Using in vitro epithelial cell culture to mimic the in vivo conditions in the oviduct. Adoption of bovine oviduct epithelium cultivation on permeable support for study of sperm binding capacity in relation to bull fertility.

Mastergradsoppgave i næringsrettet bioteknologi, Avdeling for lærerutdanning og naturvitenskap, Høgskolen i Hedmark, 2013. Master of applied and commercial biotechnology.In mammalian, fertilization is the origin to life. Researchers have found that the oviduct is the site in the female reproductive tract where capacitation of spermatozoa, fertilization and early embryonic development occurs. Fertilization-competent sperm cells that manage to reach the oviduct will interact with the oviduct epithelial cells, forming a sperm reservoir, and release themself at ovulation. An in vitro cell model system is needed to adopt increased knowledge about this interaction. In this study the main aim was to establish an in vitro bovine oviduct epithelial cell (BOEC) culture system that mimics the in vivo conditions in the oviduct. Therefore, BOECs were cultivated on membrane support and the cells were characterized by immunostaining of cell specific marker proteins and real time PCR (RT-PCR) analysis of OVGP1 gene expression. The cultivated BOECs were further used to evaluate sperm binding capacity in semen from high and low fertile NRF bulls. The statement that capacitated sperm cells are unable to bind BOECs, led to the adoption of a flow cytometry Ca2+ analysis protocol, as capacitated cells have a high level of Ca2+. Additionally, the semen used in the sperm binding capacity assay was evaluated for viability, acrosomal integrity, capacitation and DNA fragmentation. Results revealed that cultivated BOECs were a pure oviduct epithelial cell line. They grew in an increased cell height, had the ability to stay viable 5 days post-confluence and were able to bind spermatozoa. However, the OVGP1 gene expression was lost during cultivation time. The sperm binding capacity results did not show any significant differences between the bulls with high and low fertility. These findings show that the cultivation of BOECs on membrane was successfully achieved and the cells mimic the in vivo to a greater extent than BOECs on plastic. However, further optimization of the sperm binding assay is needed. The adopted protocol for Ca2+ analysis revealed a significant higher Ca2+ level in bulls with high fertility than the low fertility group. From this result it can be speculated that capacitation ability of sperm cells may have an effect on the oviduct-sperm release capacity and thus fertilization competence

Using in vitro epithelial cell culture to mimic the in vivo conditions in the oviduct. Adoption of bovine oviduct epithelium cultivation on permeable support for study of sperm binding capacity in relation to bull fertility.

Mastergradsoppgave i næringsrettet bioteknologi, Avdeling for lærerutdanning og naturvitenskap, Høgskolen i Hedmark, 2013. Master of applied and commercial biotechnology.In mammalian, fertilization is the origin to life. Researchers have found that the oviduct is the site in the female reproductive tract where capacitation of spermatozoa, fertilization and early embryonic development occurs. Fertilization-competent sperm cells that manage to reach the oviduct will interact with the oviduct epithelial cells, forming a sperm reservoir, and release themself at ovulation. An in vitro cell model system is needed to adopt increased knowledge about this interaction. In this study the main aim was to establish an in vitro bovine oviduct epithelial cell (BOEC) culture system that mimics the in vivo conditions in the oviduct. Therefore, BOECs were cultivated on membrane support and the cells were characterized by immunostaining of cell specific marker proteins and real time PCR (RT-PCR) analysis of OVGP1 gene expression. The cultivated BOECs were further used to evaluate sperm binding capacity in semen from high and low fertile NRF bulls. The statement that capacitated sperm cells are unable to bind BOECs, led to the adoption of a flow cytometry Ca2+ analysis protocol, as capacitated cells have a high level of Ca2+. Additionally, the semen used in the sperm binding capacity assay was evaluated for viability, acrosomal integrity, capacitation and DNA fragmentation. Results revealed that cultivated BOECs were a pure oviduct epithelial cell line. They grew in an increased cell height, had the ability to stay viable 5 days post-confluence and were able to bind spermatozoa. However, the OVGP1 gene expression was lost during cultivation time. The sperm binding capacity results did not show any significant differences between the bulls with high and low fertility. These findings show that the cultivation of BOECs on membrane was successfully achieved and the cells mimic the in vivo to a greater extent than BOECs on plastic. However, further optimization of the sperm binding assay is needed. The adopted protocol for Ca2+ analysis revealed a significant higher Ca2+ level in bulls with high fertility than the low fertility group. From this result it can be speculated that capacitation ability of sperm cells may have an effect on the oviduct-sperm release capacity and thus fertilization competence

Effect of antirheumatic treatment on endothelial function and levels of pentraxin 3 and selenium in patients with inflammatory arthritis

