No description available.

A novel cell culture system was designed to serve as a model of neuroinflammation. Neurons, astrocytes, and microglia derived from embryonic and perinatal rat cortical tissue were combined in a three-dimensional hydrogel utilizing a method that facilitated cell maturation and viability. Chemical challenge of the cultures with a broad pro-inflammatory stimulus resulted in the production of inflammatory cytokines and other associated molecules commensurate with the response observed in vivo and in other in vitro systems. It was hypothesized that mechanical deformation of the multitypic neural cell cultures would produce a similar response and thus validate the system as an in vitro model of traumatic brain injury-induced neuroinflammation. Mechanical injury delivered using custom-manufactured culture chambers and injury devices successfully imparted a moderate level of cell death to the cultures. It was determined that a mechanically-induced inflammatory response required chemical stimulation prior to the injury. The research presented here describes the generation and characterization of a novel in vitro culture system and its implementation in experiments designed to model secondary injury mechanisms associated with injury-induced neuroinflammation. The findings of these studies, applications of the culture system, and future research avenues are discussed.

Epidemiological studies have linked exposure to elevated levels of airborne particulate matter with increased incidences of several types of respiratory disease, hospital admissions and morbidity. Millions of tons of airborne particulate matter are generated and released into the atmosphere each year. However, particulate matter resulting from the combustion of fuel oil and coal are of particular concern, because they are generally composed of small particles that can easily penetrate deep into the lungs, and can contain significant concentrations of toxic transition metals, such as zinc, iron and vanadium. Pulmonary toxicity (i.e. damage caused to lung tissues) of particulate matter is currently evaluated via time-consuming in-vivo testing, or via in-vitro testing. Compared to in-vivo testing, in-vitro testing offers significant advantages in terms of time savings and sample throughput. Unfortunately, the number of in-vitro testing methods are currently very limited, and do not allow a thorough investigation of the mechanisms of particulate matter toxicity. In light of these issues, the goals of the study described here were three-fold: *To adapt several in-vitro toxicity assays currently used in other applications to use in measuring particulate matter toxicity on lung cell layers; *To use these adapted assays to quantify the toxicity of numerous types of oil and coal ashes with varying particle sizes and transition metal concentrations, and; *To use the same assays to quantify the toxicities of several transition metals found in coal and oil ashes to better understand their relative contributions to overall particulate matter toxicity. Three colorimetric in-vitro assays were chosen for adaptation, and proved effective in measuring adverse cellular response to particulate matter exposure. Particle size was shown to have a large effect on the overall cytotoxicity of particulate matter; fine (less than 2.5 μm aerodynamic diameter) particles proved substantially more toxic than coarse (larger than 2.5 μm aerodynamic diameter) particles. Dose-response experiments measuring the toxic effects of the transition metals zinc, vanadium and iron revealed that zinc was the most toxic; a concentration of 0.6 mM caused a 50% drop in cellular metabolism, compared to 3 mM and 4 mM for vanadium and iron respectively.

THW

No description available.

In vitro cell culture is an essential part of many cell and tissue engineering approaches. In particular, monolayer culture of mammalian cells is a key tool for applications such as cell therapy. Novel bioreactors like the Cellerator(TM) allow for expansion of cell populations on mechanically stimulated surfaces coated with proteins. This thesis constitutes a preliminary study which focused on cell-matrix interactions in the absence of stretch. The aim was to establish standard protocols for protein coating on poly (dimethyl siloxane) (PDMS) and for measuring cell proliferation. Specifically, the proliferation of rat pulmonary artery vascular smooth muscle (PAC1) cells on type I collagen and soluble fibronectin was studied. Growth curves were obtained and the doubling time for subconfluent cultures was computed. Although cell-matrix interactions do not enhance proliferation of PAC1 cells, it was found that a preliminary sulphuric acid treatment is necessary to yield a well-behaved culture.

Cell culture media used in the manufacture of biopharmaceuticals conventionally contain many animal derived components. These components can harbor adventitious agents which can be transmitted through biotherapeutics, employed in the medical treatment of immunocompromised patients. An ADCF (animal derived component free) medium formulation obviates this concern. A rational method for the rapid and efficient screening and optimization of ADCF media while preserving, if not enhancing, cellular growth and protein productivity is needed. CHO (Chinese Hamster Ovary) cells, widely used as a production platform in industry, expressing a recombinant protein, were employed as a model system. Design of Experiment (DOE) and statistical analysis were employed to assess the impact of media formulation on cellular physiology. Metabolic flux, cellular growth, and protein productivity were evaluated as the measures of ADCF media formulation success. Measurements of extracellular metabolites were determined by HPLC and enzymatic methods. Recombinant protein production was measured by HPLC. This research demonstrates the successful screening and optimization of four plant hydrolysate mixtures (2 soy and 2 wheat) as a replacement for animal derived components. / NSERC, ABIN, MABNET

