ^ In the first approach, MET transcript and protein levels were reduced by directly targeting the cellular MET transcripts using shRNA retroviral infection techniques. This direct reduction of MET mRNA leads to a reduction of MET protein levels, which caused an inhibition of growth and induction of cell death. ^ In the second approach, a global transcription inhibitor flavopiridol was used as a potential pharmacological tool to reduce MET levels. MET has a short half-life of 30 min for mRNA and 4 hours for protein; therefore using a RNA pol II inhibitor such as flavopiridol would be a viable option to reduce MET levels. When using flavopiridol in MM cell lines, there was a reduction of MET transcript and protein levels, which was associated with the induction of cell death. ^ Finally in the last strategy, MET kinase activity was suppressed by MP470, a small molecule inhibitor that binds to the ATP binding pocket in the kinase domain. At concentrations where phosphorylation of MET was inhibited there was induction of cell death in MM cell lines and primary cells from patients. In addition, in MM cell lines there was a decrease in phosphorylation of AKT (ser473) and caspase-9 (ser196); downstream of MET, suggesting that the mechanism of action for survival may be through these cascade of events. ^ Overall, this study provides a proof-of-principle that MET is important for the survival of MM cell lines as well as primary plasma cells obtained from patients. Therefore, targeting MET therapeutically may be a possible strategy to treat patients with this debilitating disease of MM. ^

SOCIAL AND BIOLOGICAL DETERMINANTS OF SHORT-TERM GROWTH VELOCITY AMONG PRESCHOOLERS OF RURAL GUATEMALA: A PATH ANALYSIS APPROACH (WEIGHT-GAIN, IRON DEFICIENCY, GASTROINTESTINAL SYMPTOMS)

This dissertation develops and tests through path analysis a theoretical model to explain how socioeconomic, socioenvironmental, and biologic risk factors simultaneously influence each other to further produce short-term, depressed growth in preschoolers. Three areas of risk factors were identified: child's proximal environment, maturational stage, and biological vulnerability. The theoretical model represented both the conceptual framework and the nature and direction of the hypotheses. Original research completed in 1978-80 and in 1982 provided the background data. It was analyzed first by nested-analysis of variance, followed by path analysis. The study provided evidence of mild iron deficiency and gastrointestinal symptomatology in the etiology of depressed, short-term weight gain. Also, there was evidence suggesting that family resources for material and social survival significantly contribute to the variability of short-term, age-adjusted growth velocity. These results challenge current views of unifocal intervention, whether for prevention or control. For policy formulations, though, the mechanisms underlying any set of interlaced relationships must be decoded. Theoretical formulations here proposed should be reassessed under a more extensive research design. It is suggested that studies should be undertaken where social changes are actually in progress; otherwise, nutritional epidemiology in developing countries operates somewhere between social reality and research concepts, with little grasp of its real potential. The study stresses that there is a connection between substantive theory, empirical observation, and policy issues. ^

The Role of Type I Insulin-Like Growth Factor Receptor Signaling in Breast Cancer Brain Metastasis

Brain metastasis is a common cause of mortality in cancer patients. Approximately 20-30% of breast cancer patients acquire brain metastasis, yet potential therapeutic targets remain largely unknown. The type I insulin-like growth factor receptor (IGF- IR) is known to play a role in the progression of breast cancer and is currently being investigated in the clinical setting for various types of cancer. The present study demonstrates that the IGF-IR signaling axis is constitutively active in brain-seeking sublines of breast cancer cells, driving an increase in in vitro metastatic properties. We demonstrate that IGF-IR signaling is activated in an autocrine manner as a result of IGFBP3 overexpression in brain-seeking cells. Transient and stable knockdown of IGF-IR results in a downregulation of IGF-IR downstream signaling through phospho-AKT, as well as decreased in vitro migration and invasion of MDA- MB-231Br brain-seeking cells. Using an in vivo experimental brain metastasis model, we show that IGF-IR ablation attenuates the establishment of brain metastases and prolongs survival. Finally, we demonstrate that the malignancy of brain-seeking cells is attenuated by pharmacological inhibition with picropodophyllin, an IGF-IR-specific tyrosine kinase inhibitor. Together, our data suggest that the IGF-IR is an important mediator of brain metastasis and its ablation delays the onset of brain metastases in our model system.

