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Baixos níveis séricos de 25(OH)D3 e 1,25(OH)2D3 (calcitriol) podem estar associados à incidência e prognóstico do câncer de mama. Além disso, vários estudos indicam que a vitamina D tenha um efeito antiproliferativo em linhagens celulares de câncer de mama expostas a concentrações supra-fisiológicas de calcitriol (1,25(OH)2D3, 100nM). A suplementação com vitamina D é indicada a mulheres pós-menopausadas para prevenção de osteoporose e observamos previamente que a suplementação de calcitirol a pacientes pós-menopausadas com câncer de mama causa redução do índice proliferativo tumoral. Entretanto, não há estudos até o momento que avaliam o efeito da vitamina D na expressão gênica global in vivo. Incluímos 31 pacientes pós-menopausadas com câncer de mama. Estas pacientes realizaram suplementação com calcitriol (0,5g/dia, dose indicada para prevenção de osteoporose) por um curto período de tempo (mediana de 32 dias). A amostra tumoral foi coletada por ocasião da biópsia (présuplementação) e da ressecção tumoral (pós-suplementação). Os perfis de expressão gênica de 16 pacientes foram analisados a partir de 100ng de RNA total no gene chip U133 Plus 2.0 Affymetrix. Observamos redução na expressão de Ki-67 após a suplementação. Dentre os genes diferencialmente expressos encontram-se EGR1, FOS, DUSP1, MMP12 e RGS1, os quais foram mais expressos em amostras pós-suplementadas. Genes modulados pela vitamina D estão associados à resposta inflamatória e à membrana. Nossos resultados indicam que a suplementação com vitamina D reduz o índice de proliferação tumoral, sendo a mesma envolvida em vias importantes na regulação da resposta inflamatória / Low 25(OH)2D3 or 1,25(OH)2D3 serum levels may be associated with breast cancer incidence and prognosis. Additionally, the antiproliferative effects of vitamin D are observed in breast cancer cell lines exposed to phamacological doses of calcitriol (1,25(OH)2D3, 100nM). Vitamin D supplementation is indicated for post-menopausal women to prevent osteoporosis and a previous study from our group observed a reduced tumor proliferative index after calcitriol supplementation on post menopausal breast cancer patients. However, there is no study that verifies the effect of vitamin D on gene expression profile in vivo so far. Thirty one post menopausal breast cancer patients were included on our analysis. They were supplemented with calcitriol after tumor biopsy (0.50g/day, indicated dose for osteoporosis prevention) for a short period of time (median 32 days). Tumor samples were collected during biopsy (before supplementation) and breast surgery (after supplementation). Gene expression profile of 16 patients was analyzed using the U133 Plus 2.0 Affymetrix Gene Chips from 100ng of total RNA. After supplementation, a reduced expression of Ki-67 was observed. Among the differentially expressed genes, EGR1, FOS, DUSP1, MMP12 and RGS1 were upregulated after calcitriol supplementation. Differentially expressed genes were involved in inflammatory response or were associated with the membrane. Our results indicate that calcitriol supplementation diminish tumor proliferation index regulating inflammatory pathways .

