Understanding the role of cleaning in the control of Salmonella Typhimurium in grower-finisher pigs: a modelling approach.

Salmonella Typhimurium (STM) infection in pigs represents a considerable food safety concern. This study used mathematical modelling to evaluate the effectiveness of cleaning (faeces removal) as a measure to control STM spread among grower-finisher pigs. A modified Susceptible-Infected-Recovered-Susceptible (SIRS) model of STM transmission through a contaminated environment was developed. Infected pigs were divided into three states according to the pathogen level being shed in their faeces. Infection transmission was evaluated using the basic reproduction number (R 0) and the prevalence of infectious pigs at slaughter age. Although increased frequency and efficiency of cleaning did reduce the prevalence of STM shedding at the time of slaughter, these efforts alone were not capable of eliminating the infection from the population. The level of STM faecal shedding by infectious pigs strongly influenced the infection spread and prevalence at slaughter. To control STM in pigs, cleaning should be combined with vaccination and/or isolation of high-level shedders.

Identifying areas of high risk of human exposure to coccidioidomycosis in Texas using serology data from dogs.

Coccidioidomycosis or Valley Fever (VF) is an emerging soil-borne fungal zoonosis affecting humans and animals. Most non-human cases of VF are found in dogs, which we hypothesize may serve as sentinels for estimating the human exposure risk. The objective of this study is to use the spatial and temporal distribution and clusters of dogs seropositive for VF to define the geographic area in Texas where VF is endemic, and thus presents a higher risk of exposure to humans. The included specimens were seropositive dogs tested at a major diagnostic laboratory between 1999 and 2009. Data were aggregated by zip code and smoothed by empirical Bayesian estimation to develop an isopleth map of VF seropositive rates using kriging. Clusters of seropositive dogs were identified using the spatial scan test. Both the isopleth map and the scan test identified an area with a high rate of VF-seropositive dogs in the western and southwestern parts of Texas (relative risk = 31). This location overlapped an area that was previously identified as a potential endemic region based on human surveys. Together, these data suggest that dogs may serve as sentinels for estimating the risk of human exposure to VF.

Effectiveness of environmental decontamination as an infection control measure.

The effectiveness of environmental decontamination (ED) as a measure in the control of infectious diseases is controversial. This work quantifies the effectiveness of ED by analysing the transmission of pathogens from the environment to susceptible hosts in a Susceptible-Infected-Susceptible model. Analysis of the model shows that ED can render a population disease-free only when the duration of infection (D) is within a certain range. As host-to-host transmission rate is increased, D falls outside this range and the higher levels of ED have a diminishing return in reducing the number of infected hosts at endemic equilibrium. To avoid this, ED can be combined with other control measures, such as treating infected individuals to push the duration of infection into the specified range. We propose decision criteria and minimum ED efforts required for control policies to be effective.

Characterization and role of NUMB in the human extravillous trophopblast.

NUMB is a multifunctional protein involved in asymmetric cell differentiation, proliferation and maintenance. Four mammalian NUMB isoforms have been identified, which utilize the phosphotyrosine binding (PTB) domain and the proline rich region (PRR) domain to regulate cell growth and differentiation in the developing nervous system. The observation that a decrease in spongiotrophoblast number and thickness of placentae of null (Numb(-/-)) mouse embryos, which died at E10.5, suggests NUMB may play a role in placental development. In this study, we demonstrated for the first time, that NUMB isoforms 1, 2, 3, and 4 are present in the human placenta and the human extravillous trophoblast (EVT) cell line HTR8/SVneo. We report three novel isoforms, NUMB 7, 8, and 9, identified by cloning of RT-PCR products and sequencing. Corresponding sequences of novel isoforms were submitted to genebank (accession numbers for each new isoform: NUMB 7- EU265736, NUMB 8- EU265737 and NUMB 9-EU265738). Western blot analysis confirmed the presence of all NUMB isofoms in human placental samples in all trimesters and in EVT cells. NUMB immunosignals were extensively localized in human extravillous trophoblasts and decidual cells at the maternal-fetal interface. NUMB 8 appeared to be the predominant isoform in placental villi. Furthermore, cell migration studies revealed NUMB isoform 1 to be involved in EVT cell migration and NUMB isoforms 2 and 4 to induce EVT apoptosis.

