8 research outputs found
The many faces of estrogen signaling
Estrogens have long been known as important regulators of the female reproductive functions; however, our understanding of the role estrogens play in the human body has changed significantly over the past years. It is now commonly accepted that estrogens and androgens have important functions in both female and male physiology and pathology. This is in part due to the local synthesis and action of estrogens that broadens the role of estrogen signaling beyond that of the endocrine system. Furthermore, there are several different mechanisms through which the three estrogen receptors (ERs), ERα, ERβ and G protein-coupled estrogen receptor 1 (GPER1) are able to regulate target gene transcription. ERα and ERβ are mostly associated with the direct and indirect genomic signaling pathways that result in target gene expression. Membrane-bound GPER1 is on the other hand responsible for the rapid non-genomic actions of estrogens that activate various protein-kinase cascades. Estrogen signaling is also tightly connected with another important regulatory entity, i.e. epigenetic mechanisms. Posttranslational histone modifications, microRNAs (miRNAs) and DNA methylation have been shown to influence gene expression of ERs as well as being regulated by estrogen signaling. Moreover, several coregulators of estrogen signaling also exhibit chromatin-modifying activities further underlining the importance of epigenetic mechanisms in estrogen signaling. This review wishes to highlight the newer aspects of estrogen signaling that exceed its classical endocrine regulatory role, especially emphasizing its tight intertwinement with epigenetic mechanisms
Policies for biosimilar uptake in Europe : an overview
Background: Across European countries, differences exist in biosimilar policies, leading to variations in uptake of biosimilars and divergences in savings all over Europe. Objectives: The aim of this article is to provide an overview of different initiatives and policies that may influence the uptake of biosimilars in different European countries. Recommendations will be formulated on how to create sustainable uptake. Methods: An overview of policies on biosimilars was obtained via a questionnaire, supplemented with relevant articles. Topics were organized in five themes: availability, pricing, reimbursement, demand-side policies, and recommendations to enhance uptake. Results: In all countries studied, biological medicines are available. Restrictions are mainly dependent on local organization of the healthcare system. Countries are willing to include biosimilars for reimbursement, but for commercial reasons they are not always marketed. In two thirds of countries, originator and biosimilar products may be subjected to internal reference pricing systems. Few countries have implemented specific incentives targeting physicians. Several countries are implementing pharmacist substitution; however, the scope and rules governing such substitution tend to vary between these countries. Reported educational policies tend to target primarily physicians, whereas fewer initiatives were reported for patients. Recommendations as proposed by the different country experts ranged from the need for information and communication on biosimilars to competitive pricing, more support for switching and guidance on substitution. Conclusions: Most countries have put in place specific supply-side policies for promoting access to biosimilars. To supplement these measures, we propose that investments should be made to clearly communicate on biosimilars and educate stakeholders. Especially physicians need to be informed on the entry and use of biosimilars in order to create trust. When physicians are well-informed on the treatment options, further incentives should be offered to prescribe biosimilars. Gainsharing can be used as an incentive to prescribe, dispense or use biosimilars. This approach, in combination with binding quota, may support a sustainable biosimilar market
The role of Wnt signalling pathway genes and TNFSF11 gene in bone homeostasis