Die sekundäre Osteoporose ist eine der häufigsten mit der Lebertransplantation verbundenen Komplikationen. Sie ist mit spezifischen chronischen Lebererkrankungen assoziiert und manifestiert sich in ihrem ausgeprägtesten Stadium in Form von Frakturen nicht selten nach der Lebertransplantation (LTX). Management und Therapie der Osteoporose stellen noch heute ein wesentliches Problem bei Lebertransplantatempfängern dar. Das erste Ziel dieser Studie bestand in der Erfassung der Bedeutsamkeit verschiedener spezifischer Lebererkrankungen, des Einflusses von anabolen Sexualhormonen und Immunosuppression auf den Knochensubstanzverlust bei Patienten mit LTX. Das zweite Ziel dieser Studie bestand in der Abschätzung des therapeutischen Effektes von Calcitriol (1,25 (OH)2D3) in niedrigen Dosierungen zu 0,25 µg und 0,5 µg als Monotherapeutikum oder in Kombination mit 1000 mg Kalzium (Ca) bei leichter oder mäßiger Osteoporose. Patienten mit schwerer Osteoporose wurden mit einer Dreifachkombination bestehend aus 0,5 µg Calcitriol, 1000 mg Ca und 25 mg Natriumfluorid behandelt. Von 860 Patienten, die sich im Zeitraum von 1988 bis 1996 einer Lebertransplantation unterzogen, wurden insgesamt 509 Patienten (256 Männer, 213 Frauen) 5 Therapiegruppen und einer Kontrollgruppe zugeteilt. Der Mineralstatus des Knochens und der therapeutische Effekt wurden mittels vor LTX und danach halbjährlich erfolgten Knochendichtemessungen (Dual Energy X-ray Absorptiometry - DEXA) an der Lendenwirbelsäule (LWS) sowie am Schenkelhals (SH) bewertet. Patienten mit primärer biliärer Zirrhose, primär sklerosierender Cholangitis und autoimmuner Zirrhose weisen präoperativ und innerhalb der ersten 6 postoperativen Monate den niedrigsten Knochenmineralbestand auf. Den Sexualhormonstatus betrachtend wiesen 17,5% aller gemessenen Testosteronserumspiegel bei Männern und 78,3% aller gemessenen Serumöstrogenspiegel bei postmenopausalen Frauen hypogonadische Werte auf. Jedoch waren die Serumtestosteronkonzentrationen bei Männern und die Serumöstrogenkonzentrationen bei postmenopausalen Frauen zwischen den mit Calcitriol therapierten Patienten und nichttherapierten Patienten (Kontrollgruppe) nicht signifikant verschieden. Die Basisimmunsuppression bestand aus Cyclosporin A und Tacrolimus kombiniert mit Prednisolon. Bei Patienten, die Tacrolimus erhielten war der Knochenverlust an der LWS signifikant geringer (p=0,0249). Diese Beobachtung wurde höchstwahrscheinlich durch deutlich erhöhte Prednisolongaben bei Patienten mit Cyclosporin A bedingt. Mit Calcitriol therapierte Patienten erhielten wesentlich mehr Prednisolon bezogen auf die kumulative Menge und den Zeitraum im Vergleich zur nichttherapierten Kontrollgruppe. Während die niedrige Dosierung von 0,25 mg zusätzlich Kalzium benötigte um bessere Resultate am SH zu erzielen, führte die Dosierung von 0,5 µg zu einem Knochendichtezuwachs von 10,17% an der LWS und 5,9% am SH ohne Kalziumzusatz und zu einem Knochendichtezuwachs von 10,0% an der LWS und 5,2% am SH mit Kalziumgabe in einem durschnittlichen Therapiezeitraum von 1,5 Jahren. Die Dreifachkombination aus 0,5 µg Calcitriol, 1000 mg Ca und 25 mg Natriumfluorid zeigte bei Patienten mit schwerwiegender Osteoporose die besten Resultate an der LWS (Zuwachsrate 10,67%) und am SH (Zuwachsrate 12,97%) nach 1,15 Jahren. Bei nichttherapierten Patienten der Kontrollgruppe wurde ein Spontanzuwachs der Knochendichte an der LWS von 2,25% und ein Knochendichteabfall am SH von 0,86% beobachtet. Die Rate atraumatischer Frakturen konnte mit 1,77% gering gehalten werden. Calcitriol ist ein wirkungsvolles, nebenwirkungsarmes Therapeutikum zum Ausgleich und zur Prävention des Knochenmasseverlustes bei Patienten mit Lebertransplantation. Natriumfluorid steigert den Mineralisationseffekt besonders am Schenkelhals. / Secondary osteoporosis is a frequent complication of endstage liver disease which often detoriates after orthotopic liver transplantation (OLT). Management and therapy of osteopenic bone disease are still a major problem in liver transplant recipients. First purpose of this study was to estimate the magnitude of various specific liver diseases, sexual hormones and immunosuppression on bone loss in patients undergoing OLT. The second aim was to evaluate the effect of calcitriol (1,25(OH)2D3) in comparatively low dosages of 0,25 µg and 0,5 µg as a single therapy or in combination with 1000 mg calcium (Ca) in light and moderate osteoporosis. Patients with severe osteoporosis received a triple combination with 0,5 µg calcitriol, 1000 mg Ca and 25 mg sodium fluoride. Out of 860 patients undergoing OLT from 1988 