Lysophosphatidic acid (LPA) is the simplest of the glycerol lipids and regulates a number of cellular processes such as morphological changes, migration, proliferation, and inhibition of apoptosis. LPA exerts these effects through activation of the G-protein coupled receptors (GPCRs) LPA1-6 and the intracellular fatty acid receptor peroxisome proliferator-activated receptor-gamma (PPARγ). The overall goal of this thesis was to determine the mechanisms by which LPA enhances cell survival by inhibiting apoptosis. The project was divided into three studies: 1) to determine the mechanism of LPA-mediated inhibition of p53 in A549 lung carcinoma cells, 2) to investigate the regulation of growth plate chondrocytes by LPA, and 3) to determine the mechanisms of LPA-mediated effects in the growth plate. In the first study, evidence is provided that LPA reduces the cellular abundance of the tumor suppressor p53 in A549 lung carcinoma cells. The LPA effect depends upon increased proteasomal degradation of p53 and it results in a corresponding decrease in p53-mediated transcription. The result of LPA-mediated inhibition of p53 in A549 cells is enhanced resistance to chemotherapeutic-induced apoptosis. In the second study, the role of LPA in resting zone chondrocytes (RC cells) was investigated. RC cells are regulated by 24,25-dihydroxyvitamin D3 [24,25(OH)[subscript2]D [subscript 3]] via a phospholipase D-dependent pathway, suggesting downstream phospholipid metabolites are involved. In this study, we showed that 24R,25(OH)[subscript 2]D[subscript 3] stimulates rat costochondral RC cells to release LPA. Additionally, we demonstrated that RC cells respond to LPA with increased proliferation, maturation, and inhibition of apoptosis. In the final study, the mechanism of LPA and 24R,25(OH)[subscript 2]D[subscript 3]-mediated inhibition of chondrocyte apoptosis was further investigated. Our data show that 24R,25(OH)[subscript 2]D[subscript 3] inhibits apoptosis through Ca⁺⁺, PLD, and PLC signaling and through LPA/Gαi/PI[subscript 3]K/mdm2-mediated degradation of p53, resulting in decreased caspase-3 activity. Collectively, our data establish LPA, vitamin D, and p53 as an anti-apoptotic signaling axis.

<p>The vitamin D metabolite 1,25(OH)2D3 has long been known to inhibit growth of prostate cancer cells and this mainly through a VDR-mediated pathway controlling target gene expression, resulting in cell cycle arrest, apoptosis and differentiation. Another major way in which 1,25(OH)2D3 inhibits cell growth in prostate cancer is via membrane-initiated steroid signalling, which triggers activation of signal cascades upon steroid binding to a receptor complex, leading to induction of genes regulating cell growth, proliferation and apoptosis. The main prostate cancer inhibiting membrane-initiated route is the TGFβ signalling pathway, elicited by the protein TGFβ. Two other important proteins downstream in this cascade are Smad2 and Smad3. In this study the early effects of 1,25(OH)2D3 on activated Smad2/3 levelsin PC-3 prostate cancer cells were examined. PC-3 cells were incubated for 3, 5, 10, 30 and 60 minutes as well as 38 hours both together with 1,25(OH)2D3 of the concentrations 10-10 and 10-7 M and without. Western Blots were then performed on supernatants from the cells treated followed by treatment of the membranes with primary antibodies against phosphorylated Smad2/3 C-terminal linker regions, alkaline phosphatase conjugated secondary antibodies and finally visualization with BCIP/ NBT tablets. As the downstream cascade protein JNK is a proposed activator of Smad2/3, this procedure was also repeated with a JNK inhibitor. This is a follow-up to an earlier study which examined the influence of 1,25(OH)2D3 on TGFβ levels using the same doses and time points and which found that 1,25(OH)2D3 initially lowered the level of active TGFβ, then increased it. The results of this study indicated a 1,25(OH)2D3 mediated induction of the same pattern in the levels of active Smad2 and 3, both with and without JNK inhibitor. The results did not indicate that 1,25(OH)2D3 activates the Smad2/3 C-terminal linker region via the JNK pathway.</p>

<p>The vitamin D metabolite 1,25(OH)2D3 has long been known to inhibit growth of prostate cancer cells and this mainly through a VDR-mediated pathway controlling target gene expression, resulting in cell cycle arrest, apoptosis and differentiation. Another major way inwhich 1,25(OH)2D3 inhibits cell growth in prostate cancer is via membrane-initiated steroid signalling, which triggers activation of signal cascades upon steroid binding to a receptor complex, leading to induction of genes regulating cell growth, proliferation and apoptosis. The main prostate cancer inhibiting membrane-initiated route is the TGFβ signalling pathway, elicited by the protein TGFβ. Another important protein downstream in this cascade is Smad2. In this study the early effects of 1,25(OH)2D3 on activated Smad2 levels in PC-3 prostate cancer cells were examined. PC-3 cells were incubated for 5, 10, 30 and 60 minutes as well as 24 and 40 hours both together with 1,25(OH)2D3 of the concentrations 10-10 and 107 M and without. An ELISA assay scanning for activated Smad2 was then performed on supernatants from both treated and untreated cells. This is a follow-up to an earlier study which examined the influence of 1,25(OH)2D3 on TGFβ levels using the same doses and similar time points and which found that 1,25(OH)2D3 initially lowered the level of active TGFβ, then increased it. The results of this study showed a statistically insignificant, time delayed 1,25(OH)2D3 mediated induction of the same pattern in the levels of active Smad2.</p> / Project Work in Biomedicine, Advanced Level, 7.5 ECTS