Na razvoj osteoporoze, ki je večfaktorska bolezen, vplivajo številni genetski in negenetski dejavniki. Na osnovi poznavanja fiziologije kosti poskušamo identificirati kandidatne gene, ki bi lahko vplivali na razvoj osteoporoze. V kostni premeni imata pomembno vlogo signalna pot Wnt, ki sodeluje v procesih osteoblastogeneze in osteoklastogeneze in pri kateri so ključni proteini LRP5, LRP6, Dickkopf-1, β-katenin in SOST, in sistem RANKL/RANK/OPG, ki ima ključno vlogo v osteoklastogenezi in aktivaciji zrelih osteoklastov. Geni, ki kodirajo ključne proteine signalne poti Wnt in sistema RANKL/RANK/OPG, bi lahko bili kandidatni geni za razvoj osteoporoze. V okviru doktorske naloge smo te gene preučevali in funkcijsko vrednotili njihov vpliv na razvoj osteoporoze pri ženskah pred in po menopavzi ter pri starejših moških.V prvi stopnji smo v skupini žensk v menopavzi ovrednotili vlogo naslednjih polimorfizmov: BsmI, FokI in Cdx2 v genu VDR, A1330V v genu LRP5, I1062V v genu LRP6, -1397_-1396insGGA v genu SOST in K3N v genu OPG, ki so jih v povezavi z razvojem osteoporoze že preučevali. Pri zdravih pomenopavznih ženskah smo dokazali značilno povezanost polimorfizma v genu LRP5 z mineralno kostno gostoto (MKG) v predelu kolka in ledvene hrbtenice (p=0,010 in 0,033). Pri pomenopavznih ženskah z osteoporozo so imeli na MKG ledvene hrbtenice pomemben vpliv polimorfizmi BsmI in Cdx2 v genu VDR (p=0,015 in p=0,047) in polimorfizem K3N v genu OPG (p=0,021). Preostali preučevani polimorfizmi niso bili povezani z MKG. Noben od preučevanih polimorfizmov ni pokazal povezave z biokemijskimi označevalci kostne premene. Mutacije E232K v genu DKK1 ter D32Y, G34V in N287S v genu CTNNB1 v naši skupini niso bile polimorfne, zato njihove povezanosti z MKG in biokemijskimi označevalci nismo
preučevali. V drugi stopnji smo pri ključnem proteinu osteoklastogeneze RANKL, ki ga kodira gen TNFSF11, iskali nove polimorfizme v promotorju gena, ki bi lahko vplivali na njegovo izražanje. S sekveniranjem smo odkrili polimorfizme -290C>T, -643C>T, -693G>C in -1594G>A ter preliminarno ovrednotili njihovo povezanost z MKG. V skupini 115 pomenopavznih žensk je polimorfizem -290C>T pokazal značilno povezanost z MKG vratu stegnenice (p=0,022). Pri pomenopavznih ženskah brez osteoporoze je bila pri polimorfizmu -693G>C po enem letu izguba MKG kolka in vratu stegnenice statistično značilna (p=0,011 in p=0,037). Drugih povezav MKG z identificiranimi polimorfizmi v promotorju gena TNFSF11 nismo našli. V naslednji stopnji smo pri identificiranih polimorfizmih v promotorju gena TNFSF11 izvedli haplotipsko in funkcijsko analizo ter jih ovrednotili še na večji skupini 404 pomenopavznih žensk. S haplotipsko analizo smo opredelili dva najpogostejša haplotipa, CCG in TTC, ki se pojavljata pri 44,3 % oz. 49,3 % preiskovank. Pomnožene fragmente promotorja gena TNFSF11, ki so vsebovali haplotip CCG ali TTC, smo klonirali v poročevalski plazmid pGL3-basic ter ga skupaj s plazmidom pRL-TK s transfekcijo vnesli v celice HEK293. Izvedli smo dvojni luciferazni poročevalski test. Analiza poročevalskega gena je pokazala značilno večjo luciferazno aktivnost pri haplotipu CCG glede na haplotip TTC (p=0,018). Oba haplotipa sta bila pri pomenopavznih osteoporoznih ženskah značilno povezana z MKG ledvene hrbtenice (p=0,005 pri TTC in p=0,007 pri CCG), medtem ko pri MKG kolka in vratu stegnenice ni bilo povezave. Pokazali smo povezanost MKG ledvene hrbtenice s -290C>T, -643C>T in -693G>C (vrednosti p: 0,001, 0,041 in 0,013). Povezavo z MKG vratu stegnenice smo pokazali le pri -693G>C (p=0,049). Povezave biokemijskih označevalcev z genotipskimi podskupinami pri promotorskih polimorfizmih gena TNFSF11 nismo našli. V skupini pomenopavznih žensk smo ovrednotili tudi kombiniran vpliv genov TNFSF11, VDR in OPG ter LRP5, LRP6 in SOST na MKG. Pokazali smo, da polimorfizem -290C>T v genu TNFSF11 in mutacija K3N v genu OPG skupaj značilno vplivata na MKG kolka
(p=0,041). Pri neosteoporoznih ženskah je bila kombinacija polimorfizmov K3N v genu OPG in Cdx2 v genu VDR povezana z MKG ledvene hrbtenice, kolka in vratu stegnenice, kombinacija -290C>T-K3N pa z MKG vratu stegnenice. V premenopavznem obdobju na MKG v predelu kolka po naših ugotovitvah vpliva le polimofizem K3N v genu OPG, pri ostalih preučevanih polimorfizmih v genih TNFSF11, VDR, LRP5, LRP6 in SOST pa povezave z MKG nismo opazili. Predvideva se, da se genetski vplivi pri osteoporozi kažejo tudi pri hitrosti zniževanja MKG po 35. letu, zato smo iskali povezanost sprememb MKG po enem in treh letih z genotipom. Rezultati kažejo, da so spremembe MKG po enem letu v okviru napake meritve, po treh letih pa smo značilni vpliv na spremembo MKG opazili le pri polimorfizmu K3N v genu OPG. Pri premenopavznih ženskah povezanosti genotipov z biokemijskimi označevalci nismo pokazali. V okviru farmakogenetike zdravljenja osteoporoze smo preučevali uspešnost zdravljenja z alendronatom in raloksifenom v povezavi z genotipi genov TNFSF11, VDR, LRP5, LRP6, SOST in OPG. Pri nobeni od učinkovin nismo pokazali značilnega vpliva genotipa na prirast MKG po enem letu zdravljenja z zdravilom, prav tako pa nismo v času enoletnega zdravljenja dokazali vpliva genotipa na spremembe biokemijskih označevalcev. Pri starejših moških smo pokazali povezanost polimorfizmov A1330V v genu LRP5 z MKG vratu stegnenice (p=0,007), -1397_-1396insGGA v genu SOST z MKG kolka in ledvene hrbtenice (p=0,022 in p=0,046) ter