to 1996, 509 (256 males, 213 females) were assigned to 5 treatment groups as well as to a control group. Bone mineral status and the effect of therapy were estimated by bone mineral density (BMD) measurements with dual energy X-ray absorptiometry of lumbar spine (LS) and femoral neck (FN) before and every six month after OLT. Primary biliary cirrhosis, primary sclerosing cholangitis and autoimmune cirrhosis were associated with a low pre-existing bone mineralisation and most severe decrease of bone mass during the early post-transplantation period. Considering hormonal measuring performed during the study period 17,5% of all measured testosterone levels in men and 78,3% of all measured oestrogen levels in postmenopausal women were in hypogonadic range. Testosterone and oestrogen levels were not significant different among with calcitriol treated and non-treated patients. Baseline immunosuppression consisted of cyclosprin A or tacrolimus initially combined with corticosteroids. Patients treated with tacrolimus had significant less bone mass reduction in the lumbar spine than patients treated with cyclosporine (p=0,0249). This observation was certainly caused by less application of prednisolone. With calcitriol treated patients received considerably more prednisolone and over a longer period of time than non-treated controls. Bone mineralisation essentially increased under calcitriol therapy in all treatment groups. Whereas the low dose of 0,25 µg needed a complementation of Ca especially to achieve better results in the FN, the dosage of 0,5 µg led to BMD improvement of 10,17% in LS and 5,9% in FN without Ca and to an improvement of 10,0 % in LS and 5,2% in FN with Ca supplementation in an average period of 1,5 years. The triple combination with 0,5 µg calcitriol, 1000 mg Ca and 25 mg sodium fluoride which was used in cases of severe osteoporosis showed the best results with a BMD augmentation rate of 10,67% in LS and 12,79% in FN after a period of 1,15 years. In the untreated controls we only found spontaneous BMD improvement of 2,25% in LS and a further bone loss of 0,86% in FN. A small fracture rate of 1,77% was observed. Calcitriol therapy effectively prevents posttransplant bone loss and augments bone mineralisation in osteoporotic patients. Moreover it minimizes the incidence of atraumatic fractures. Additional sodium fluoride increases the bone density in LS and has a special effect on FN. Side effects are negligible.

O estudo investigou a influência do tratamento de 10 semanas com paricalcitol (0,1µg/kg 5x/semana) e calcitriol (0,03µg/kg 5x/semana) em modelo de aterosclerose espontânea utilizando camundongos ApoE -/- sham e uninefrectomizados (UNX). Resultados: a densidade capilar por comprimento no tecido cardíaco foi significativamente mais baixa nos animais UNX Controle quando comparados aos shams, o que não ocorreu nos animais UNX tratados com paricalcitol e calcitriol. Nas aortas, a relação parede/lúmen foi significativamente menor no grupo Sham Controle quando comparada à dos grupos UNX Controle e UNX Calcitriol, sendo que nesses últimos, foram evidenciadas calcificações vasculares acompanhadas por células positivas para Runx-2 (cbfa-1). Além disso, foi evidenciada uma menor expressão de TGFß nas aortas dos animais do grupo UNX Paricalcitol em relação aos grupos UNX Controle e UNX Calcitriol. Conclusões: Ambos os tratamentos preveniram alterações na capilarização cardíaca induzidas pela UNX. O tratamento com calcitriol na dose empregada induziu significativas calcificações vasculares, o que não ocorreu com o paricalcitol. / The study investigated the influence of a 10-week treatment with Paricalcitol (0.1µg/kg, 5x/week) or Calcitriol (0.03µg/kg, 5x/week) on cardiovascular disease in spontaneously atherosclerotic ApoE -/- mice submitted to uninephrectomy (UNX). Results: capillary length density of the heart was significantly lower in UNX Control, but not in UNX Paricalcitol and UNX Calcitriol animals, when compared to shams. In the aortas, a significantly lower wall/lumen ratio was observed in the Sham Control group when compared to UNX Control and UNX Calcitriol groups. In the latter, vascular calcifications accompanied by a significant presence of Runx-2 (cbfa-1) positive cells was observed. TGFß aorta expression was significantly higher in UNX Control and UNX Calciriol groups when compared to UNX Paricalcitol. Conclusions: Both treatments were able to prevent the reduction in heart capillarization induced by the UNX model. Treatment with Calcitriol at the employed dose and duration, though, induced significant vascular calcifications.