For å forbedre behandlingen av aterosklerose og dens akselererte form ved inflammatorisk artritt (IA), og av IA selv, er det viktig å forbedre innsikten i patofysiologien av disse tilstandene, og finne optimale biomarkører for overvåking av IA-aktivitet og kardiovaskulær (KV) risiko. Derfor fokuserte vi i dette PhD arbeidet på tre parametere som har vært mistenkt for å være involvert i patofysiologi av KV sykdommer (KVD) og / eller inflammatoriske sykdommer: Endotelfunksjon (EF), pentraxin 3 (PTX3) og selen. Svekket EF er et av de første trinnene ved ateroskleroseutvikling, og er en verdifull biomarkør av KV risiko. PTX3, et viktig molekyl i immunsystemet har vært foreslått som en potensiell nyttig biomarkør for både betennelse og KV risiko. Den produseres blant annet direkte i det betente vevet og dens nivåer responderer raskt på endringer i sykelige tilstander i kroppen. Underskudd på selen ser ut til å øke KV risiko og betennelse. Vi undersøkte pasienter fra PSARA, en prospektiv observasjons-studie som inkluderte 140 pasienter med revmatoid artritt, psoriasis artritt eller ankyloserende spondylitt som var i ferd med å starte metotrexat og / eller anti-TNF behandling på grunn av aktiv sykdom. Vi vurderte pasientene ved oppstart og etter 6 ukers og 6 måneders behandling. PTX3 verdien hos IA pasientene var høy under hele oppfølgingen (lå ovenfor referanseområdet). Selv om andre inflammatoriske biomarkører ble redusert av antirevmatisk behandling, forble PTX3 nivået uendret. Derfor kan de høye PTX3-nivåene i IA gjenspeile en pågående subklinisk immunreaksjon som ikke responderer på anti-revmatisk behandling. Hos IA-pasienter med endoteldysfunksjon forbedret EF seg raskt med antirevmatisk behandling, uavhengig av endring i sykdomsaktivitet. Derfor kan også andre virkemåter av DMARDs, i tillegg til de anti-inflammatoriske effektene, bidra til deres aterobeskyttende effekter. Selen-nivået (72 μg/L) var innenfor referanseområdet, men under grensen på 80-85 μg/L som anses som optimal for KV helse. Dermed er det nødvendig med ytterligere forskning for å avklare om selen-mangel bidrar til økt KV risiko ved IA, eller om den tidligere påviste assosiasjon mellom lavt selen nivå og KV risiko skyldes underliggende inflammasjon. Selen-nivået økte med behandling, muligens på grunn av hemning av selens forbruksfremmende proinflammatoriske prosesser. Våre resultater kan bidra til bedre innsikt i patogenesen av IA/ akselerert KV-risiko og i virkemåten av to av de mest vanlige anti-revmatiske behandlingene, noe som kan muliggjøre utvikling av bedre strategier for behandling av disse tilstandene

Using in vitro epithelial cell culture to mimic the in vivo conditions in the oviduct. Adoption of bovine oviduct epithelium cultivation on permeable support for study of sperm binding capacity in relation to bull fertility.

In mammalian, fertilization is the origin to life. Researchers have found that the oviduct is the site in the female reproductive tract where capacitation of spermatozoa, fertilization and early embryonic development occurs. Fertilization-competent sperm cells that manage to reach the oviduct will interact with the oviduct epithelial cells, forming a sperm reservoir, and release themself at ovulation. An in vitro cell model system is needed to adopt increased knowledge about this interaction. In this study the main aim was to establish an in vitro bovine oviduct epithelial cell (BOEC) culture system that mimics the in vivo conditions in the oviduct. Therefore, BOECs were cultivated on membrane support and the cells were characterized by immunostaining of cell specific marker proteins and real time PCR (RT-PCR) analysis of OVGP1 gene expression. The cultivated BOECs were further used to evaluate sperm binding capacity in semen from high and low fertile NRF bulls. The statement that capacitated sperm cells are unable to bind BOECs, led to the adoption of a flow cytometry Ca2+ analysis protocol, as capacitated cells have a high level of Ca2+. Additionally, the semen used in the sperm binding capacity assay was evaluated for viability, acrosomal integrity, capacitation and DNA fragmentation. Results revealed that cultivated BOECs were a pure oviduct epithelial cell line. They grew in an increased cell height, had the ability to stay viable 5 days post-confluence and were able to bind spermatozoa. However, the OVGP1 gene expression was lost during cultivation time. The sperm binding capacity results did not show any significant differences between the bulls with high and low fertility. These findings show that the cultivation of BOECs on membrane was successfully achieved and the cells mimic the in vivo to a greater extent than BOECs on plastic. However, further optimization of the sperm binding assay is needed. The adopted protocol for Ca2+ analysis revealed a significant higher Ca2+ level in bulls with high fertility than the low fertility group. From this result it can be speculated that capacitation ability of sperm cells may have an effect on the oviduct-sperm release capacity and thus fertilization competence