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 65-82).

published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

Background: The interest of vitamin D has increased in the last years. That is because there is so many possible positive effects of vitamin D and also because many individuals has vitamin D deficiency. Modern man spends much time indoors which leads to lower levels of vitamin D. People who have emigrated from a sunny climate to a Nordic climate often gets a deficiency due to a more pigmented skin which requires a larger amount of UVB to get an adequate synthesis of vitamin D. Aim: The aim with this study is to compare and evaluate how similar the instrument mini VIDAS measures 25(OH)D total against the current existing method cobas e 602. A discussion about if 25(OH)D total has a place in primary health care is included in the study. Method: The comparison was based on 39 samples. The samples was analyzed on cobas e 602 and mini VIDAS. A precision test was performed. External controls from DEQAS was also included in the study. The results have been presented with simple linear regression analysis, mean value, SD and CV. Results: The comparison between cobas e 602 and mini VIDAS gave a coefficient of determination of 81,34 %. mini VIDAS was closest to the external controls target values. Conclusion: There is no obvious conclusions about if mini VIDAS fulfills the requirement to be introduced to primary health care. The coefficient of determination of 81,34 % should be at least 95 %. However is mini VIDAS closer to the external controls target values then cobas e 602. There is factors that implies that 25(OH)D total has a place in primary health care with regards to demand, use and because many individuals has vitamin D deficiency. The instrument is also user-friendly to a primary health care laboratory. / Bakgrund: Intresset för vitamin D har ökat de senare åren. Det beror dels på att det finns många eventuella positiva effekter av vitamin D och dels på att många individer har brist på vitamin D. Nutidens människa spenderar mycket tid inomhus vilket leder till lägre nivåer av vitamin D. Personer som har utvandrat från ett soligt klimat till nordiskt klimat får ofta brist på grund av en mer pigmenterad hud som behöver större mängd UVB för att få en adekvat syntes av vitamin D. Syfte: Syftet med den här studien är att jämföra och utvärdera hur lika det patientnära instrumentet mini VIDAS mäter 25(OH)D total mot befintlig metod cobas e 602. Diskussion om analysen 25(OH)D total har en plats i primärvården ingår även i studien. Metod: Jämförelsen baserades på 39 st prover. Proven analyserades på cobas e 602 och mini VIDAS. Ett precisionsförsök gjordes. Externkontroller från DEQAS inkluderades även i studien. Resultaten har presenterats genom enkel linjär regressionsanalys, medelvärde, SD och CV. Resultat: Jämförelsen mellan cobas e 602 och mini VIDAS gav en förklaringsgrad på 81,43 %. mini VIDAS var närmst externkontrollernas målvärden. Slutsats: Det går inte att dra självklara slutsatser ifall mini VIDAS uppfyller kraven att införas i primärvården. Förklaringsgraden som är på 81,43 % bör vara minst 95 %. Däremot överensstämmer mini VIDAS med externkontrollerna bättre än cobas e 602. Det finns faktorer som tyder på att analysen 25(OH)D har en plats i primärvården med avseende på efterfrågan, användningsområde och antal individer med brist. Instrumentet är dessutom användarvänligt för ett primärvårdslaboratorium.