Der Hedgehog (Hh)-Signalweg ist an der Entstehung verschiedener Tumorentitäten beteiligt. Es wird vermutet, dass die Signalwegskomponente Patched (Ptch) physiologischerweise als Oxysterolpumpe dient und ein Molekül - vermutlich ein Vitamin D3 Derivat - sezerniert, welches den Hh Signalweg durch Bindung an das Transmembranprotein Smoothened (Smo) hemmt. Durch inaktivierende Ptch Mutationen wird die Sekretion dieses Moleküls vermutlich gestört, was zu einer Aktivierung des Signalwegs führt. Vorarbeiten lassen vermuten, dass es sich bei dem sezernierten Vitamin D3-Derivat um 1α,25(OH)2D3 (Calcitriol) handeln könnte. Da Calcitriol seine antitumorale Wirkung neben der Hemmung der Hh-Signalkaskade auch über die Aktivierung des Vitamin-D-Rezepor (Vdr) -Signalwegs vermitteln kann, sollten in dieser Arbeit mögliche Schnittpunkte der Hh-Signalkaskade, des Vdr-Signalwegs und des Calcitriol Metabolismus untersucht werden. Zunächst ergaben die Untersuchungen, dass Ptch-defiziente Rhabdomyosarkom Zellen, verglichen mit Wildtyp-Ptch Zellen, eine verminderte Calcitriol-Synthese aufweisen, die vermutlich durch eine herabgesetzte Aktivität der 1α Hydroxylase bedingt ist. Die dadurch verminderte Calcitriol Konzentration ist wahrscheinlich die Ursache der gleichzeitig erhöhten Hh Signalwegsaktivität in den Zellen. Dies bestätigten weitere Untersuchungen mit dem 1α Hydroxylaseinhibitor Itraconazol an Ptch-/- Zellen. Weiterhin konnte gezeigt werden, dass Itraconazol mit Calcitriol hinsichtlich der Hemmung des Hh-Signalwegs kooperiert. Die verstärkte Hemmung ist aber unabhängig von einer Itraconazol-abhängigen Herabsetzung der 24 Hydroxylaseaktivität. Zudem zeigten die Untersuchungen, dass Calcitriol (neben der Aktivierung des Vdr Signalwegs) den Hh-Signalweg in Ptch / Zellen effizienter hemmt als Cyclopamin. Weiterhin konnte gezeigt werden, dass Calcitriol auch mit Cyclopamin bezüglich der Hemmung der Hh-Signalkaskade kooperiert. Weitere Untersuchungen bekräftigen die Hypothese, dass Calcitriol, ähnlich wie der bekannte Smo-Inhibitor Cyclopamin, an Smoothened (Smo) bindet. Daneben weisen die Untersuchungen darauf hin, dass 1α,25(OH)2D3 den Hh-Signalweg zusätzlich über einen nicht-kanonischen, Suppressor of fused-unabhängigen Weg hemmen könnte. Transfektionsuntersuchungen an Gli-Knock-Out-Zellen zeigten, dass Gli3 möglicherweise die Vdr-Expression und dadurch auch die Calcitriol/Vdr-vermittelte Proliferationshemmung von Calcitriol reguliert. Schließlich konnte im Tiermodell für BCC die antitumorale Wirkung von 100 ng/kg/d Calcitriol gezeigt werden. Die Studie zeigte die effektive Wirkung von Calcitriol, wenn die Substanz ab einem frühen Tumorstadium verabreicht wird. Daneben scheint die Dauer der Behandlung eine entscheidende Rolle für die antiproliferative in vivo Wirkung von Calcitriol zu spielen.