Tumor necrosis factor inhibitors are associated with reduced complement activation in spondylarthropathies: An observational study

Background The complement system is involved in pathogenesis of cardiovascular disease, and might play a role in accelerated atherogenesis in spondylarthropathies (SpA). Hence, we examined complement activation in SpA, and its relationship to antirheumatic treatment, inflammatory and cardiovascular markers. Methods From PSARA, a prospective observational study, we examined 51 SpA patients (31 psoriatic arthritis (PsA), and 20 ankylosing spondylitis (AS)), starting tumor necrosis factor (TNF) inhibitor alone (n = 25), combined with methotrexate (MTX) (n = 10), or MTX monotherapy (n = 16). Complement activation was determined by the soluble terminal complement complex (sC5b-9), inflammation by erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), and endothelial function by finger plethysmography (Endopat) at baseline, after 6 weeks and 6 months of treatment. Results SpA patients had sC5b-9 levels at (PsA) or above (AS) the upper limit of the estimated reference range. Median sC5b-9 levels decreased significantly from baseline to 6 weeks, with no significant difference between the AS and PsA group. Notably, a significant reduction in sC5b-9 was observed after administration of TNF inhibitor ± MTX, whereas no significant changes were observed in patients treated with MTX alone. Between 6 weeks and 6 months, sC5b-9 remained stable across all subgroups. Reduction in sC5b-9 was independently related to decreased ESR and CRP, and to increased high density cholesterol and total cholesterol. Reduction in sC5b-9 from baseline to 6 weeks was associated with improved EF in age and gender adjusted analyses. Conclusion TNF-inhibition, but not MTX monotherapy, led to rapid and sustained reduction of complement activation in SpA. Thus, the observed decrease in cardiovascular morbidity in patients treated with TNF-inhibitors might be partly due to its beneficial effect on complement. Trial registration Clinical Trials (NCT00902005), retrospectively registered on the 14th of May 2009

Antirheumatic therapy is not associated with changes in circulating N-terminal pro-brain natriuretic peptide levels in patients with autoimmune arthritis

Background: Patients with autoimmune arthritis (AA) are at increased risk for impaired cardiac function and heart failure. This may be partly due to the effect of inflammation in heart function. The impact of antirheumatic drugs on cardiac dysfunction in AA remains controversial. Therefore, we aimed to examine effects of antirheumatic treatment on serum N-terminal pro-brain natriuretic peptide (NT-proBNP) in AA patients and its relationship to inflammatory markers. Methods: We examined 115 patients with AA (64 rheumatoid arthritis (RA), 31 psoriatic arthritis and 20 ankylosis spondylitis) starting with methotrexate (MTX) monotherapy or tumor necrosis factor inhibitors (TNFi) with or without MTX co-medication. NT-proBNP (measured in serum by ECLIA from Roche Diagnostics), and other clinical and laboratory parameters were evaluated at baseline, after 6 weeks and 6 months of treatment. Results: NT-proBNP levels did not change significantly after 6 weeks and 6 months of antirheumatic therapy (pbaseline-6weeks = 0.939; pbaseline-6months = 0.485), although there was a modest improvement from 6 weeks to 6 months in the MTX only treatment group (median difference = -18.2 [95% CI = -32.3 to -4.06], p = 0.013). There was no difference in the effects of MTX monotherapy and TNFi regimen on NT-proBNP levels. The changes in NT-proBNP after antirheumatic treatment positively correlated with changes in C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). Baseline NT-proBNP levels were related to baseline CRP and ESR levels, and some other established markers of disease activities in crude analyses. Conclusion: Circulating levels of NT-proBNP were related to established inflammatory markers at baseline, and the changes in NT-proBNP after antirheumatic treatment were positively related to these markers. Nevertheless, antirheumatic therapy did not seem to affect NT-proBNP levels compared to baseline, even though inflammatory markers significantly improved

Methotrexate and anti-tumor necrosis factor treatment improves endothelial function in patients with inflammatory arthritis

