Transcriptional analysis of persistent Chlamydia pneumoniae infection in vitro

Chlamydia pneumoniae can cause acute respiratory infections including pneumonia. Repeated and persistent Chlamydia infections occur and persistent C. pneumoniae infection may have a role in the pathogenesis of atherosclerosis and coronary heart disease and may also contribute to the development of chronic inflammatory lung diseases like chronic obstructive pulmonary disease (COPD) and asthma. In this thesis in vitro models for persistent C. pneumonia infection were established in epithelial and monocyte/macrophage cell lines. Expression of host cell genes in the persistent C. pneumoniae infection model of epithelial cells was studied by microarray and RT-PCR. In the monocyte/macrophage infection model expression of selected C. pneumoniae genes were studied by RT-PCR and immunofluorescence microscopy. Chlamydia is able to modulate host cell gene expression and apoptosis of host cells, which may assist Chlamydia to evade the host cells' immune responses. This, in turn, may lead to extended survival of the organism inside epithelial cells and promote the development of persistent infection. To simulate persistent C. pneumoniae infection in vivo, we set up a persistent infection model exposing the HL cell cultures to IFN-gamma. When HL cell cultures were treated with moderate concentration of IFN-gamma, the replication of C. pneumoniae DNA was unaffected while differentiation into infectious elementary bodies (EB) was strongly inhibited. By transmission electron microscopy small atypical inclusions were identified in IFN-gamma treated cultures. No second cycle of infection was observed in cells exposed to IFN-gamma , whereas C. pneumoniae was able to undergo a second cycle of infection in unexposed HL cells. Although monocytic cells can naturally restrict chlamydial growth, IFN-gamma further reduced production of infectious C. pneumoniae in Mono Mac 6 cells. Under both studied conditions no second cycle of infection could be detected in monocytic cell line suggesting persistent infection in these cells. As a step toward understanding the role of host genes in the development and pathogenesis of persistent C. pneumoniae infection, modulation of host cell gene expression during IFN-gamma induced persistent infection was examined and compared to that seen during active C. pneumoniae infection or IFN-gamma treatment. Total RNA was collected at 6 to 150 h after infection of an epithelial cell line (HL) and analyzed by a cDNA array (available at that time) representing approximately 4000 human transcripts. In initial analysis 250 of the 4000 genes were identified as differentially expressed upon active and persistent chlamydial infection and IFN-gamma treatment. In persistent infection more potent up-regulation of many genes was observed in IFN-gamma induced persistent infection than in active infection or in IFN-gamma treated cell cultures. Also sustained up-regulation was observed for some genes. In addition, we could identify nine host cell genes whose transcription was specifically altered during the IFN-gamma induced persistent C. pneumoniae infection. Strongest up-regulation in persistent infection in relation to controls was identified for insulin like growth factor binding protein 6, interferon-stimulated protein 15 kDa, cyclin D1 and interleukin 7 receptor. These results suggest that during persistent infection, C. pneumoniae reprograms the host transcriptional machinery regulating a variety of cellular processes including adhesion, cell cycle regulation, growth and inflammatory response, all of which may play important roles in the pathogenesis of persistent C. pneumoniae infection. C. pneumoniae DNA can be detected in peripheral blood mononuclear cells indicating that the bacterium can also infect monocytic cells in vivo and thereby monocytes can assist the spread of infection from the lungs to other anatomical sites. Persistent infection established at these sites could promote inflammation and enhance pathology. Thus, the mononuclear cells are in a strategic position in the development of persistent infection. To investigate the intracellular replication and fate of C. pneumoniae in mononuclear cells we analyzed the transcription of 11 C. pneumoniae genes in Mono Mac 6 cells during infection by real time RT-PCR. Our results suggest that the transcriptional profile of the studied genes in monocytes is different from that seen in epithelial cells and that IFN-gamma has a less significant effect on C. pneumoniae transcription in monocytes. Furthermore, our study shows that type III secretion system (T3SS) related genes are transcribed and that Chlamydia possesses a functional T3SS during infection in monocytes. Since C. pneumoniae infection in monocytes has been implicated to have reduced antibiotic susceptibility, this creates opportunities for novel therapeutics targeting T3SS in the management of chlamydial infection in monocytes.