Calcitriol [1α,25(OH)2D3], the hormonally active form of vitamin D3 (D3) is produced in both renal and extrarenal tissues. Our findings demonstrate that physiological doses of UVB radiation at 300 nm induce the conversion of 7-dehydrocholesterol (7-DHC) via preD3 and D3 into calcitriol in the pmol range in epidermal keratinocytes. The hydroxylation of photosynthesized D3 to calcitriol is strongly suppressed by ketoconazole, a known inhibitor of cytochrome P450 mixed function oxidases. The UVB-induced formation of calcitriol in human skin is demonstrable in vivo by the microdialysis technique. These results suggest that human skin is an autonomous source of hormonally active calcitriol. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

O estudo investigou a influência do tratamento de 10 semanas com paricalcitol (0,1µg/kg 5x/semana) e calcitriol (0,03µg/kg 5x/semana) em modelo de aterosclerose espontânea utilizando camundongos ApoE -/- sham e uninefrectomizados (UNX). Resultados: a densidade capilar por comprimento no tecido cardíaco foi significativamente mais baixa nos animais UNX Controle quando comparados aos shams, o que não ocorreu nos animais UNX tratados com paricalcitol e calcitriol. Nas aortas, a relação parede/lúmen foi significativamente menor no grupo Sham Controle quando comparada à dos grupos UNX Controle e UNX Calcitriol, sendo que nesses últimos, foram evidenciadas calcificações vasculares acompanhadas por células positivas para Runx-2 (cbfa-1). Além disso, foi evidenciada uma menor expressão de TGFß nas aortas dos animais do grupo UNX Paricalcitol em relação aos grupos UNX Controle e UNX Calcitriol. Conclusões: Ambos os tratamentos preveniram alterações na capilarização cardíaca induzidas pela UNX. O tratamento com calcitriol na dose empregada induziu significativas calcificações vasculares, o que não ocorreu com o paricalcitol. / The study investigated the influence of a 10-week treatment with Paricalcitol (0.1µg/kg, 5x/week) or Calcitriol (0.03µg/kg, 5x/week) on cardiovascular disease in spontaneously atherosclerotic ApoE -/- mice submitted to uninephrectomy (UNX). Results: capillary length density of the heart was significantly lower in UNX Control, but not in UNX Paricalcitol and UNX Calcitriol animals, when compared to shams. In the aortas, a significantly lower wall/lumen ratio was observed in the Sham Control group when compared to UNX Control and UNX Calcitriol groups. In the latter, vascular calcifications accompanied by a significant presence of Runx-2 (cbfa-1) positive cells was observed. TGFß aorta expression was significantly higher in UNX Control and UNX Calciriol groups when compared to UNX Paricalcitol. Conclusions: Both treatments were able to prevent the reduction in heart capillarization induced by the UNX model. Treatment with Calcitriol at the employed dose and duration, though, induced significant vascular calcifications.