Background: Inflammatory arthritis (IA), including rheumatoid arthritis (RA), ankylosing spondylitis (AS) and psoriatic arthritis (PsA), leads to increased cardiovascular disease occurrence probably due to atherosclerosis. One of the first stages in atherogenesis is endothelial dysfunction (ED). Therefore, we aimed to compare endothelial function (EF) in patients with IA, and to examine the effects of methotrexate (MTX) monotherapy and antitumor necrosis factor (anti-TNF) treatment with or without MTX comedication (anti-TNF±MTX) on EF. Methods: From the PSARA observational study, all patients with RA (n=64), PsA (n=29), and AS (n=20) were evaluated for EF. In patients with ED at baseline (n=40), we evaluated changes in the Reactive Hyperemic Index (RHI) after 6 weeks and 6 months of antirheumatic therapy. Results: In IA patients with ED, RHI significantly improved after 6 weeks (p<0.001) and 6 months (p<0.001) of treatment, independent of changes in disease activity parameters. After 6 months, RHI had improved more in the MTX group than in the anti-TNF±MTX group, and the difference remained statistically significant after adjustments for potential confounders. Among patients with active RA, AS, and PsA, those with AS appeared to have the worst endothelial function, although they were the youngest. Conclusion: Treatment with MTX and anti-TNF±MTX was associated with a relatively fast improvement of EF in IA patients with ED, independent of change in disease activity. Therefore, modes of action other than the antiinflammatory effect may contribute to the EF improvement. After 6 months, the EF improvement was more pronounced in the MTX group than in the anti-TNF±MTX group

Anti-rheumatic treatment is not associated with reduction of pentraxin 3 in rheumatoid arthritis, psoriatic arthritis and ankylosing spondylitis

Background: Pentraxin 3 is proposed to be a marker of inflammation and cardiovascular risk, but its role in inflammatory rheumatic diseases (IRDs) is still uncertain. Therefore, we wanted to examine if anti-rheumatic treatment reduced serum PTX3 (s-PTX3) levels in IRDs, and if s-PTX3 levels were related to other markers of inflammation and to endothelial function (EF). Methods: We examined s-PTX3, EF and established inflammatory biomarkers in 114 IRD patients from the PSARA study before and after 6 weeks and 6 months of treatment with methotrexate (MTX) or anti-tumor necrosis factor alpha (anti-TNF) therapy with or without MTX co-medication. Results: s-PTX3 levels in all IRD diagnoses were above the upper limit of the reference range. In contrast to established inflammatory markers, in particular CRP and ESR, s-PTX3 levels did not change significantly after 6 weeks and 6 months of anti-rheumatic therapy. There was no difference in change in s-PTX3 levels from baseline to 6 weeks and 6 months between MTX monotherapy and anti-TNF regimens. CRP, ESR and EF were not related to changes in s-PTX3 neither in crude nor adjusted analyses. Conclusion: IRD patients have increased s-PTX3 levels, which, in contrast to other inflammatory markers, do not seem to improve within 6 months of therapy with MTX and/or anti-TNF. Thus, s-PTX3 might reflect a persisting immune process, even a causal factor of inflammation, not inhibited by the standard anti-rheumatic treatment. Furthermore, even though s-PTX3 is thought to be a strong predictor of cardiovascular prognosis, it was not related to EF

Antirheumatic treatment is associated with reduced serum Syndecan-1 in Rheumatoid Arthritis

The endothelial glycocalyx (EG) is essential for proper function of the endothelium and for vascular integrity, but its role in premature atherogenesis in rheumatoid arthritis (RA) has not been studied yet. EG impairment can play a role in pathogenesis of vascular disease, and one of its characteristics is shedding of syndecan-1 from endothelial cells. Syndecan-1 shedding is mediated by matrix metalloproteinase-9 (MMP-9) and counteracted by tissue inhibitor of metalloproteinases (TIMP)-1. Cardiovascular disease risk in RA is reversible by disease modifying antirheumatic drugs (DMARDs), but the exact modes of action are still unclear. Therefore, we examined effects of DMARDs on syndecan-1, MMP-9 and TIMP-1 in RA patients, and searched for associations between these parameters and inflammatory activity. From the observational PSARA study, we examined 39 patients starting with methotrexate (MTX) monotherapy (in MTX naïve patients, n = 19) or tumor necrosis factor inhibitors (TNFi) in combination with MTX (in MTX non-responders, n = 20) due to active RA. Serum syndecan-1, MMP-9 and TIMP-1 were measured at baseline and after six weeks of treatment. Serum syndecan-1 (p = 0.008) and TIMP-1 (p<0.001) levels decreased after six weeks of anti-rheumatic treatment. Levels of MMP-9 also decreased, but the difference was not statistically significant. The improvement in syndecan-1 levels were independent of changes in inflammatory activity. There was no significant difference in changes in syndecan-1 levels from baseline to 6 weeks between the MTX and TNFi groups, however the change was significant within the MTX group. Six weeks of antirheumatic treatment was associated with reduction in serum levels of syndecan-1, which might reflect reduced syndecan-1 shedding from EG. Thus, it is possible that EG-preserving properties of DMARDs might contribute to their cardioprotective effects. These effects may be at least partly independent of their anti-inflammatory actions. Our findings do not support the notion that syndecan-1 shedding in RA is mediated mainly by increased MMP-9 or decreased TIMP-9 serum concentration