Rapid fabrication and chemical patterning of polymer microstructures and their applications as a platform for cell cultures.

Much of the current knowledge regarding biological processes has been obtained through in-vitro studies in bulk aqueous solutions or in conventional Petri-dishes, with neither methodology accurately duplicating the actual in-vivo biological processes. Recently, a number of innovative approaches have attempted to address these shortcomings by providing substrates with controlled features. In particular, tunable surface chemistries and topographical micro and nanostructures have been used as model systems to study the complex biological processes. We herein report a versatile and rapid fabrication method to produce a variety of microstructured polymer substrates with precise control and tailoring of their surface chemistries. A poly(dimethylsiloxane) (PDMS) substrate, produced by replication over a master mold with specific microstructures, is modified by a fluoro siloxane derivative to enhance its anti-adhesion characteristics and used as a secondary replication mold. A curable material, deposited by spin coating on various substrates, is stamped with the secondary mold and crosslinked. The removal of the secondary mold produces a microstructured surface with the same topographical features as the initial master mold. The facile chemical patterning of the microstructured substrates is demonstrated through the use of microcontact printing methods and these materials are tested as a platform to guide cell attachment, growth and proliferation. The master mold and flexible fluorinated PDMS stamps can be used in a repeated manner without any degradation of the anti-adhesion characteristics opening the way to the development of high-throughput fabrication methods that can yield reliable and inexpensive microstructured and chemically patterned substrates.

Array analysis of the genes regulated during neuronal differentiation of human embryonal cells.

Recent advances in genetic technology have provided a new platform on which the simultaneous analysis of a large number of genes is possible in a rapid and efficient fashion. To assess the differential expression of human genes during neuronal differentiation, we compared the transcript profiles of undifferentiated, partially differentiated, and fully differentiated NT2/D1 cultures with cDNA expression arrays. Approximately 75 genes (13% of the gene array pool) were differentially expressed during neuronal development of NT2/D1 cells. Genes coding for pyruvate kinase M2 isozyme, clathrin assembly proteins, calmodulin, fibronectin, laminin, thymosin beta-10, and many others were upregulated as NT2/D1 cells differentiated into neurons. In contrast, several kinases, phosphatases, and G-protein coupled receptor genes showed downregulation upon neuronal differentiation. The information provided here is an invaluable reference for characterizing the phenotype of these cells. This information can also be used in cell therapy and transplantation in which the graft microenvironment and interaction with the host tissue is crucial.

The effects of bone morphogenetic protein 2 and 4 (BMP2 and BMP4) on gap junctions during neurodevelopment.

Nervous system deficits account for the third largest group of fatal birth defects (after heart and respiratory problems) in North America. Although considerable advance has been made in neuroscience research, the early events involved in neurogenesis remain to be elucidated. More specifically, the effects of signaling molecules on intercellular communication during neurodevelopment have not yet been studied. The development of the central nervous system is regulated, at least in part, by signaling molecules such as bone morphogenetic proteins (BMPs). In this study, we have used the embryonal mouse P19 cell line to examine the effects of BMP2 and BMP4 on gap junctional communication as well as neuronal and astrocytic differentiation. The undifferentiated P19 cells show high levels of the gap junction protein, connexin43 (Cx43), and functional intercellular coupling. However, Cx43 expression and dye coupling decrease as these cells differentiate into neurons and astrocytes. In contrast, cells treated with BMP2 or BMP4 lose their capacity to differentiate into neurons but not astrocytes, while they maintain extensive gap junctional communication. The very few neurons that remain in the BMP-treated cultures are coupled (a characteristic not seen in the control neurons). Together, our data suggest that BMPs may play a critical role in morphogenesis of P19 cells while they affect gap junctions.

The effects of gap junction blockage on neuronal differentiation of human NTera2/clone D1 cells.