Osteoporosis literally means 'porous bone" and is characterized by an increase in bone fragility and susceptibility to fracture, which typically involves the wrist, spine and hip (South African Medical Association (SAMA) Working Group, 2000). In South Africa osteoporosis and fractures are more common in whites than in blacks. African-American men experience hip fractures at a rate of only half of that of Caucasian men. The bone mass in Africans were found to be 6 - 12 % higher than in Caucasians at all ages. A higher peak bone density at skeletal maturity in African-Americans were found, so that despite comparable age related bone loss, African Americans reach the fracture threshold less frequently than whites. Age-related bone loss that begins later, is less severe, or occurs in different skeletal sites in African-Americans than whites (Luckey et al., 1996). American whites have a higher bone turnover than American blacks, but in contrast to this American data. South African blacks may have a higher bone turnover and lower bone density than whites (Daniels et a/., 1995). If it is compared with Caucasians a lower rate of hip fracture in South African blacks were found, despite lower bone density at all ages (Villa, 1994). The lower fracture rate in blacks than in whites is because of greater bone mass and higher bone turnover leading to more frequent renewal of damaged bone. Blacks excrete less urinary calcium, and show no skeletal sensitivity towards the parathyroid hormone. Few studies focus on older black South African men and osteoporosis. Objectives The aim of this study was to investigate the relationship of calcium intake, vitamin D status, anthropometry and physical activity and bone density in black South African men. Methods A case-control study design was used, in which variables associated with bone density were compared. The case group were men with fractures of the proximal femur, the proximal humerus or the distal radius and an equal number of age-matched healthy black men (with not more than a 5-year age difference) with no fracture (the proximal femur and humerus and distal radius) previously, was recruited as a control group. Bone density was measured with DEXA. Fat percentage was measured with a Tanita scale. Biochemical analyses were done. Questionnaires were used to gather demographic, activity and dietary information. To our knowledge, this is the first case-control study on osteoporotic fractures in South African black men. Results Both the groups' bone mineral densities were lower than recommended. The bone density of the case group for lumbar and hip regions was 0.86 and 0.88 and the control group's bone density for lumbar region was 0.95 and hip region 0.91. The control group was more physically active and had a better nutritional status than the case group. The control group's calcium intake was higher but the vitamin D status was lower than the case group. Both calcium and vitamin D status were not statistically significant (pc0.5), between the two groups. Body mass indices of the groups were the same. The serum albumin was higher in the control group than in the case group. The case group serum calcium was higher than the control group. Both serum albumin and serum calcium were statistical significant between the two groups. There were no statistically significant differences in any of the other biochemical variables between the two groups. Serum phosphate and serum vitamin D were statistical significant for bone density of the hip and lumbar regions. Conclusion To conclude it seems logical to suggest a healthy diet with optimal macro- and micro nutrient intake. Maintain ideal body weight and body fat percentage and recommend regular but moderate-weight-bearing exercise from a young age throughout adult life, as part of a strategy to prevent and treat osteoporosis. In the present study black South African men present with low bone mineral density, but other studies indicated a lower rate of hip fracture in South African blacks, despite lower bone density at all ages. It can be recommended that other factors may play a role in black South African men with osteoporosis. Factors such as serum phosphorus, 25-hydroxy-vitamin D, body mass index (BMI), physical activity index (PAI), animal protein, total fat intake and dietary calcium are important determinants of BMD in older South African blacks, as shown in the present study. Osteoporosis is a multi factorial problem and must be treated that way. / Thesis (M.Sc. (Dietetics))--North-West University, Potchefstroom Campus, 2004.

1,25-dihydroxyvitamin D3 (Vitamin D) is a potent immunomodulator capable of generating regulatory T cells (Tregs) and contributing to immune tolerance. Additionally, vitamin D has been shown to promote mucosal immunity when used as a vaccine adjuvant. We show here that pretreatment of an area of skin with the synthetic vitamin D analog calcipotriol combined with transcutaneous immunization results in the induction of CD4⁺CD25⁺ Tregs capable of inhibiting the elicitation of a contact hypersensitivity response. We also demonstrate that topical calcipotriol has significant effects on the immune response to subcutaneously injected vaccines, and compare it with another common topical immunosuppressant, the corticosteroid betamethasone-17-valerate (BMV). Functionally, calcipotriol and BMV treatment both result in the suppression of CD8⁺ T cell priming in response to subcutaneous vaccination, despite the topical co-administration of the potent Th1 inducing TLR9 agonist unmethylated CpG DNA. The effects of calcipotriol on the humoral response are subtler as we observe marginally increased production of antigen-specific IgG1 immunoglobulins along with a strong suppression of the IgG2a isotype. This is in contrast to pretreatment with BMV, which instead suppresses the production of IgG1 and IgA antibodies. In the draining lymph nodes of calcipotriol treated animals, we see no change in the percentage of Foxp3⁺ CD4⁺ T cells post-immunization, but show that tolerance is transferable with the adoptive transfer of CD4⁺CD25⁺ cells. Despite a decrease in the percentage of antigen-bearing APCs in the DLN of calcipotriol treated animals, the DCs maintain high expression of co-stimulatory markers and can induce CD4⁺ T cell proliferation ex vivo. Our data indicate that calcipotriol has distinct effects on immune responses to subcutaneous vaccines consistent with its role as an immunomodulator, although the mechanism(s) through which it is acting remain unclear. We believe that further research is warranted into its potential use as part of a treatment modality for allergy and autoimmune disorders.

Change particularities of the blood serum levels of vitamin D, calcitonin and parathyroid hormone were analysed in healthy cows of different feeding, age and productivity and in sick cows with metabolic diseases. It was investigated how biochemical indicators vary in cows with metabolic diseases (parturient paresis, osteomalacia and mastitis) and the most informative indicators for diagnostics of these diseases (for levels of calcium, phosphorus and magnesium) were determined. Obtained findings were processed statistically. The method of electrochemiluminescence analysis used for analysis of the blood serum levels of PTH in humans was applied. Compared with radioimmunic method, this method is not expensive and quite fast; in addition, this method is sensitive and reliable, because it permits to detect low levels of PTH in the blood serum (up to 0.127 pmol/l). The method of chemiluminescence immunometric analysis used to find the level of CT in humans was tested. Also the method of immunoferment analysis (ELISA) was tested to measure the blood serum level of 25-OH vitamin D in cattle using human antibodies.

No description available.