RESUMO
OBJECTIVE: Infection triggers inflammation that, in turn, enhances the expression of contractile-associated factors in myometrium and increases the risk of preterm delivery. In this study, we assessed vitamin D regulation of inflammatory markers, contractile-associated factors, steroid hormone receptors, and NFκB pathway proteins in human uterine myometrial smooth muscle (UtSM) cells that were cultured in an inflammatory environment. STUDY DESIGN: Inflammatory environment was simulated for UtSM cells by coculturing them with monocyte lineage (THP1) cells. We measured the expression of inflammatory markers, contractile-associated factors, steroid hormone receptors, and NFκB pathway proteins in UtSM cells that were cultured with THP1 cells in the presence and absence of vitamin D by real time polymerase chain reaction and Western blot analysis. RESULTS: Monocytes secreted monocyte inflammatory protein-1α and -1ß, interleukin (IL)-1ß and 6, and tumor necrosis factor-α into the conditioned medium. In the UtSM cells that had been cocultured with THP1 cells, there was a significant (P < .05) increase in the expression of inflammatory markers IL-1ß, -6, and -13 and tumor necrosis factor-α; the contractile-associated factors connexin-43, Cox-2, and prostaglandin F2α receptor; the estrogen receptor α, and progesterone receptors A and B. Vitamin D treatment of cocultures decreased (P < .05) the expression of inflammatory markers and contractile-associated factors in UtSM cells. Similarly, vitamin D decreased estrogen receptor α and progesterone receptors A-to-B ratio in UtSM cells that were cocultured with THP1 cells. In addition, vitamin D treatment significantly (P < .05) decreased monocyte-induced p-IκBα in cytosol and NFκB-p65 in the nucleus and increased IκBα in cytosol in UtSM cells. CONCLUSION: Our results suggest that vitamin D treatment decreases inflammation-induced cytokines and contractile-associated factors in the uterine myometrial smooth muscle cells through the NFκB pathway.
Assuntos
Anti-Inflamatórios/farmacologia , Contração Muscular/efeitos dos fármacos , Miométrio/citologia , NF-kappa B/metabolismo , Vitamina D/farmacologia , Análise de Variância , Western Blotting , Linhagem Celular , Técnicas de Cocultura , Conexina 43/genética , Conexina 43/metabolismo , Ciclo-Oxigenase 2/metabolismo , Citocinas/genética , Citocinas/metabolismo , Citosol/metabolismo , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Proteínas I-kappa B/metabolismo , Inibidor de NF-kappaB alfa , Fosforilação , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Ocitocina/genética , Receptores de Ocitocina/metabolismo , Receptores de Progesterona/metabolismo , Receptores de Prostaglandina/genética , Receptores de Prostaglandina/metabolismoRESUMO
Infection during pregnancy triggers inflammation, which can increase myometrial contractions and the risk of premature labor and delivery. In this study, we assessed the effects of vitamin D, an anti-inflammatory ligand on cytokines, chemokines, toll-like receptors, and contractile-associated proteins on immortalized human myometrial smooth muscle (UtSM) cells stimulated with lipopolysaccharide (LPS), a bacterial endotoxin, or interleukin (IL)-1ß and measured Toll-like receptor (TLR)-10 expression in pregnant myometrial tissues. A superarray analysis revealed downregulation of the chemokines monocyte chemoattractant protein (MCP)-1, Chemokine (C-X-C motif) ligand (CXCL)-10, CXCL-11, and chemokine (C-X3-C motif) ligand (CX3CL)-1; the proinflammatory cytokines IL-13 and tumor necrosis factor (TNF)-α; the TLR-4 and -5 and triggering receptor expressed on myeloid cells (TREM)-2 and upregulation of the anti-inflammatory cytokine IL-10, as well as Toll interacting protein (TOLLIP) and TREM-1 in vitamin D-treated UtSM cells. In the presence of LPS, vitamin D caused dose-dependent decreases in the messenger RNA expression of MCP-1, IL-1ß, IL-13, TNF-α, TLR-4, and TLR-5, the contractile-associated proteins connexin 43, the oxytocin receptor, and the prostaglandin receptor but caused increases in IL-10 and TLR-10 in UtSM cells. The TLR-10 expression was higher in human myometrial tissue obtained from women at term not in labor compared to labor. Vitamin D also attenuated IL-1ß-induced MCP-1, IL-6, connexin 43, cyclooxygenase (COX)-2, and prostaglandin receptor expression. Western analysis showed that vitamin D decreased MCP-1, TLR-4, and connexin 43 in the presence of LPS and decreased connexin 43 in the presence of IL-1ß. Our results suggest that vitamin D can potentially decrease infection-induced increases in cytokines and contractile-associated proteins in the myometrium.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Proteínas Contráteis/antagonistas & inibidores , Proteínas Contráteis/fisiologia , Miométrio/fisiologia , Receptores Toll-Like/metabolismo , Vitamina D/farmacologia , Linhagem Celular Transformada , Células Cultivadas , Feminino , Humanos , Miométrio/citologia , Miométrio/efeitos dos fármacos , Gravidez , Receptores Toll-Like/biossíntese , Receptores Toll-Like/fisiologiaRESUMO