Gap junctions are intercellular channels which provide for the passage of small ions and molecules (MW <1200 D) among adjacent cells. The NTera2/clone D1 (NT2/D1) cells are CNS precursors which differentiate into NT2-N neurons upon treatment with retinoic acid (RA) and antiproliferative agents. In this study, the effects of gap junction blockers 18 alpha-glycyrrhetinic acid (GRA) and carbenoxolone (CBX) have been compared with those of oleanolic acid (OLA) and glycyrrhizic acid (GZA), GRA analogs with no blocking effects. Both control and experimental cultures showed reduction of Cx43 protein after 4 weeks of RA induction. A major reduction was also observed in expression of cytokeratin, vimentin, and nestin in control cells at this time point while the cultures treated with the blockers did not show any significant change. The average number of MAP2-positive NT2-N differentiated neurons per field of view in the cultures treated with the blockers was less than 7% of that of control cultures. NT2-N cells were negative for Cx43, cytokeratin, vimentin, and nestin. The blockers did not appear to be operating through inhibition of RA signaling, as their presence did not affect the expression of retinoic acid receptors (RARalpha and RARgamma) nor did they inhibit RA-mediated gene transcription. These results, together, show that the blockage of gap junctions interferes with neuronal differentiation of NT2/D1 cells.

Gap junction blockage interferes with neuronal and astroglial differentiation of mouse P19 embryonal carcinoma cells.

During embryonic development, cells not only increase in number, they also undergo specialization and differentiate into diverse cell types that are organized into different tissues and organs. Nervous system development, for example, involves a complex series of events such as neuronal and astroglial differentiation that are coordinated among adjacent cells. The organization of growth and differentiation may be mediated, at least partly, by exchange of small ions and molecules via intercellular gap junction channels. These structures are mode of connexons (hemichannels), which are hexameric assemblies of the gap junction proteins, connexins. We investigated the role of intercellular communication in neuronal and astroglial differentiation by using a gap junction blocking agent, carbenoxolone (CBX), in comparison to its inactive (control) analog, glycyrrhizic acid (GZA). We used the mouse P19 embryonal carcinoma cell line, which differentiates into neurons and astrocytes upon retinoic acid (RA) induction. Our results show that both GZA- and CBX-treated cells express alpha 1 connexin (connexin43). The level of alpha 1 connexin decreases upon RA induction. CBX treated cells show significant reduction in both neuronal (5-fold) and astrocytic (13-fold) differentiation compared with those of control. These results clearly indicate that the blockage of gap junction-mediated intercellular communication interferes with differentiation of P19 cells into neurons and astrocytes.

Consequences of impaired gap junctional communication in glial cells.

Astrocytes are characterized by extensive gap junctional intercellular communication (GJIC) mediated by channels composed primarily of connexin43. To examine some of the functions of this intercellular communication in glial cells, we have used three approaches. The first involves transfection of glioma cells, which are deficient in connexin expression and gap junctional communication, with connexin cDNAs to examine changes in cellular phenotype following increased gap junctional communication. Using differential display, we have identified several genes which appear to be regulated by GJIC. The second is to study astrocytes cultured from embryonic mice with a null mutation in the connexin43 gene. These homozygous null astrocytes are devoid of connexin43 and also deficient in intercellular dye transfer. Markers of glial differentiation appear similar in all genotypes. Measurement of intercellular calcium concentration following mechanical stimulation of confluent astrocytes revealed that the number of cells affected by a rise in intracellular calcium was reduced in homozygous cultures compared to wild type. The growth rate of astrocytes lacking connexin43 was reduced compared to wild-type astrocytes. The third approach employs the use of gap junction blockers in a model of neuronal and glial differentiation, namely P19 mouse embryonal carcinoma cells treated with retinoic acid. In this case, blocking GJIC during the differentiation protocol prevents the appearance of neuronal and astrocytic phenotypes. Taken together, these data suggest an important role for GJIC in glial function and differentiation.

Human NT2/D1 cells differentiate into functional astrocytes.

As the most numerous cell type in the brain, astrocytes are coupled via gap junction channels. It is believed that communication among astrocytes is normally regulated by extracellular ions, neurotransmitters and neuromodulators. However, the level of astrocytic coupling is altered in abnormal conditions such as stroke, brain trauma and Alzheimer's disease. A well established human progenitor cell line, NT2/D1, has been previously differentiated into pure neuronal cultures. In the current study, for the first time, we report the differentiation of NT2/D1 cells into astrocytes, which express connexin43 and are coupled via gap junctions. Thus, human NT2/D1 cells are not merely committed neuronal progenitors but, similar to the embryonal stem cells, they can give rise to both lineages.

