Selection of reference genes for normalization of quantitative real-time PCR in organ culture of the rat and rabbit intervertebral disc

<p>Abstract</p> <p>Background</p> <p>The accuracy of quantitative real-time RT-PCR (qRT-PCR) is often influenced by experimental artifacts, resulting in erroneous expression profiles of target genes. The practice of employing normalization using a reference gene significantly improves reliability and its applicability to molecular biology. However, selection of an ideal reference gene(s) is of critical importance to discern meaningful results. The aim of this study was to evaluate the stability of seven potential reference genes (Actb, GAPDH, 18S rRNA, CycA, Hprt1, Ywhaz, and Pgk1) and identify most stable gene(s) for application in tissue culture research using the rat and rabbit intervertebral disc (IVD).</p> <p>Findings</p> <p><it>In vitro</it>, four genes (Hprt1, CycA, GAPDH, and 18S rRNA) in rat IVD tissue and five genes (CycA, Hprt1, Actb, Pgk1, and Ywhaz) in rabbit IVD tissue were determined as most stable for up to 14 days in culture. Pair-wise variation analysis indicated that combination of Hprt1 and CycA in rat and the combination of Hprt1, CycA, and Actb in rabbit may most stable reference gene candidates for IVD tissue culture.</p> <p>Conclusions</p> <p>Our results indicate that Hprt1 and CycA are the most stable reference gene candidates for rat and rabbit IVD culture studies. In rabbit IVD, Actb could be an additional gene employed in conjunction with Hprt1 and CycA. Selection of optimal reference gene candidate(s) should be a pertinent exercise before employment of PCR outcome measures for biomedical research.</p

Planar Cell Polarity Effector Proteins Inturned and Fuzzy Form a Rab23 GEF Complex

A subset of Rab GTPases have been implicated in cilium formation in cultured mammalian cells [1-6]. Rab11 and Rab8, together with their GDP-GTP exchange factors (GEFs), TRAPP-II and Rabin8, promote recruitment of the ciliary vesicle to the mother centriole and its subsequent maturation, docking, and fusion with the cell surface [2-5]. Rab23 has been linked to cilium formation and membrane trafficking at mature cilia [1, 7, 8]; however, the identity of the GEF pathway activating Rab23, a member of the Rab7 subfamily of Rabs, remains unclear. Longin-domain-containing complexes have been shown to act as GEFs for Rab7 subfamily GTPases [9-12]. Here, we show that Inturned and Fuzzy, proteins previously implicated as planar cell polarity (PCP) effectors and in developmentally regulated cilium formation [13, 14], contain multiple longin domains characteristic of the Mon1-Ccz1 family of Rab7 GEFs and form a specific Rab23 GEF complex. In flies, loss of Rab23 function gave rise to defects in planar-polarized trichome formation consistent with this biochemical relationship. In cultured human and mouse cells, Inturned and Fuzzy localized to the basal body and proximal region of cilia, and cilium formation was compromised by depletion of either Inturned or Fuzzy. Cilium formation arrested after docking of the ciliary vesicle to the mother centriole but prior to axoneme elongation and fusion of the ciliary vesicle and plasma membrane. These findings extend the family of longin domain GEFs and define a molecular activity linking Rab23-regulated membrane traffic to cilia and planar cell polarity

Design and baseline characteristics of the finerenone in reducing cardiovascular mortality and morbidity in diabetic kidney disease trial

Background: Among people with diabetes, those with kidney disease have exceptionally high rates of cardiovascular (CV) morbidity and mortality and progression of their underlying kidney disease. Finerenone is a novel, nonsteroidal, selective mineralocorticoid receptor antagonist that has shown to reduce albuminuria in type 2 diabetes (T2D) patients with chronic kidney disease (CKD) while revealing only a low risk of hyperkalemia. However, the effect of finerenone on CV and renal outcomes has not yet been investigated in long-term trials. Patients and Methods: The Finerenone in Reducing CV Mortality and Morbidity in Diabetic Kidney Disease (FIGARO-DKD) trial aims to assess the efficacy and safety of finerenone compared to placebo at reducing clinically important CV and renal outcomes in T2D patients with CKD. FIGARO-DKD is a randomized, double-blind, placebo-controlled, parallel-group, event-driven trial running in 47 countries with an expected duration of approximately 6 years. FIGARO-DKD randomized 7,437 patients with an estimated glomerular filtration rate >= 25 mL/min/1.73 m(2) and albuminuria (urinary albumin-to-creatinine ratio >= 30 to <= 5,000 mg/g). The study has at least 90% power to detect a 20% reduction in the risk of the primary outcome (overall two-sided significance level alpha = 0.05), the composite of time to first occurrence of CV death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for heart failure. Conclusions: FIGARO-DKD will determine whether an optimally treated cohort of T2D patients with CKD at high risk of CV and renal events will experience cardiorenal benefits with the addition of finerenone to their treatment regimen. Trial Registration: EudraCT number: 2015-000950-39; ClinicalTrials.gov identifier: NCT02545049