FokI v genu VDR z MKG vratu stegnenice in ledvene hrbtenice (p=0,040 in p=0,036). V okviru naloge smo pokazali, da MKG glede na starost in spol uravnavajo različni genetski dejavniki. Identificirani novi polimorfizmi v promotorju gena TNFSF11 imajo funkcijsko vlogo pri uravnavanju MKG, kar je korak naprej k pojasnitvi dejanskega biološkega pomena polimorfizma. Pri farmakogenetiki osteoporoze na uspešnost zdravljenja z alendronatom ali raloksifenom preučevani geni niso imeli vpliva, zato bodo v prihodnje izziv predstavljali drugi geni, vpleteni v razvoj osteoporoze ali v samo farmakokinetiko preučevanih zdravil.Osteoporosis is a multifactorial disease influenced by numerous environmental and genetic factors. On the basis of bone physiology, candidate genes for the development of osteoporosis are being identified. In bone remodelling, Wnt signalling pathway has an important role, influencing osteoblastogenesis and osteoclastogenesis. The key proteins in Wnt pathway are LRP5, LRP6, SOST, β-catenin and Dickkopf-1. The RANKL/RANK/OPG system plays a crucial role in osteoclast biology. The genes, that code for key proteins in the Wnt signalling pathway and in the RANKL/RANK/OPG system, could also be candidate genes for the development of osteoporosis. In the thesis, these genes were studied and functionally evaluated for their influence on the development of osteoporsis in pre- and postmenopausal women as well as in elderly men.
In postmenopausal women, we first evaluated the role of the polymorphisms BsmI, FokI and Cdx2 in VDR, A1330V in LRP5, I1062V in LRP6, -1397_-1396insGGA in SOST and K3N in OPG, that have already been studied in osteoporosis. In healthy postmenopausal women, a significant association was found for A1330V in LRP5 and bone mineral density in total hip (BMD-th) and lumbar spine (BMD-ls) (p=0.010 and 0.033). In osteoporotic postmenopausal women, a significant association was established for BMD-ls with BsmI and Cdx2 in the VDR gene (p=0.015 and p=0.047), as well as with K3N in the OPG gene (p=0.021). Other polymorphisms were not associated with BMD. None of the studied polymorphisms were associated with biochemical markers of bone turnover. The mutations E232K in the DKK1 gene, and D32Y, G34V and N287S in the CTNNB1 gene were not polymorphic and therefore not associated with BMD and biochemical markers of bone turnover.
Secondly, we searched for new polymorphisms in the promoter of the TNFSF11 gene, which codes for the key protein of osteoclastogenesis RANKL and could influence its expression. Using sequencing method, the polymorphisms -290C>T, -643C>T, -693G>C and -1594G>A were identified and associated with BMD. In 115 postmenopausal women, polymorphism -290C>T showed significant association with femoral neck BMD (BMDfn) (p=0.022). In 43 non-osteoporotic postmenopausal women, the polymorphism - 693G>C was associated with the statistically significant loss in BMD-th and BMD-fn after 1 year (p=0.011 and p=0.037). No other association of the identified polymorphisms with BMD have been established. In the next step, the identified polymorphisms in the TNFSF11 gene promoter were involved in the haplotype and functional analysis, and further evaluated in a larger group of 404 postmenopausal women. Using haplotype analysis, two most common haplotypes were inferred: CCG and TTC (occuring in 44.3 % and 49.3 % of women, respectively). Amplified fragments of the TNFSF11 gene promoter, containing CCG or TTC haplotype, were cloned into pGL3-basic reporter plasmid, which was co-transfected with pRL-TK plasmid into HEK293 cells. Dual Luciferase Reporter Assay was performed. Reporter gene analysis showed significantly higher luciferase activity in CCG than in TTC haplotype (p=0.018). In osteoporotic postmenopausal women, both showed association with BMD-ls (p=0.005 and 0.007 for TTC and CCG, respectively), whereas in femoral neck there was no association with BMD. Association with BMD-ls was established in -290C>T, - 643C>T and -693G>C (p values: 0.001, 0.041 and 0.013). Association with BMD-fn was shown in -693G>C (p=0.049). No association was found between genotype subgroups of TNFSF11 gene promoter polymorphisms and biochemical markers of bone turnover. In postmenopausal women, the combined influence of TNFSF11, VDR and OPG genes, as well as LRP5, LRP6 and SOST genes on BMD was evaluated. The combination of
polymorphisms -290C>T in the TNFSF11 gene and K3N in the OPG gene have a significant influence on BMD-th (p=0,041). In non-osteoporotic women, the combination K3N(OPG)-Cdx2(VDR) was associated with BMD-ls, BMD-th and BMD-fn, and the combination -290C>T(TNFSF11)-K3N(OPG) with BMD-fn. In the premenopause, only K3N polymorphism was associated with BMD in the hip area, whereas in the TNFSF11, VDR, LRP5, LRP6 and SOST gene polymorphisms no association with BMD was found. It is assumed, that genetic factors influence the rate of
