Pro‑differentiating compounds for human intervertebral disc cells are present in Violina pumpkin leaf extracts

The intervertebral disc degeneration (IDD) is closely associated with inflammation, oxidative stress and loss of the discogenic phenotype which current therapies are unable to reverse. Here, the effects of acetone extract from Violina pumpkin (Cucurbita moschata) leaves on degenerated intervertebral disc (IVD) cells was investigated. IVD cells were isolated from degenerated disc tissue of patients undergoing spinal surgery, and exposed to acetone extract and three major thin layer chromatography subfractions. We found that the cells benefit from exposure in particular to subfraction 7 consisting almost entirely of p-Coumaric acid. Subfraction 7-treated cells showed a significant increase of discogenic transcription factors (SOX9, TRPS1), extracellular matrix components (aggrecan, collagen type II), cellular homeostasis and stress response regulators (FOXO3a, Nrf2, SOD2, SIRT1). Migratory ability and the expression of OCT4, two important markers related to the presence and activity of stem cells also increased. Moreover, subfraction 7 counteractes H2O2-triggered cell damage preventing in particular the increase of the pro-inflammatory and antichondrogenic microRNA, miR-221. This strengthens the hypothesis that adequate stimuli can support resident cells to repopulate the degenerate IVD and restart the anabolic machinery. Taken together, the data we obtained contribute to the discovery of molecules potentially effective in slowing the progression of IDD, a disease for which there is currently no effective treatment. Moreover, the enhancement of a part of plant, the pumpkin leaves, considered a waste product in the Western world, demonstrating that it contains substances with potential beneficial effects on human health

Assembly of the Inner Perivitelline Layer, a Homo log of the Mammalian Zona Pellucida: An Immunohistochemical and Ultrastructural Study

The avian inner perivitelline layer (IPVL), a homologous structure to the mammalian zona pellucida, is deposited between the granulosa cells and the oocyte cell membrane during folliculogenesis. The glycoprotein meshwork of the IPVL forms a 3-dimensional matrix and possesses important functions in the fertilization process: it contributes to the binding of avian spermatozoa to the oocyte and induces acrosomal exocytosis. In contrast to the zona pellucida of mammals, the IPVL does not prevent the physiological polyspermy found in birds. Previous studies have shown that in the Japanese quail (Cotumix japonica) at least 5 glycoproteins are constituents of the IPVL (ZP1, ZP2, ZP3, ZP4, and ZPD). In this study, we investigated the spatiotennporal assembly pattern of the IPVL during folliculogenesis using immunohistochemical and ultrastructural methods. The obtained results clearly show that these glycoproteins are incorporated into the IPVL at distinct points during follicular development, supporting the hypothesis that ZP2 and ZP4 form a type of prematrix into which ZP1, ZP3, and ZPD are integrated at a later stage of development. Copyright (C) 2011 S. Karger AG, Base

High doses of medroxyprogesterone as the cause of disappearance of adherence of the zona pellucida to an oocyte

The zona pellucida (ZP) is an external glycoprotein membrane of oocytes of mammals and embryos in the early stage of their development. ZP first appears in growing ovarian follicles as an extracellular substance between the oocyte and granular cells. The zona pellucid markedly affects the development and maturation of the oocyte. The morphology of the ZP-oocyte complex allows a more precise determination of the oocyte maturity. According to numerous experimental studies, ZP is essential for preimplantation embryonic development of humans and other mammals. It prevents dispersion of blastomeres and enhances their mutual interactions. ZP is a dynamic structure responsible for the provision of nutrients to early forms of oocytes in mammals. The aim of the present study was untrastructural evaluation of the ZP-oocyte contact during inhibited ovulation. Female white rats (Wistar strain) received a suspension of medroxyprogesterone acetate (MPA) in incremental intramuscular bolus doses of 3.7 mg (therapeutic dose), 7.4 mg and 11.1 mg. The animals were decapitated 5 days after the administration of MPA. Ovarian sections were evaluated under a transmission electron microscope (TEM) Zeiss EM 900. Morphometric analysis of ZP was conducted using the cell imaging system by Olympus. In females exposed to therapeutic doses of MPA, ZP showed the structure of granular-fibrous reticulum of a medium electron density with single cytoplasmic processes originating from the surrounding structures. The oocyte cell membrane generated single, delicate processes directed toward ZP. Microvilli of the oocyte were short and thin. In the group receiving 7.4 mg of MPA, ZP had the structure of a delicate, loose granular-fibrous reticulum, and the oocyte cell membrane generated single microvilli directed toward ZP. In both those groups, the close ZP-oocyte contact was observed. Otherwise, in the group exposed to the highest MPA doses (11.1 mg), thicker and more numerous oocyte microvilli were found, which did not penetrate ZP matrix. They were dense, irregularly separated contour, forming a barrier between ZP and oocyte. The present findings are likely to suggest that MPA has inhibiting effects on the synthesis of binding proteins and causes the loss of the oocyte contact with ZP