Assessment of In-House Natural Product and Synthetic Compound Libraries Based on In vitro Inhibition of Cholinesterases

The first line medication for mild to moderate Alzheimer s disease (AD) is based on cholinesterase inhibitors which prolong the effect of the neurotransmitter acetylcholine in cholinergic nerve synapses which relieves the symptoms of the disease. Implications of cholinesterases involvement in disease modifying processes has increased interest in this research area. The drug discovery and development process is a long and expensive process that takes on average 13.5 years and costs approximately 0.9 billion US dollars. Drug attritions in the clinical phases are common due to several reasons, e.g., poor bioavailability of compounds leading to low efficacy or toxic effects. Thus, improvements in the early drug discovery process are needed to create highly potent non-toxic compounds with predicted drug-like properties. Nature has been a good source for the discovery of new medicines accounting for around half of the new drugs approved to market during the last three decades. These compounds are direct isolates from the nature, their synthetic derivatives or natural mimics. Synthetic chemistry is an alternative way to produce compounds for drug discovery purposes. Both sources have pros and cons. The screening of new bioactive compounds in vitro is based on assaying compound libraries against targets. Assay set-up has to be adapted and validated for each screen to produce high quality data. Depending on the size of the library, miniaturization and automation are often requirements to reduce solvent and compound amounts and fasten the process. In this contribution, natural extract, natural pure compound and synthetic compound libraries were assessed as sources for new bioactive compounds. The libraries were screened primarily for acetylcholinesterase inhibitory effect and secondarily for butyrylcholinesterase inhibitory effect. To be able to screen the libraries, two assays were evaluated as screening tools and adapted to be compatible with special features of each library. The assays were validated to create high quality data. Cholinesterase inhibitors with various potencies and selectivity were found in natural product and synthetic compound libraries which indicates that the two sources complement each other. It is acknowledged that natural compounds differ structurally from compounds in synthetic compound libraries which further support the view of complementation especially if a high diversity of structures is the criterion for selection of compounds in a library.

In vitro meristem cloning of Eucalyptus tereticornis Sm

Rapid multiplication of axillary meristems and direct shoot development occurred from nodal explants of mature Eucalyptus tereticornis Sm. with 5.3 mgrM NAA, 1.1 mgrM IAA and 4.4 mgrM BA in Murashige-Skoog medium. Repeated subcultures of the second generation shoot cultures into low cytokinin-auxin containing media (0.44�0.88 mgrM BA+0.1 mgrM NAA) yielded axillary microshoots in large numbers. Half-strength MS liquid medium with 4.9 mgrM IBA, 5.5 mgrM IAA and 5.3 mgrM NAA for four days, half-strength semi-solid hormonefree MS medium with charcoal, and MS liquid medium without charcoal and hormones, in sequence, induced rooting of shoots in the dark. This system is suitable for the mass propagation of this difficult-to-root eucalypt.

The effects of organic environmental toxicants on hard tissue formation in developing tooth : An in vitro study in mice