Role and regulation of c-KIT gene expression in tumor growth and metastasis of human melanoma

The purpose of this study was to investigate the role of the c-KIT receptor in the progression of human melanoma and the mechanism(s) for the regulation of c-KIT gene expression in human melanoma.^ The molecular changes associated with the transition of melanoma cells from radial growth phase (RGP) to vertical growth phase (VGP) (metastatic phenotype) are not well-defined. Expression of the tyrosine-kinase receptor c-KIT progressively decreases during local tumor growth and invasion of human melanomas. To provide direct evidence that the metastasis of human melanoma is associated with the loss of c-KIT expression, highly metastatic A375SM cells, which express very low or undetectable levels of c-KIT, were tranduced with the human c-KIT gene. We demonstrated that enforced c-KIT expression in highly metastatic human melanoma cells significantly suppressed their tumorigenicity and metastatic propensity in nude mice. In addition, we showed that the ligand for c-KIT, SCF, induces apoptosis in human melanoma cells expressing c-KIT under both in vitro and in vivo conditions. These results suggest that loss of c-KIT receptor may allow malignant melanoma cells to escape SCF/c-KIT-mediated apoptosis, thus contributing to tumor growth and eventually metastasis.^ Furthermore, we investigated the possible mechanism(s) for the down-regulation of c-KIT gene expression in malignant melanoma. Sequence analysis of the c-KIT promoter indicated that this promoter contains several consensus binding-site sequences including three putative AP2 and two Myb sites. Although Myb was shown to be associated with c-KIT expression in human hemotopoietic cells, we found no correlation between c-KIT expression and Myb expression in human melanoma cell lines. In contrast, we showed that c-KIT expression directly correlates with expression of AP2 in human melanoma cells. We found that highly metastatic cells do not express the transcription factor AP2. Expression of AP2 in A375SM cells (c-KIT-negative and AP2-negative) was enough to restore luciferase activity driven by the c-KIT promoter in a dose-dependent manner. On the other hand, co-expression of the dominant-negative form of AP2 (AP2B) in Mel-501 cells (c-KIT-positive and AP2-positive) resulted in two-fold reduction in luciferase activity. Electrophoretic mobility shift assays revealed that the c-KIT promoter contains functional AP2 binding sites which could associate with AP2 protein. Endogenous c-KIT gene expression levels were elevated in AP2 stably-transfected human melanoma A375SM cells. Expression of exogenous AP2 in A375SM cells inhibited their tumorigenicity and metastatic potential in nude mice. The c-KIT ligand, SCF, also induced apoptosis in the AP2 stably-transfected A375SM cells. The identification of AP2 as an important regulator for c-KIT expression suggests that AP2 may have tumor growth and metastasis inhibitory properties, possibly mediated through c-KIT/SCF effects on apoptosis of human melanoma cells. Since AP2 binding sites were found in the promoters of other genes involved in the progression of human melanoma, such as MMP2 (72 kDa collagenase), MCAM/MUC18 and P21/WAF-1, our findings suggest that loss of AP2 expression might be a crucial event in the development of malignant melanoma. ^

Axl -Gas6 signaling counteracts adenovirus type 5 E1A-mediated cell growth suppression and proapoptotic activity