BMD changes after 35 years of age. Therefore, the BMD changes were studied after 1 and 3 years. The data show, that the BMD changes after 1 year are lower than the least significant change and after 3 years, significant association with BMD was only found in K3N polymorphism. In premenopausal women, no association was found between genotype subgroups and biochemical markers of bone turnover. To study phamacogenetics of osteoporosis, the impact of polymorphisms in the TNFSF11, VDR, LRP5, LRP6, SOST and OPG genes on the efficacy of treatment was studied in women, treated with alendronate, and in those, treated with raloxifene. After one year of treatment, neither for alendronate nor for raloxifene a significant influence of genotype on the changes in BMD or biochemical markers has been shown. In elderly men, the following associations were shown: in A1330V in the LRP5 gene with BMD-fn (p=0.007), for -1397_-1396insGGA in the SOST gene with BMD-k and BMD-ls (p=0.022 and p=0.046), and for FokI in the VDR gene with BMD-fn and BMD-ls (p=0.040 and p=0.036).
We have shown that different genetic factors influence BMD in respect of age and gender. The next step in the clarification of the biological function is the functional evaluation of the identified polymorphisms in the TNFSF11 gene promoter that could play a role in the regulation of BMD. In the pharmacogenetics study of osteoporosis, no influence of the genes under study on the efficacy of alendronate or raloxifene was shown. In the future, farmacogenetics of osteoporosis will focus on other genes, involved in the development of osteoporosis or pharmacokinetics of the drug
Risk factors for symptomatic osteonecrosis in childhood ALL: A retrospective study of a Slovenian pediatric ALL population between 1970 and 2004
Policies for biosimilar uptake in Europe: an overview
Across European countries, differences exist in biosimilar policies, leading to variations in uptake of biosimilars and divergences in savings all over Europe.status: publishe
Analysis of Association of LRP5, LRP6, SOST, DKK1, and CTNNB1 Genes with Bone Mineral Density in a Slovenian Population
Policies for biosimilar uptake in Europe: An overview
markdownabstract__Background__ Across European countries, differences exist in biosimilar policies, leading to variations in uptake of biosimilars and divergences in savings all over Europe.
__Objectives__ The aim of this article is to provide an overview of different initiatives and policies that may influence the uptake of biosimilars in different European countries. Recommendations will be formulated on how to create sustainable uptake.
__Methods__ An overview of policies on biosimilars was obtained via a questionnaire, supplemented with relevant articles. Topics were organized in five themes: availability, pricing, reimbursement, demand-side policies, and recommendations to enhance uptake.
__Results__ In all countries studied, biological medicines are available. Restrictions are mainly dependent on local organization of the healthcare system. Countries are willing to include biosimilars for reimbursement, but for commercial reasons they are not always marketed. In two thirds of countries, originator and biosimilar products may be subjected to internal reference pricing systems. Few countries have implemented specific incentives targeting physicians. Several countries are implementing pharmacist substitution; however, the scope and rules governing such substitution tend to vary between these countries. Reported educational policies tend to target primarily physicians, whereas fewer initiatives were reported for patients. Recommendations as proposed by the different country experts ranged from the need for information and communication on biosimilars to competitive pricing, more support for switching and guidance on substitution.
__Conclusions__ Most countries have put in place specific supply-side policies for promoting access to biosimilars. To supplement these measures, we propose that investments should be made to clearly communicate on biosimilars and educate stakeholders. Especially physicians need to be informed on the entry and use of biosimilars in order to create trust. When physicians are well-informed on the treatment options, further incentives should be offered to prescribe biosimilars. Gainsharing can be used as an incentive to prescribe, dispense or use biosimilars. This approach, in combination with binding quota, may support a sustainable biosimilar market
Policies for biosimilars in different countries in Europe (April 2017).
<p>Policies for biosimilars in different countries in Europe (April 2017).</p