Dioxins are organic toxicants that are known to impair tooth development, especially dental hard tissue formation. The most toxic dioxin congener is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Further, clinical studies suggest that maternal smoking during pregnancy can affect child s tooth development. One of the main components of tobacco smoke is the group of non-halogenated polycyclic aromatic hydrocarbons (PAHs), a representative of which is 7,12-dimethylbenz[a]anthracene (DMBA). Tributyltin (TBT), an organic tin compound, has been shown to impair bone mineralization in experimental animals. In addition to exposure to organic toxicants, a well-established cause for enamel hypomineralization is excess fluoride intake. The principal aim of this thesis project was to examine in vitro if, in addition to dioxins, other organic environmental toxicants, like PAHs and organic tin compounds, have adverse effects on tooth development, specifically on formation and mineralization of the major dental hard tissues, the dentin and the enamel. The second aim was to investigate in vitro if fluoride could intensify the manifestation of the detrimental developmental dental effects elicited by TCDD. The study was conducted by culturing mandibular first and second molar tooth germs of E18 NMRI mouse embryos in a Trowell-type organ culture and exposing them to DMBA, TBT, and sodium fluoride (NaF) and/or TCDD at various concentrations during the secretory and mineralization stages of development. Specific methods used were HE-staining for studying cell and tissue morphology, BrdU-staining for cell proliferation, TUNEL-staining for apoptosis, and QPCR, in situ hybridization and immunohistochemistry for the expressions of selected genes associated with mineralization. This thesis work showed that DMBA, TBT, TCDD and NaF interfere with dentin and enamel formation of embryonic mouse tooth in vitro, and that fluoride can potentiate the harmful effect of TCDD. The results suggested that adverse effects of TBT involve altered expression of genes associated with mineralization, and that DMBA and TBT as well as NaF and TCDD together primarily affect dentin mineralization. Since amelogenesis does not start until mineralization of dentin begins, impaired enamel matrix secretion could be a secondary effect. Dioxins, PAHs and organotins are all liposoluble and can be transferred to the infant by breast-feeding. Since doses are usually very low, developmental toxicity on most of the organs is difficult to indentify clinically. However, tooth may act as an indicator of exposure, since the major dental hard tissues, the dentin and the enamel, are not replaced once they have been formed. Thus, disturbed dental hard tissue formation raises the question of more extensive developmental toxicity.

A role for calcium in gonadotrophin-releasing hormone (GnRH) stimulated secretion of chorionic gonadotrophin by first trimester human placental minces in vitro

In vitro studies using first-trimester human placental minces have shown that stimulation of human chorionic gonadotrophin (hCG) secretion by gonadotrophin-releasing hormone (GnRH) is dependent upon the presence of extracellular calcium. Addition of GnRH to first-trimester placental minces in vitro was found to stimulate 45Ca2+ uptake into placental minces, and the process was associated with an increase in immunoreactive hCG in the medium. Addition of GnRH to placental minces preloaded with 45Ca2+ stimulated the efflux of 45Ca2+ within one minute. The calmodulin inhibitors chlorpromazine andtrifluoperazine inhibited the basal uptake and efflux of 45Ca2+ suggesting the involvement of calmodulin in the mobilization of calcium in the placenta.

CSR in forest industry – Case study of reporting and implementation of social responsibility in three international companies

Corporate Social Responsibility (CSR) has become increasingly important topic in forest industries, and other global companies, in recent years. Globalisation, faster information delivery and demand for sustainable development have set new challenges for global companies in their business operations. Also the importance of stakeholder relations, and pressure to become more transparent has increased in the forest industries. Three dimensions of corporate responsibility economic, environmental and social, are often included in the concept of CSR. Global companies mostly claim that these dimensions are equally important. This study analyses CSR in forest industry and has focus on reporting and implementation of social responsibility in three international companies. These case-companies are Stora Enso, SCA and Sappi, and they have different geographical base, product portfolios and therefore present interesting differences about forest industry strategy and CSR. Global Reporting Initiative (GRI) has created the most known and used reporting framework in CSR reporting. GRI Guidelines have made CSR reporting a uniform function, which can also be measured between companies and different sectors. GRI Guidelines have also made it possible to record and control CSR data in the companies. In recent years the use of GRI Guidelines has increased substantially. Typically CSR reporting on economic and environmental responsibility have been systematic in the global companies and often driven by legistlation and other regulations. However the social responsibility has been less regulated and more difficult to compare. Therefore it has previously been often less focused in the CSR reporting of the global companies. The implementation and use of GRI Guidelines have also increased dialogue on social responsibility issues and stakeholder management in global companies. This study analyses the use of GRI´s framework in the forest industry companies´ CSR reporting. This is a qualitative study and the disclosure of data is empricially analysed using content analysis. Content analysis has been selected as a method for this study because it makes it possible to use different sources of information. The data of this study consists of existing academic literature of CSR, sustainability reports of thecase-companies during 2005-2009, and the semi-structured interviews with company representatives. Different sources provide the possibility to look at specific subject from more than one viewpoint. The results of the study show that all case-companies have relatively common themes in their CSR disclosure, and the differences rise mainly from their product-portfolios, and geographic base. Social impacts to local communities, in the CSR of the companies, were mainly dominated by issues concerning creating wealth to the society and impacting communities through creation of work. The comparability of the CSR reporting, and especially social indicators increased significally from 2007 onwards in all case-companies. Even though the companies claim that three dimensions of CSR economic, environmental and social are equally important economic issues and profit improvement still seem to drive most of the operations in the global companies. Many issues that are covered by laws and regulations are still essentially presented as social responsibility in CSR. However often the unwelcome issues in companies like closing operations are covered just briefly, and without adequate explanation. To make social responsibility equally important in the CSR it would demand more emphasis from all the case-companies. A lot of emphasis should be put especially on the detail and extensiveness of the social reponsibility content in the CSR.