In vitro cell and tissue culture systems were developed for sugarcane in order to utilise current transformation techniques to introduce genes to South African sugarcane varieties, which would be difficult, if not impossible to achieve in conventional breeding programmes. Embryogenic calli were initiated in the dark from stem explants of sugarcane varieties NCo376 and N13, on a MS medium containing sucrose (20-50 g/l), 2,4-D (2-4 mg/l), casein (1 g/l), inositol (100 mg/l) and agar (9g/l). After 2 months the somatic embryos were cultured in a light/dark photoperiod for a further 2 months. The best combination of sucrose and 2,4-D for callus initiation, and subsequent plant regeneration, was 20 g/l and 2 mg/l, respectively. Plant yields ranged from 16 to 36 plants per gram fresh weight callus, and the yields were not significantly increased by the addition of activated charcoal to the regeneration medium. When plantlets reached a height of 10 cm, they were transferred to autoclaved soil in pots, hardened-off and placed in the glasshouse. Suspension cultures were initiated from friable NCo376 calli in liquid MS medium shaken at 100 rev/min in the dark at 27°C, and were subcultured every 3-7 days. Protoplasts from various sources (leaf, calli and suspension cultures) were obtained after enzymatic digestion in cellulase (20-30 g/l), macerozyme (0,2 g/l), hemicellulase (5 g/l), and sorbitol (0,55 M) in a calcium and magnesium salt solution. Protoplasts cultured for 48 h resulted in a loss in viability of 84%. The potential of the seed as a recipient for direct gene uptake was investigated, as this eliminated the need for in vitro culture and plant regeneration. Uptake of [3H] pBR322 DNA by seeds was demonstrated, and seeds with the testa removed exhibited higher initial uptake rates than those with intact seed coats. However, transient expression, using the GUS reporter gene (coding for bacterial B-glucuronidase) carried on plasmid pBI221, could not be conclusively shown using the histochemical GUS assay, due to GUS activity generated by either microbial contamination or endogenous plant GUS activity. Neither microwaving to eradicate contaminants nor the addition of methanol (20%) to the GUS incubation buffer were successful in overcoming positive results observed in control seeds. An alternative approach to sugarcane transformation, using PEG-mediated DNA uptake and subsequent transient expression of GUS by protoplasts was investigated, but microbial contamination was a persistant problem and no positive results were observed. Further examination and elimination of endogenous contamination is required before transformation studies can be continued. / Thesis (M.Sc.)-University of Natal, Durban, 1992.

A novel technique was developed to immobilize plant cells. The cells are deposited on a surface of man-made fibrous material which provides for strong binding of the plant tissue biomass growing in the submerged culture. It was shown that the plant cells need to be fully viable for the attachment process to occur. / The scale-up of this technique to laboratory size specifically designed bioreactors was performed successfully. The cell immobilizing matrix was formed into a vertical spirally wound configuration to provide for a high immobilizing area-to-volume ratio (0.8-1.2 cm$ sp{-1}$). A modified airlift (riser-to-downcomer area ratio of 0.03 and vessel height-to-diameter (H/D ratio of 3) and a low H/D ($ sim$1.5) mechanically stirred vessel delivered the optimum bioreactor performance characterized by low foaming of the broth and highly efficient plant cell attachment and retention ($ geq$96%). / The growth of Catharantus roseus plant cells was investigated in these bioreactors. This process was found not to be mass transfer limited above minimal mild mixing and aeration levels ensuring sufficient supply of nutrients, especially oxygen (k$ sb{ rm L}$a $ sim$ 10-15 h$ sp{-1}$) to the immobilized biomass. / The gentle surface immobilization technique developed in this work did not hinder the biosynthesis potential of the SIPC. In fact, it appeared to induce a partial secretion of some valuable compounds into the culture medium. The mildness, easiness, efficiency, mass transfer characteristics, scale-up potential and biomass loading capacity (11-13 g d.w./L) of the surface immobilization technique make it superior to all other immobilization techniques used to culture plant cells. In addition, its bioreactor overall biomass concentration compares favourably to suspended plant cell concentrations attainable in bioreactors (15-20 g d.w./L).