BACKGROUND AND PURPOSE: Struvite in kidney stones is an important marker for infection. In kidney stone samples, struvite is known to be prone to chemical breakdown, but no data exist on the stability of samples stored in dry form. The objective of this study was to examine stability of struvite under increasingly poor conditions of storage. MATERIALS AND METHODS: Samples of struvite kidney stones were broken to obtain 38 pieces averaging 67 mg in weight, and these were randomized into four storage conditions: Airtight containers stored in the dark, open containers in the dark, open containers in ambient light, and open containers at elevated temperature (40°C). Pieces were left for 6 months, and then analyzed for changes using micro CT and Fourier transform infrared spectroscopy (FT-IR). RESULTS: Initial samples proved to be struvite, indicating no transformation in the large specimens that had been stored in airtight containers in the dark for more than 6 years before this study. Pieces of struvite taken from these large specimens appeared unchanged by micro CT and FT-IR after being stored in closed containers for 6 months, but 8 of 9 pieces in open containers showed the presence of newberyite in surface layers, as did 10 of 10 pieces in open containers out in ambient light. All pieces stored at 40°C showed transformation of struvite, with 60% of the pieces showing the presence of amorphous phosphates, indicating complete breakdown of struvite in the surface layers of the pieces. CONCLUSION: We conclude that struvite in dry kidney stone samples is stable when the specimens are stored in airtight containers at room temperature, even after several years.
Assuntos
Infecções/diagnóstico , Compostos de Magnésio , Fosfatos , Cálculos Urinários/microbiologia , Humanos , Espectroscopia de Infravermelho com Transformada de Fourier , Estruvita , Tomografia Computadorizada por Raios X , Cálculos Urinários/diagnóstico por imagemRESUMO
Mutations among genes that participate in the canonical Wnt signaling pathway can lead to drastically different skeletal phenotypes, ranging from severe osteoporosis to severe osteosclerosis. Many high-bone-mass (HBM) causing mutations that occur in the LRP5 gene appear to impart the HBM phenotype, in part, by increasing resistance to soluble Wnt signaling inhibitors, including sclerostin. Sost loss-of-function mutant mice (Sost knock-out) and Lrp5 gain-of-function mutant mice (Lrp5 HBM knock-in) have high bone mass. These mutants potentially would be predicted to be phenocopies of one another, because in both cases, the sclerostin-Lrp5 interaction is disrupted. We measured bone mass, size, geometry, architecture, and strength in bones from three different genetic mouse models (Sost knock-out, Lrp5 A214V knock-in, and Lrp5 G171V knock-in) of HBM. We found that all three mouse lines had significantly elevated bone mass in the appendicular skeleton and in the cranium. Sost mutants and Lrp5 A214V mutants were statistically indistinguishable from one another in most endpoints, whereas both were largely different from the Lrp5 G171V mutants. Lrp5 G171V mutants preferentially added bone endocortically, whereas Lrp5 A214V and Sost mutants preferentially added bone periosteally. Cranial thickness and cranial nerve openings were similarly altered in all three HBM models. We also assessed serum serotonin levels as a possible mechanism accounting for the observed changes in bone mass, but no differences in serum serotonin were found in any of the three HBM mouse lines. The skeletal dissimilarities of the Lrp5 G171V mutant to the other mutants suggest that other, non-sclerostin-associated mechanisms might account for the changes in bone mass resulting from this mutation.
Assuntos
Desenvolvimento Ósseo , Mutação , Tamanho do Órgão , Transdução de Sinais/genética , Proteínas Wnt/metabolismo , Animais , Fenômenos Biomecânicos , Masculino , Camundongos , Camundongos Mutantes , Fenótipo , Serotonina/sangue , Tomografia Computadorizada por Raios X/métodosRESUMO
Urinary kidney injury molecule (KIM-1) is a sensitive quantitative biomarker for early detection of kidney tubular injury. The objective of the present work was to analytically validate this urinary renal injury biomarker. Duo-set reagents from R&D were used to develop the ELISA and validate the assay's linearity, intra-run precision, inter-run precision, lower limit of quantification, recovery, dilutional verification, reference range, stability, and length of run. The reference range data suggests that the healthy population falls within the assay range (59 - 2146 pg/mL) and upper limit of quantitation for this assay is 17168 pg/mL for the patient population. This is a robust assay to detect urinary levels of KIM-1, which serves as a non-invasive sensitive, reproducible, and potentially high-throughput method to detect early kidney injury in drug development studies.