In vitro expression of belladonna mottle virus genome

In vitro translation of belladonna mottle virus BDMV(I) genomic RNA in a rabbit reticulocyte lysate system produced proteins of Mr 210,000, 150,000 and 78,000 which form the non-structural proteins. The coat protein, on the other hand, was expressed from a subgenomic RNA which was found to be encapsidated in the empty capsids forming the top component viral particles. The implications of subgenomic RNA encapsidation in viral replication and assembly are discussed.

In vitro protection of umbilical cord blood-derived primitive hematopoietic stem progenitor cell pool by mannose-specific lectins via antioxidant mechanisms.

BACKGROUND: Earlier we reported that an oral administration of two mannose-specific dietary lectins, banana lectin (BL) and garlic lectin (GL), led to an enhancement of hematopoietic stem and progenitor cell (HSPC) pool in mice. STUDY DESIGN AND METHODS: Cord blood derived CD34+ HSPCs were incubated with BL, GL, Dolichos lectin (DL), or artocarpin lectin (AL) for various time periods in a serum- and growth factor free medium and were subjected to various functional assays. Reactive oxygen species (ROS) levels were detected by using DCHFDA method. Cell fractionation was carried out using lectin-coupled paramagnetic beads. RESULTS: CD34+ cells incubated with the lectins for 10 days gave rise to a significantly higher number of colonies compared to the controls, indicating that all four lectins possessed the capacity to protect HSPCs in vitro. Comparative analyses showed that the protective ability of BL and GL was better than AL and DL and, therefore, further experiments were carried out with them. The output of long-term culture-initiating cell (LTC-IC) and extended LTC-IC assays indicated that both BL and GL protected primitive stem cells up to 30 days. The cells incubated with BL or GL showed a substantial reduction in the ROS levels, indicating that these lectins protect the HSPCs via antioxidant mechanisms. The mononuclear cell fraction isolated by lectin-coupled beads got enriched for primitive HSPCs, as reflected in the output of phenotypic and functional assays.CONCLUSION: The data show that both BL and GL protect the primitive HSPCs in vitro and may also serve as cost-effective HSPC enrichment tools.

Putative neuroprotective compounds in in vitro models of dopaminergic neurodegeneration