Urogenital chlamydia infection, caused by the bacterium Chlamydia trachomatis (CT), is the most common sexually transmitted bacterial infection in Sweden. In 2008 it was estimated by WHO that there were 105.7 million new cases of CT worldwide, an increase by 4.2 million cases (4.1%) compared to 2005. If untreated, CT infections can progress to serious reproductive health problems, especially in women. These complications include subfertility/infertility, ectopic pregnancy and chronic pain. The CT infection is often asymptomatic and reliable diagnostic methods and contact tracing are important tools for identifying infected individuals. CT infection is classified in the Swedish Communicable Diseases Act as a serious disease; consequently, written reporting and contact tracing are compulsory. Previous or ongoing CT infection is not uncommon in infertile couples, especially in women with tubal factor infertility (TFI). We have tested 244 infertile couples for CT antibodies, and CT IgG positive couples were tested for CT DNA in urine. The prevalence of CT antibodies was higher in infertile men and women, and ongoing CT infection was common. Our results support a role of CT in infertility and underscore the importance of prevention of CT infection. Contact tracing was studied during using questionnaires. A total of 544 questionnaires was sent to tracers in a Swedish county and 534 (98%) were completed. Centralized contact tracing performed by experienced tracers is effective; on average 65% of sexual contacts found by contact tracing are CT-infected. Our data show that it is worthwhile to extend the tracing period beyond 6 months as 30% of reported sexual contacts between months 7-12 were CT-infected. Contact tracing may be performed face-to-face at the clinic or by telephone. Because of the severe consequences of CT infection there is a need for useful methods for both primary and secondary prevention of CT and other sexually transmitted infections (STIs). An important sub-population for CT/STI-prevention is the “core group”, i.e. a subpopulation with high incidence of STIs combined with risky sexual behaviours. This subpopulation contributes particularly to the spread of STIs in the population. Therefore, we have developed and evaluated a brief standardised but flexible manual-based single-session intervention based on motivational interviewing (MI) for the reduction of high risk sexual behaviour. Women (n=105) and men (n=119) at high risk of contracting CT infection were randomly eighter offered brief MI counselling or standard care. Our findings support the effectiveness of brief MI-based counselling in reducing high-risk sexual behaviour and incident CT infection in women (p<0.01) but not in men. Our results suggest that gender aspects need to be considered and that men and women should be treated differently for achieving maximal risk-reduction. Whereas it might be sufficient to include information and motivation when performing risk-reducing counselling on women, counsellors may also add other components, such as behavioural skills and booster sessions, when counselling is performed on men. / Klamydiainfektion orsakas av Chlamydia trachomatis och är den vanligaste sexuellt överförda bakterieinfektionen. WHO har uppskattat att det år 2008 var 105,7 miljoner nya fall av klamydia i världen, en ökning med 4,2 miljoner fall (4,1 %) jämfört med år 2005. Klamydiainfektion är ett folkhälsoproblem och klassificeras i den svenska smittskyddslagen som en allmänfarlig sjukdom varför det är obligatoriskt att smittspåra och göra en skriftlig anmälan till smittskyddsläkaren och Smittskyddsinstitutet. Klamydiainfektionen ger oftast inga symtom och tillförlitliga diagnostiska metoder och smittspårning är viktiga ”redskap” för att hitta smittade personer. Om klamydiainfektionen inte behandlas kan den leda till allvarliga hälsoproblem, speciellt hos kvinnor. Bland komplikationer efter klamydiainfektion ingår ofrivillig barnlöshet, utomkvedshavandeskap och kronisk buksmärta. Tecken på tidigare eller pågående klamydiainfektion är vanliga hos ofrivilligt barnlösa par, speciellt hos kvinnor med skadade äggledare som orsak till barnlösheten. Våra resultat ger stöd för betydelsen av klamydia vid ofrivillig barnlöshet och understryker vikten av förebyggande åtgärder mot klamydia samt klamydiaprovtagning av både män och kvinnor vid utredning av ofrivillig barnlöshet. Centraliserad klamydiasmittspårning utförd av erfarna smittspårare är effektiv och i genomsnitt är 65 % av spårade sexuella kontakter klamydiasmittade. Våra data visar att det lönar sig att förlänga smittspårningsperioden från 6 till 12 månader eftersom betydligt fler klamydiasmittade kontakter då hittas. Den så kallade ”Västerbottensmodellen” med en smittspårningsperiod på 12 månader rekommenderas nu av Socialstyrelsen. Kontaktspårning kan utföras antingen på mottagningen eller per telefon. På grund av risk för allvarliga konsekvenser av klamydia finns det behov av metoder för att förebygga klamydiasmitta. En viktig grupp för prevention är den så kallade ”kärngruppen", alltså de personer som har en hög förekomst av klamydia och andra sexuellt överförda infektioner i kombination med sexuellt riskbeteende. Denna grupp bidrar särskilt till spridningen av sexuellt överförda infektioner bland befolkningen. Därför har vi utvecklat och utvärderat en kort samtalsmetod som bygger på metoden motiverande samtal (MI, motivational interviewing) för att minska sexuellt risktagande. Våra fynd visar att kort MI-baserad rådgivning för att minska sexuellt riskbeteende och klamydiainfektion fungerar bra på kvinnor men inte lika bra på män. Resultaten tyder på att genusaspekter måste beaktas och att kvinnor och män ska behandlas på olika sätt för att uppnå maximal riskminskning. Det kan vara tillräckligt att fokusera på information och motivation vid rådgivning av kvinnor men för rådgivning av män kan man behöva komplettera med beteendemässiga färdigheter och/eller upprepad MI-baserad rådgivning för att nå god effekt.