The adenovirus type 5 E1A (abbreviated E1A) has previously been known as an immortalization oncogene because E1A is required for transforming oncogenes, such as ras and E1B, to transform cells in primary cultures. However, E1A has also been shown to downregulate the overexpression of the Her-2/neu oncogene, resulting in suppression of transformation and tumorigenesis induced by that oncogene. In addition, E1A is able to promote apoptosis induced by anticancer drugs, irradiation, and serum deprivation. Many tyrosine kinases, such as the EGF receptor, Her-2/Neu, Src, and Axl are known to play a role in oncogenic signals in transformed cells. To study the mechanism underlying the E1A-mediated tumor-suppressing function, we exploited a modified tyrosine kinase profile assay (Proc. Natl. Acad. Sci, 93, 5958–5962, 1996) to identify potential tyrosine kinases regulated by E1A. RT-PCR products were synthesized with two degenerate primers derived from the conserved motifs of various tyrosine kinases. A tyrosine kinase downregulated by E1A was identified as Axl by analyzing the Alu I-digested RT-PCR products. We isolated the DNA fragment of interest, and found that E1A negatively regulated the expression of the transforming receptor tyrosine kinase Axl at the transcriptional level. To study whether downregulation of the Axl receptor is involved in E1A-mediated growth suppression, we transfected axl cDNA into E1A-expressing cells (ip1-E1A) to establish cells that overexpressed Axl (ip1-E1A-Axl). The Axl ligand Gas6 triggered a greater mitogenic effect in these ip1-E1A-Axl cells than in the control cells ip1-E1A and protected the Axl-expressing cells from serum deprivation-induced apoptosis. Further study showed that Akt is required for Axl-Gas6 signaling to prevent ip1-E1A-Axl cells from serum deprivation-induced apoptosis. These results indicate that downregulation of the Axl receptor by E1A is involved in E1A-mediated growth suppression and E1A-induced apoptosis, and thereby contributes to E1A's anti-tumor activities. ^

Modulation of replicative senescence of skeletal muscle satellite cells by insulin -like growth factor-I (IGF-I)

Growth and regeneration of postnatal skeletal muscle requires a population of mononuclear myogenic cells, called satellite cells to add/replace myonuclei, which are postmitotic. Wedged between the sarcolemma and the basal lamina of the skeletal muscle fiber, these cells function as the stem cells of mature muscle fibers. Like other normal diploid cells, satellite cells undergo cellular senescence. Investigations of aging in both rodents and humans have shown that satellite cell self-renewal capacity decreases with advanced age. As a consequence, this could be a potential reason for the characteristically observed age-associated loss in skeletal muscle mass (sarcopenia). This provided the rationale that any intervention that can further increase the proliferative capacity of these cells should potentially be able to either delay, or even prevent sarcopenia. ^ Using clonogenicity assays to determine a cell's proliferation potential, these studies have shown that IGF-I enhances the doubling potential of satellite cells from aged rodents. Using a transgenic model, where the mice express the IGF-I transgene specifically in their striated muscles, some of the underlying biochemical mechanisms for the observed increase in replicative life span were delineated. These studies have revealed that IGF-I activates the PI3/Akt pathway to mediate downregulation of p27KIP1, which consequently is associated with an increase in cyclin E-cdk2 kinase activity, phosphorylation of pRb, and upregulation of cyclin A protein. However, the beneficial effects of IGF-I on satellite cell proliferative potential appears to be limited as chronic overexpression of IGF-I in skeletal muscles did not protect against sarcopenia in 18-mo old mice, and was associated with an exhaustion of satellite cell replicative reserves. ^ These results have shown that replicative senescence can be modulated by environmental factors using skeletal muscle satellite cells as a model system. A better understanding of the molecular basis for enhancement of proliferative capacity by IGF-I will provide a rational basis for developing more effective counter-measures against physical frailty. However, the implications of these studies are that these beneficial effects of enhanced proliferative potential by IGF-I may only be over a short-term period, and other alternative approaches may need to be considered. ^