Suppression of tumorigenicity of human lung carcinoma cells after transfection with connexin43.

The human lung carcinoma cell line PG is defective in gap junctional intercellular communication (GJIC). Connexin43 (Cx43) mRNA, which is expressed in normal human lung cells, is undetectable in these tumor cells. To explore if up-regulation of Cx43 gene expression will suppress malignancy of PG cells, Cx43 cDNA was co-transfected with pSV2neo cDNA into PG cells. Control cells were transfected with the blank vector plus neo cDNA. Several stable Cx43 transfectant clones, which acquired high levels of Cx43 expression and the capacity of GJIC, were compared with control clones and the parental cell line, both of which lacked Cx43 expression and GJIC. The control clones resembled the parental cells in exhibiting high cell growth rate, weak attachment to the substratum and a high frequency of colony formation in soft agar. In contrast to the control cells, Cx43 transfected clones showed reduced growth rate, enhanced attachment to the substratum and inhibition of colony formation in soft agar. In vivo results from nude mice experiments showed high tumorigenicity with control clones and inhibition of tumorigenicity in Cx43 transfected clones. The consistency between in vitro and in vivo results strongly suggests a tumor suppressing effect of the Cx43 gene in human lung carcinoma cells.

Gap junctional communication in the developing central nervous system.

The development of the central nervous system is a complex process involving multiple interactions between various cell types undergoing mitosis, migration, differentiation, axonal outgrowth, synaptogenesis and programmed cell death. For example, neocortical development is characterized by a series of transient events that ultimately leads to the formation of a discrete pattern of laminar and columnar organization. While neuron-glial cell-cell interactions have been shown to be involved in neuronal migration, recent observations that neurons are extensively coupled by gap junctions in the developing neocortex have implicated this phenomenon in the process of neocortical differentiation. The present review will examine the putative role of gap junctional intercellular communication in development of the central nervous system, with specific reference to recent studies in the development of the cerebral cortex.

Reduction of connexin43 expression and dye-coupling during neuronal differentiation of human NTera2/clone D1 cells.

Gap junctions are plasma membrane specializations that allow direct communication among adjoining cells. We used a human pluripotential teratocarcinoma cell line, NTera-2/clone D1 (NT2/D1), as a model to study gap junctions in CNS neurons and their neuronal precursors. These cells were differentiated following retinoic acid (RA) treatment for 4 weeks and antiproliferative agents for 3 weeks, respectively, to yield post-mitotic CNS neuronal (NT2-N) cells. The cytoplasmic RNA was isolated from NT2/D1 cells both before and during RA treatment and from differentiated neurons (NT2-N cells). These RNA samples were examined using Northern blot analysis with cDNA probes specific for connexin26, -32, and -43. Connexin26 and -32 mRNAs were absent in NT2/D1 and NT2-N cells. Connexin43 mRNA was expressed at high levels in NT2/D1 cells before RA treatment, but it decreased significantly during RA induction. There was no detectable connexin43 mRNA in NT2-N cells. Western blot analysis confirmed the expression of connexin43 protein in NT2/D1 cells before and during RA treatment. The protein profile detected in Western blot analysis indicated two bands representing different phosphorylation states of connexin43. Our immunocytochemistry results did not show connexin26 and -32 immunoreactivity in NT2/D1 and NT2-N cells. However, we detected connexin43 immunoreactivity in NT2/D1 cells with a decreasing pattern upon RA induction. Both Western blotting and immunocytochemistry confirmed the absence of connexin43 protein in NT2-N cells. NT2/D1 cells passed calcein readily to an average of 18 cells, confirming the functionality of gap junctions in these cells. The extent of dye-coupling decreased about 78% when NT2/D1 cells were RA treated for 4 weeks. NT2-N differentiated neurons did not pass dye to the adjacent cells. We conclude that both connexin43 expression and dye coupling capacity decrease during neuronal differentiation of NT2/D1 cells.