Baixos níveis séricos de 25(OH)D3 e 1,25(OH)2D3 (calcitriol) podem estar associados à incidência e prognóstico do câncer de mama. Além disso, vários estudos indicam que a vitamina D tenha um efeito antiproliferativo em linhagens celulares de câncer de mama expostas a concentrações supra-fisiológicas de calcitriol (1,25(OH)2D3, 100nM). A suplementação com vitamina D é indicada a mulheres pós-menopausadas para prevenção de osteoporose e observamos previamente que a suplementação de calcitirol a pacientes pós-menopausadas com câncer de mama causa redução do índice proliferativo tumoral. Entretanto, não há estudos até o momento que avaliam o efeito da vitamina D na expressão gênica global in vivo. Incluímos 31 pacientes pós-menopausadas com câncer de mama. Estas pacientes realizaram suplementação com calcitriol (0,5g/dia, dose indicada para prevenção de osteoporose) por um curto período de tempo (mediana de 32 dias). A amostra tumoral foi coletada por ocasião da biópsia (présuplementação) e da ressecção tumoral (pós-suplementação). Os perfis de expressão gênica de 16 pacientes foram analisados a partir de 100ng de RNA total no gene chip U133 Plus 2.0 Affymetrix. Observamos redução na expressão de Ki-67 após a suplementação. Dentre os genes diferencialmente expressos encontram-se EGR1, FOS, DUSP1, MMP12 e RGS1, os quais foram mais expressos em amostras pós-suplementadas. Genes modulados pela vitamina D estão associados à resposta inflamatória e à membrana. Nossos resultados indicam que a suplementação com vitamina D reduz o índice de proliferação tumoral, sendo a mesma envolvida em vias importantes na regulação da resposta inflamatória / Low 25(OH)2D3 or 1,25(OH)2D3 serum levels may be associated with breast cancer incidence and prognosis. Additionally, the antiproliferative effects of vitamin D are observed in breast cancer cell lines exposed to phamacological doses of calcitriol (1,25(OH)2D3, 100nM). Vitamin D supplementation is indicated for post-menopausal women to prevent osteoporosis and a previous study from our group observed a reduced tumor proliferative index after calcitriol supplementation on post menopausal breast cancer patients. However, there is no study that verifies the effect of vitamin D on gene expression profile in vivo so far. Thirty one post menopausal breast cancer patients were included on our analysis. They were supplemented with calcitriol after tumor biopsy (0.50g/day, indicated dose for osteoporosis prevention) for a short period of time (median 32 days). Tumor samples were collected during biopsy (before supplementation) and breast surgery (after supplementation). Gene expression profile of 16 patients was analyzed using the U133 Plus 2.0 Affymetrix Gene Chips from 100ng of total RNA. After supplementation, a reduced expression of Ki-67 was observed. Among the differentially expressed genes, EGR1, FOS, DUSP1, MMP12 and RGS1 were upregulated after calcitriol supplementation. Differentially expressed genes were involved in inflammatory response or were associated with the membrane. Our results indicate that calcitriol supplementation diminish tumor proliferation index regulating inflammatory pathways .

Aktuelle Studien legen nahe, dass ein niedriger Vitamin D-Status assoziiert mit steigenden Breitengraden, mit dem Auftreten von Autoantikörpern und damit einhergehenden Autoimmunerkrankungen positiv korreliert. Trifft UV-B-Strahlung auf die Haut, entsteht aus 7-Dehydrocholesterol nach enzymatischen Prozessen in Leber und Niere, die bioaktive Form von Vitamin D, 1α,25-Dihydroxyvitamin D3 (Calcitriol). Den finalen Stoffwechselschritt katalysiert die 1α-Hydroxylase CYP27B1. Die biologische Wirkung von Calcitriol wird über dessen Bindung an den nukleären Vitamin D-Rezeptor vermittelt, was letztlich zur Transkription der Zielgene führt. Die T-Zell-abhängige Sensibilisierung von Vitamin D-defizienten Cyp27b1-KO- und Wildtyp-Tieren mit Ovalbumin zeigte verstärkte humorale Immunantworten mit erhöhten Konzentrationen von Gesamt- sowie