Activation of c -Met contributes to growth and metastasis of human colorectal carcinoma

c-Met is the protein tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF) and mediates several normal cellular functions including proliferation, survival, and migration. Overexpression of c-Met correlates with progression and metastasis of human colorectal carcinoma (CRC). The goals of this study were to determine if overexpression of c-Met directly contributes to tumorigenicity and liver metastatic potential of colon cancer, and what are the critical pathways regulated by c-Met in this process. The studies used two colon tumor cell lines, KM12SM and KM20, which express high levels of constitutively active c-Met and are highly metastatic in nude mice. To examine the effects of c-Met overexpression, subclones of theses lines with reduced c-Met expression were obtained following transfection with a c-Met specific targeting ribozyme. Reduction of c-Met in KM12SM cells abolished liver metastases when cells were injected intrasplenically in an experimental metastasis assay. However, c-Met downregulation in theses clones was unstable. Three stable KM20 clones with a 25–35% reduction in c-Met protein levels but 60–90% reduction in basal c-Met autophosphorylation and kinase activity were obtained. While HGF increased c-Met kinase activity in the clones with reduced c-Met, the activity was less than that observed in parental or control transfected cells. Correlating with the reduction in c-Met kinase activity, subclones with reduced c-Met expression had significantly reduced in vitro growth rates, soft-agar colony forming abilities, and increased apoptosis. HGF/SF treatment did not affect anchorage-dependent growth or soft-agar colony forming abilities. Further, c-Met downregulation significantly impaired the ability of HGF/SF to induce migration. To examine the effects of reduced c-Met on tumor formation, parental and c-Met reduced KM20 cells were grown subcutaneously and intrahepatically in nude mice. c-Met downregulation delayed, but did not abolish growth at the subcutaneous site. When these cells were injected intrahepatically, both tumor incidences and size were significantly reduced. To further understand the molecular basis of c-Met in promoting tumor growth, the activation of several signaling intermediates that have been implicated in c-Met mediated growth, survival and migration were compared between KM20 parental cells and subclones with reduced c-Met expression levels. The expression and activity (as determined by phosphorylation) of AKT and Erk1/2 were unaltered. In contrast, Src kinase activity, as measured by immune complex kinase assay, was reduced 2–5 fold following c-Met downregulation. As Src has been implicated in growth, survival and migration, Src activation in c-Met overexpressing lines is likely contributing to the tumorigenic and metastatic capabilities of colon tumor cell lines that overexpress c-Met. Collectively, these results suggest that c-Met overexpression plays a causal role in the development of CRC liver metastases, and that c-Src and c-Met inhibitors may be of potential therapeutic benefit for late-stage colon cancer. ^

Chlorophyll a content, protist biomass and experimental plankton growth and mortality in West Greenland water samples

We evaluated the role of microzooplankton (sensu latto, grazers <500 µm) in determining the fate of phytoplankton production (PP) along a glacier-to-open sea transect in the Greenland subarctic fjord, Godthabfjord. Based on the distribution of size fractionated chlorophyll a (chl a) concentrations we established 4 zones: (1) Fyllas Bank, characterized by deep chl a maxima (ca. 30 to 40 m) consisting of large cells, (2) the mouth and main branch of the fjord, where phytoplankton was relatively homogeneously distributed in the upper 30 m layer, (3) inner waters influenced by glacial melt water and upwelling, with high chl a concentrations (up to 12 µg/l) in the >10 µm fraction within a narrow (2 m) subsurface layer, and (4) the Kapisigdlit branch of the fjord, ice-free, and characterized with a thick and deep chl a maximum layer. Overall, microzooplankton grazing impact on primary production was variable and seldom significant in the Fyllas Bank and mouth of the fjord, quite intensive (up to >100% potential PP consumed daily) in the middle part of the main and Kapisigdlit branches of the fjord, and rather low and unable to control the fast growing phytoplankton population inhabiting the nutrient rich waters in the upwelling area in the vicinity of the glacier. Most of the grazing impact was on the <10 µm phytoplankton fraction, and the major grazers of the system seem to be >20 µm microzooplankton, as deducted from additional dilution experiments removing this size fraction. Overall, little or no export of phytoplankton out of the fjord to the Fyllas Bank can be determined from our data.