Parkinson´s disease (PD) is a debilitating age-related neurological disorder that affects various motor skills and can lead to a loss of cognitive functions. The motor symptoms are the result of the progressive degeneration of dopaminergic neurons within the substantia nigra. The factors that influence the pathogenesis and the progression of the neurodegeneration remain mostly unclear. This study investigated the role of various programmed cell death (PCD) pathways, oxidative stress, and glial cells both in dopaminergic neurodegeneration and in the protective action of various drugs. To this end, we exposed dopaminergic neuroblastoma cells (SH-SY5Y cells) to 6-OHDA, which produces oxidative stress and activates various PCD modalities that result in neuronal degeneration. Additionally, to explore the role of glia, we prepared rat midbrain primary mixed-cell cultures containing both neurons and glial cell types such as microglia and astroglia and then exposed the cultures to either MPP plus or lipopolysaccharide. Our results revealed that 6-OHDA activated several PCD pathways including apoptosis, autophagic stress, lysosomal membrane permeabilization, and perhaps paraptosis in SH-SY5Y cells. Furthermore, we found that minocycline protected SH-SY5Y cells from 6-OHDA by inhibiting both apoptotic and non-apoptotic PCD modalities. We also observed an inconsistent neuroprotective effect of various dietary anti-oxidant compounds against 6-OHDA toxicity in vitro in SH-SY5Y cells. Specifically, quercetin and curcumin exerted neuroprotection only within a narrow concentration range and a limited time frame, whereas resveratrol and epigallocatechin 3-gallate provided no protection whatsoever. Lastly, we found that molecules such as amantadine may delay or even halt the neurodegeneration in primary cell cultures by inhibiting the release of neurotoxic factors from overactivated microglia and by enhancing the pro-survival actions of astroglia. Together these data suggest that the strategy of dampening oxidative species with anti-oxidants is less effective than preventing the production of toxic factors such as oxidative and pro-inflammatory molecules by pathologically activated microglia. This would subsequently prevent the activation of various PCD modalities that cause neuronal degeneration.

Candida-hiivojen asetaldehydin tuotto etanoli- ja glukoosi-inkubaatiossa in vitro

Yläruoansulatuskanavan syöpien tärkeimpiä riskitekijöitä ovat tupakointi, alkoholin suurkulutus ja huono suuhygienia. Näiden tekijöiden vaikutuksesta sylkeen erittyy korkeita pitoisuuksia asetaldehydiä, jonka Kansainvälinen syöväntutkimuslaitos (IARC) on luokitellut 1-ryhmän karsinogeeniksi. Suuri osa syljen asetaldehydistä on suun mikrobien tuottamaa. Tiedetään, että suun mikrobiomiin kuuluvat bakteerit ja Candida albicans -hiivat kykenevät tuottamaan mutageenisiä määriä asetaldehydiä. C. albicansin aiheuttaman kroonisen mukosiitin onkin todettu olevan karsinogeeninen. Muiden kandidalajien (non- albicans Candida, NAC) määrän on todettu kasvavan etenkin suusyöpähoitoja saavilla potilailla ja toisinaan osalle näistä potilaista kehittyy uusi primäärikarsinooma kandidamukosiitin läheisyyteen. NAC-lajien kykyä tuottaa asetaldehydiä ei kuitenkaan ole aiemmin tutkittu. Tutkimuksen tavoitteena oli selvittää pystyvätkö NAC-lajit tuottamaan karsinogeenisiä määriä asetaldehydiä etanoli- ja glukoosi-inkubaatiossa in vitro. Kaikkiaan kolmenkymmenen (n=30) kliinisen ja kantapankkiNAC-kannan kyky tuottaa asetaldehydiä etanoli- ja glukoosi-inkubaatiossa mitattiin kaasukromatografilla. Yksi C. albicans kantapankkikanta oli mukana kontrollina. Kaikki kandidahiivat tuottivat merkittäviä määriä asetaldehydiä etanoli-inkubaatiossa in vitro. C. tropicalis –kannat tuottivat eniten (252,3 µM) ja C. krusei –kannat vähiten (54,6 µM) asetaldehydiä etanolista. NAC-lajeista ainoastaan C. glabrata tuotti merkittäviä määriä asetaldehydiä glukoosia fermentoimalla. Suuontelon kolonisoituminen merkittävään asetaldehydituotantoon pystyvällä NAC-lajilla kuten C. glabratalla voi altistaa suun limakalvon paikallisesti korkeille määrille asetaldehydiä, mikä voi johtaa suusyövän kehittymiseen.