spezifischem IgG1, IgE und IgA bei den Cyp27b1-KO-Mäusen. Die Untersuchung der Leukopoese der Cyp27b1-KO-Tiere zeigte, dass die untersuchten lymphatischen Organe verminderte Gesamtzellzahlen gegenüber Wildtyp-Tieren aufwiesen. Die Präsenz und Verteilung in den jeweiligen Zellkompartimenten offenbarte keine wesentlichen Abweichungen zwischen Cyp27b1-KO- und Wildtyp-Mäusen. In einem Krankheitsmodell mit dem Helminthen Heligmosomoides polygyrus bakeri fielen die Cyp27b1-KO-Tiere durch dem Wildtyp gegenüber erhöhten Gesamt- sowie spezifischen IgE-Werten auf. Zwischen beiden Genotypen zeigten sich keine Unterschiede bei parasitologischen Parametern wie der Wurmlast, der Eianzahl pro Gramm Faeces sowie der Fekundität. Die Ergebnisse dieser Arbeit bestätigen, dass endogen-produziertes Vitamin D einen Einfluss auf die Funktionsweise von Lymphozyten hat. Dies äußert sich in verstärkten IgE-abhängigen Immunantworten bei Cyp27b1-KO-Tieren. In einem Parasiteninfektionsmodell wurden erneut verstärkte IgE-Antworten beobachtet, jedoch waren keine pathophysiologischen Konsequenzen in Bezug auf die Wurmabwehr nachweisbar. / Current studies demonstrate that low vitamin D levels associated with higher latitudes correlate with the occurrence of autoantibodies and linked diseases. Following UV-B radiation of the skin, numerous enzymatic reactions in liver and kidneys causes 7-dehydrocholesterol to turn into the bioactive 1α,25-dihydroxyvitamin D3 (calcitriol). The final and crucial step is thereby performed by the enzyme CYP27B1, an 1α-hydroxylase. The effect of calcitriol is mediated through binding to the vitamin D receptor, resulting in the transcription of target genes. T cell-dependent sensitization of Cyp27b1-wildtype and Cyp27b1-KO mice with ovalbumin revealed an increased humoral immune response in Cyp27b1-KO mice reflected by elevated concentrations of total and specific IgG1, IgE and IgA. Analysis of the leukopoiesis showed a diminished total cell count in bone marrow, thymus, spleen and peritoneal cavity in Cyp27b1-KO compared to Cyp27b1-wildtype mice. However, appearance and distribution of the analyzed cell compartments were comparable. A disease model using the intestinal nematode Heligmosomoides polygyrus bakeri demonstrated enhanced secretion of total and specific IgE in Cyp27b1-KO mice, which confirmed our previous findings. However, this showed no effect on parasite rejection, as seen in comparable results for worm burden, eggs per gram faeces and fecundity of female worms in Cyp27b1-wildtype and Cyp27b1-KO mice. Our work verified the role of endogenous vitamin D for lymphocyte development revealed by increased IgE-dependent immune responses in Cyp27b1-KO mice. Infection with H.p. bakeri confirmed enhanced IgE-responses, however, these results revealed no benefit in parasite clearance.

1,25(OH)2D3 em concentrações elevadas (10-100nM) exerce efeito antiproliferativo e altera o perfil de expressão gênica de linhagens de câncer de mama. Entretanto, o estudo de linhagens não considera as interações epitéliomesênquima, as quais regulam o desenvolvimento do tumor. Além disso, elevadas concentrações de 1,25(OH)2D3 induzem hipercalcemia in vivo. Nossa proposta foi avaliar as ações de uma dose relativamente baixa de 1,25(OH)2D3, concentração que pode ser alcançada in vivo (0,5nM), e uma concentração farmacológica (100nM) em cultura organotípica de câncer de mama, modelo próximo ao fisiológico que simula as condições in vivo, no perfil de expressão gênica.Para isto, avaliamos a integridade da via pela indução do gene alvo CYP24A1. Inicialmente foram avaliadas amostras de 5 pacientes. Biópsias de câncer de mama foram seccionadas, cultivadas e tratadas por 24h com 1,25(OH)2D3 0.5nM ou 100nM. A cultura organotípica manteve-se viável com preservação das características teciduais e índice de proliferação. O perfil de expressão gênica foi determinado pela análise do chip de microarray (U133 Plus 2.0, Affymetrix). Foram regulados 394 genes (Repeated Measures