Increased carbon dioxide availability alters phytoplankton stoichiometry and affects carbon cycling and growth of a marine planktonic herbivore

Rising levels of CO2 in the atmosphere have led to increased CO2 concentrations in the oceans. This enhanced carbon availability to the marine primary producers has the potential to change their nutrient stoichiometry, and higher carbon to nutrient ratios are expected. As a result, the quality of the primary producers as food for herbivores may change. Here, we present experimental work showing the effect of feeding Rhodomonas salina grown under different pCO2 (200, 400 and 800 µatm) on the copepod Acartia tonsa. The rate of development of copepodites decreased with increasing CO2 availability to the algae. The surplus carbon in the algae was excreted by the copepods, with younger stages (copepodites) excreting most of their surplus carbon through respiration, and adult copepods excreting surplus carbon mostly as DOC. We consider the possible consequences of different excretory pathways for the ecosystem. A continued increase in the CO2 availability for primary production, together with changes in the nutrient loading of coastal ecosystems, may cause changes in the trophic links between primary producers and herbivores.

Impact of dissolved inorganic carbon concentrations and pH on growth of the chemolithoautotrophic epsilonproteobacterium GD1

Epsilonproteobacteria have been found globally distributed in marine anoxic/sulfidic areas mediating relevant transformations within the sulfur and nitrogen cycles. In the Baltic Sea redox zones, chemoautotrophic epsilonproteobacteria mainly belong to the Sulfurimonas gotlandica GD17 cluster for which recently a representative strain, S. gotlandica GD1T, could be established as a model organism. In this study, the potential effects of changes in dissolved inorganic carbon (DIC) and pH on S. gotlandica GD1T were examined. Bacterial cell abundance within a broad range of DIC concentrations and pH values were monitored and substrate utilization was determined. The results showed that the DIC saturation concentration for achieving maximal cell numbers was already reached at 800 µmol/l, which is well below in situ DIC levels. The pH optimum was between 6.6 and 8.0. Within a pH range of 6.6-7.1 there was no significant difference in substrate utilization; however, at lower pH values maximum cell abundance decreased sharply and cell-specific substrate consumption increased.

Sources and proxy potential of long chain alkyl diols in lacustrine environments

Long chain 1,13- and 1,15-alkyl diols form the base of a number of recently proposed proxies used for climate reconstruction. However, the sources of these lipids and environmental controls on their distribution are still poorly constrained. We have analyzed the long chain alkyl diol (LCD) composition of cultures of ten eustigmatophyte species, with three species from different families grown at various temperatures, to identify the effect of species composition and growth temperature on the LCD distribution. The results were compared with the LCD distribution of sixty-two lake surface sediments, and with previously reported LCD distributions from marine environments. The different families within the Eustigmatophyceae show distinct LCD patterns, with the freshwater family Eustigmataceae most closely resembling LCD distributions in both marine and lake environments. Unlike the other two eustigmatophyte families analyzed (Monodopsidaceae and Goniochloridaceae), C28 and C30 1,13-alkyl diols and C30 and C32 1,15-alkyl diols are all relatively abundant in the family Eustigmataceae, while the mono-unsaturated C32 1,15-alkyl diol was below detection limit. In contrast to the marine environment, LCD distributions in lakes did not show a clear relationship with temperature. The Long chain Diol Index (LDI), a proxy previously proposed for sea surface temperature reconstruction, showed a relatively weak correlation (R2 = 0.33) with mean annual air temperature used as an approximation for annual mean surface temperature of the lakes. A much-improved correlation (R2 = 0.74, p-value<0.001) was observed applying a multiple linear regression analysis between LCD distributions and lake temperatures reconstructed using branched tetraether lipid distributions. The obtained regression model provides good estimates of temperatures for cultures of the family Eustigmataceae, suggesting that algae belonging to this family have an important role as a source for LCDs in lacustrine environments, or, alternatively, that the main sources of LCDs are similarly affected by temperature as the Eustigmataceae. The results suggest that LCDs may have the potential to be applicable as a palaeotemperature proxy for lacustrine environments, although further calibration work is still required.