Acute toxicity of methyl isocyanate in rabbit: In vitro and in vivo effects on rabbit erythrocyte membrane

Methyl isocyanate (MIC) interaction with the rabbit erythrocyte membrane increased the fluidity of the membrane and decreased the osmotic fragility of erythrocytes both in vitro and in vivo in rabbits intoxicated with MIC subcutaneously. MIC inhibited both acetylcholinesterase (AChE) and adenosine triphosphatase (ATPase) activities of erythrocytes dose-dependently in vitro, while in vivo a decreased trend in ATPase activity with unaltered AChE activity was observed. MIC also caused significant decrease in plasma sodium level with corresponding increase in potassium level in rabbits. The observed effects are due to MIC, per se, as the hydrolysis products of MIC, methylamine and N,Nprime-dimethylurea did not affect the erythrocyte fluidity and enzymes activities both in vitro and in vivo while they increased the osmotic fragility of erythrocytes in vivo in rabbits administered subcutaneously in equimolar concentration to MIC dosage. Inhibition of Na+-K+-dependent ATPase with altered permeability to cations and also probably water transport of plasma membrane due to MIC interaction are envisaged.

Effect of 80 kDa protein of porcine follicular fluid on gonadotropin stimulated progesterone production in rat granulosa cells in vitro

We reported the presence of a 80 kDa polypeptide in porcine follicular fluid that inhibited the binding of 125I-radiolabelled hFSH as well as hCG to the rat ovarian gonadotropin receptors. In the present study, the biological activity of the receptor binding inhibitor is determined using an in vitro bioassay procedure. Granulosa cells isolated from PMSG primed immature rat ovaries respond to exogenously added gonadotropins in terms of progesterone production. Addition of fractions containing the gonadotropin receptor binding inhibitory activity inhibited progesterone production stimulated by the gonadotropins in a dose-dependent fashion. The receptor binding inhibitory activity was also capable of inhibiting progesterone production stimulated by PMSG, which has both FSH- and LH-like activities in rats. In contrast, progesterone production stimulated by dbcAMP was not inhibited by the receptor binding inhibitor. This result indicates that the site of action of the inhibitor is proximal to the formation of the cAMP. The above observations point out to a possible role for this factor in modulating gonadotropin activity at the ovarian level.

Inhibition of in Vitro Aminoacylation and Translation by RNA Reactive Antibodies

Antibodies were raised against guanosine-BSA, GMP-BSA and tRNA-mBSA conjugates separately in rabbits. Binding characteristics of these antibodies to various RNAs were studied using a sensitive avidin-biotin micro ELISA. These antibodies inhibited in vitro aminoacylation of tRNA in a dose dependent manner. This inhibition was reversed by the addition of the respective homologous haptens thereby showing the specificity of these antibodies. In vitro translation of endogenous mRNAs in rabbit reticulocyte lysate was also inhibited by these antibodies in a dose dependent manner.

Cytotoxic T lymphocytes raised against Japanese encephalitis virus: effector cell phenotype, target specificity and in vitro virus clearance.

Several H-2 defined cell lines were examined for their ability to support infection and replication of Japanese encephalitis virus (JEV) before their use in in vitro and in vivo stimulation protocols for generating cytotoxic T lymphocytes (CTLs) against JEV. Among II different cell lines tested, two H-2(d) macrophage tumour lines (P388D1, RAW 264.7), an H-2(d) hybridoma (Sp2/0), an H-2K(k)D(d) neuroblastoma (Neuro 2a), and H-2(k) fibroblast cell line (L929) were found to support JEV infection and replication. These cell lines were used to generate anti-JEV CTLs by using in vivo immunization followed by in vitro stimulation of BALB/c mice. We observed that not only syngeneic and allogeneic infected cells but also JEV-infected xenogeneic cells could prime BALB/c mice for the generation of JEV-specific CTLs upon subsequent in vitro stimulation of splenocytes with JEV-infected syngeneic cells. Although infected xenogeneic cells were used for immunization, the anti-JEV effecters that were generated lysed infected syngeneic targets but not JEV-infected xenogeneic or allogeneic target cells in a 5h Cr-51 release assay. These anti-JEV effecters recognized syngeneic target cells infected with West Nile virus to a lesser extent and were shown to be Lyt-2.2(+) T cells. The results of unlabelled cold target competition studies suggested alterations in the cell surface expression of viral antigenic determinants recognized by these CTLs. We further demonstrate that the JEV-specific CTLs generated could virtually block the release of infectious virus particles from infected P388D1 and Neuro 2a cells in vitro.