A vascular smooth muscle-specific CD4+ T cell line that induces pulmonary vasculitis in MRL-+/+ mice

We established a T cell line, MV1, specific for rat vascular smooth muscle antigen from the regional lymph nodes of immunized MRL/Mp-+/+ mice. Adoptive transfer of MV1 T cells induced vasculitis lesions in the lungs of the syngeneic recipient mice pre-treated with cyclophosphamide. Flow cytometric analysis showed that MV1 was a CD4+ T cell line. The T cells proliferated in the presence of the vascular smooth muscle antigen and mitomycin C-treated syngeneic spleen cells. The cross experiments using an ovalbumin-specific T cell line demonstrated that MV1 was specific for vascular smooth muscle antigen. The antigen-specific proliferation of MV1 was CD4-dependent, which was consistent with the flow cytometric analysis. In addition, MV1 T cells, upon activation with anti-CD3 antibody or antigen-specific activation, killed A20.2J mouse B lymphoma cells. MV1 T cells also killed a CD95 (Fas)-transfected T lymphoma line, but not its parental Fas-negative cell line. These findings indicate that MV1 T cells killed target cells via a Fas ligand (FasL)/Fas pathway. The cytotoxicity of MV1 T cells may play an important role in the development of vasculitis in this model. Although the antigenic epitopes of MV1 and the lung specificity of vasculitis remain to be clarified, MV1-induced vasculitis should serve as an experimental model of human pulmonary vasculitis

In vitro interaction between muscle-derived stem cells and nucleus pulposus cells

Background context: Current therapies for intervertebral disc degeneration (IDD) are aimed at treating the clinical symptoms arising from IDD rather than directly treating the underlying problem. Pathophysiology of IDD is characterized by a progressive decrease in proteoglycan content and cell density in the nucleus pulposus (NP). A cell-based therapy is a promising concept that uses various cell types to repopulate the disc in an attempt to slow, stop, or reverse the progressive loss of proteoglycans. Stem cells appear to be an excellent candidate for this purpose, based on their ability to differentiate into various connective tissue lineages. The muscle tissue could serve as a good source of adult stem cells because of its vast abundance through out the human body. Purpose: To examine the interaction between the nucleus pulposus cells (NPCs) and the muscle-derived stem cells (MDSCs) in vitro. Study design: NPCs and MDSCs were cocultured and proteoglycan production and cell proliferation were evaluated. Methods: Various ratios of human NPCs were cocultured for 2 weeks with murine MDSCs (transduced with retro/LacZ) in a monolayer culture. Each well contained an admixture of cells with NPC-to-MDSC ratios of 0:100, 25:75, 50:50, 75:25, 100:0. Glycosaminoglycan (GAG) content (1,9 dimethylmethylene blue [DMMB]), newly synthesized proteoglycan (35S incorporation), and DNA content were measured, and cultures were stained with 5-bromo-4-chloro-3-indolyl-β-D-galactosidase (X-Gal) for cell counting. Results: The NPC-to-MDSC ratio of 75:25 resulted in a significant increase in GAG content compared with NPCs alone. All coculture ratios showed increase in GAG content in comparison with MDSC culture alone. In addition, cocultures showed a significant increase in 35S incorporation normalized to DNA content in comparison with MDSC alone. Conclusions: The data from this study shows a synergistic effect between MDSCs and NPCs resulting in an upregulated proteoglycan synthesis and NPCs proliferation in vitro. © 2008 Elsevier Inc. All rights reserved

Anti-neutrophil cytoplasmic antibodies (ANCA) in ulcerative colitis: anti-cathepsin G and a novel antibody correlate with a refractory type

We analysed the clinical significance of ANCA in patients with ulcerative colitis. On either an indirect immunofluorescence assay or an ELISA with fixed neutrophils, 71% (25/35) of the patients were positive for ANCA. However, only half of them reacted with either cathepsin G or lactoferrin. Western blot assays revealed positive bands in 40% (10/25) of the antibody-positive patients. The sizes of the bands detected were ≈58, 47, 44, 40, and 28 kD. No significant correlation was found between the ANCA positivity and various variables, i.e. disease activity, extent of lesion, or treatment of the disease. The anti-cathepsin G and 28-kD positivity, however, significantly correlated with a refractory type of ulcerative colitis