ANOVA, p<0.05, variação de expressão ³ 1,5) incluindo genes envolvidos em resposta imune e metabolismo celular primário. Foram selecionados 7 genes vitamina D induzidos (cuja variação de expressão foi ³ 2 e concordância no sentido da regulação). Análises complementares foram realizadas em um segundo grupo de pacientes (n=16) cujas amostras foram processadas da mesma maneira por qPCR. Estes genes eram: CD14, IL33, BMP6; DPP4, CA2, SHE e IL1RL1. Observou-se indução da expressão de CA2, DPP4 e CD14 mediante tratamento com 1,25(OH)2D3 100nM e CA2, também pela concentração 0,5nM. Além disso, avaliamos a expressão de genes candidatos num modelo in vitro de transformação mamária composto por células HME, HMELT, HMELT+Ras e também a linhagem MCF7, sob as mesmas condições de tratamento (por qPCR, western blotting, microscopia confocal e ELISA). Todos genes candidatos foram regulados positivamente pela vitamina D no modelo de progressão após o tratamento (RT-qPCR). Dentre eles, CD14, IL1RL1 e SHE também foram induzidos em células MCF7. A fração solúvel de CD14 mostrouse significativamente aumentada, assim como houve indução da proteína CA2 nas linhagens HME and HMELT tratadas com a VD. Concluindo, temos que a via de sinalização da vitamina D é funcional em secção de tecido tumoral cultivadas ex vivo, modelo que preserva as interações epitélio-estroma e simula as condições in vivo. Dentre os diversos genes modulados pela 1,25(OH)2D3, encontram-se CYP24A1, CA2, DPP4 e CD14, os quais podem representar biomarcadores da ação deste hormônio no câncer de mama. / High 1,25(OH)2D3 (VD) concentrations (10-100nM) exerts antiproliferative effects and modifies gene expression profile in breast cancer (BC) cell lines. However, studies conducted in cell lines disconsider stromal-epithelium interactions, which are known to regulate breast cancer development. Besides, high VD concentrations may cause hypercalcemia in vivo. Our aim was to evaluate the effects of a relatively low (0,5nM) concentrations of 1,25(OH)2D3 which can be attained in vivo, and pharmacological concentrations (100nM) in an organ culture model, a physiological model which mimics in vivo conditions, by means of differential gene expression profile. Vitamin D pathway integrity was evaluated by the expression of the target gene, CYP24A1. Freshly excised human BC tumor samples were sliced and cultivated in complete culture media containing vehicle, 0.5nM or 100nM 1,25(OH)2D3 for 24 hours. Organotypic culture remained viable with preserved tissue architecture and proliferation index for at least 24 hours. Affymetrix (U133 Plus 2.0) gene expression profiles obtained in five separate tissue samples for each treatment revealed 394 regulated genes (Repeated Measures ANOVA, p<0.05, fold induction ³ 1,5). Biological functions over-represented included immune response and primary cellular metabolism. Expression of seven candidate genes (CD14, IL33, BMP6; DPP4, CA2, SHE and IL1RL1; fold induction ³ 2) was further evaluated in a large number of samples (n=16) using qPCR. Among them, CA2, DPP4 and CD14 were induced by 1,25(OH)2D3 100nM. CA2 was also induced after 1,25(OH)2D3 0.5nM treatment. Expression of candidate genes was also assessed in a model of mammary epithelial cell transformation HME, HMELT, HMELT+Ras and also MCF7 cells treated with VD by qPCR, western blotting, confocal microscopy and ELISA assays. The seven genes were confirmed upregulated by VD (RT-qPCR analysis) in the cell transformation model. Among them, CD14, IL1RL1 and SHE were also modulated in MCF7 cells. A significant increase in soluble CD14 and induction of CA2 protein levels was also detected in HME and HMELT VD treated cells. In conclusion, VD signaling pathway is functional in BC slices cultured ex vivo, a model which preserves stromalepithelial interactions and mimics in vivo conditions. Several genes regulated by 1,25(OH)2D3 were identified in this model and CYP24A1, CA2, DPP4 and CD14 may represent biomarkers of vitamin D action in human breast cancer.