Efficacy and Safety of High-Dose Rifampin in Pulmonary Tuberculosis. A Randomized Controlled Trial

Rationale: We examined whether increased rifampin doses could shorten standard therapy for tuberculosis without increased toxicity. Objectives: To assess the differences across three daily oral doses of rifampin in change in elimination rate of Mycobacterium tuberculosis in sputum and frequency of rifampin-related adverse events. Methods: We conducted a blinded, randomized, controlled phase 2 clinical trial of 180 adults with new smear-positive pulmonary tuberculosis, susceptible to isoniazid and rifampin. We randomized 1:1:1 to rifampin at 10, 15, and 20 mg/kg/d during the intensive phase. We report the primary efficacy and safety endpoints: change in elimination rate of M. tuberculosis log10 colony-forming units and frequency of grade 2 or higher rifampin-related adverse events. We report efficacy by treatment arm and by primary (area under the plasma concentration–time curve [AUC]/minimum inhibitory concentration [MIC]) and secondary (AUC) pharmacokinetic exposure. Measurements and Main Results: Each 5-mg/kg/d increase in rifampin dose resulted in differences of −0.011 (95% confidence interval, −0.025 to +0.002; P = 0.230) and −0.022 (95% confidence interval, −0.046 to −0.002; P = 0.022) log10 cfu/ml/d in the modified intention-to-treat and per-protocol analyses, respectively. The elimination rate in the per-protocol population increased significantly with rifampin AUC0–6 (P = 0.011) but not with AUC0–6/MIC99.9 (P = 0.053). Grade 2 or higher rifampin-related adverse events occurred with similar frequency across the three treatment arms: 26, 31, and 23 participants (43.3%, 51.7%, and 38.3%, respectively) had at least one event (P = 0.7092) up to 4 weeks after the intensive phase. Treatment failed or disease recurred in 11 participants (6.1%). Conclusions: Our findings of more rapid sputum sterilization and similar toxicity with higher rifampin doses support investigation of increased rifampin doses to shorten tuberculosis treatment

Urokinase redistribution from the secreted to the cell-bound fraction in granulosa cells of rat preovulatory follicles

Plasminogen activators (PAs) have been shown to be synthesized in ovarian follicles of several mammalian species, where they contribute to the ovulation process. The type of PA secreted by granulosa cells is species-specific. In fact, whereas in the rat, gonadotropins stimulate tissue-type PA (tPA) production, the same hormonal stimulation induces urokinase PA (uPA) secretion in mouse cells. To investigate in more detail the hormonal regulation of this system, we used the rat ovary as a model in which we analyzed the production of PAs by theca-interstitial (TI) and granulosa cells obtained from preovulatory follicles after gonadotropin stimulation. In untreated rats, uPA was the predominant enzyme in both TI and granulosa cells. After hormonal stimulation, an increase in uPA and tPA activity was observed in both cell types. Surprisingly, only tPA mRNA increased in a time-dependent manner in both cell types, while uPA mRNA increased only in TI cells and actually decreased in granulosa cells. These divergent results between uPA enzyme activity and mRNA levels in granulosa cells were explained by studying the localization of the enzyme. Analysis of granulosa cell lysates showed that after hormonal stimulation, 60-70% of the uPA behaved as a cell-associated protein, suggesting that uPA, already present in the follicle, accumulates on the granulosa cell surface through binding to specific uPA receptors. The redistribution of uPA in granulosa cells and the differing regulation of the two PAs by gonadotropins in the rat ovary suggest that the two enzymes might have different functions during the ovulation process. Moreover, the ability of antibodies anti-tPA and anti-uPA to significantly inhibit ovulation only when coinjected with hCG confirmed that the PA contribution to ovulation occurs at the initial steps