Preventive effects of siRNA targeting PPARγ gene on steroid-induced osteonecrosis in rabbits

Purposes/Aim: Glucocorticoid steroids can induce expression of PPARγ gene and enhance adipogenesis by bone marrow mesenchymal stem cells (BMSCs), which may result in osteonecrosis of the femoral head. Currently, there are no medications available to prevent steroid-induced osteonecrosis. We hypothesized that siRNA targeting PPARγ gene may prevent steroid-induced adipogenesis and osteonecrosis in rabbit. The purpose of this study was to evaluate the preventive effects of siRNA targeting PPARγ gene on steroid-induced adipogenesis and osteonecrosis.

Methods: Forty-eight healthy New Zealand rabbits were randomized into four groups with Group M treated with dexamethasone only, Group S with dexamethasone and a recombinant adenovirus shuttle vector carrying siRNA targeting PPARγ gene, Group Con with dexamethasone and a vector carrying irrelative sequence, and Group N with no treatment serving as control. Expressions of the PPARγ, osteocalcin and Runx2 genes, as well as histopathologic changes were evaluated.

Results: The levels of PPARγ gene expression were decreased while the levels of osteocalcin and Runx2 gene expression were increased in rabbits treated with dexamethasone and recombinant adenovirus shuttle vector carrying siRNA targeting PPARγ gene (Group S), compared to rabbits treated either with dexamethasone alone (Group M) or with both dexamethasone and a vector carrying irrelative sequence (Group Con). The marrow necrosis, adipocyte hypertrophy and proliferation, diminished hematopoiesis, thinner and sparse trabeculae, and increased empty osteocyte lacunae in the femoral head were observed in Group M and Group Con rabbits. However, no such changes were seen in Group S rabbits that were treated with dexamethasone and a recombinant adenovirus shuttle vector carrying siRNA targeting PPARγ gene.

Conclusion: siRNA targeting PPARγ gene can inhibit adipogenic differentiation of BMSCs and prevent steroid-induced osteonecrosis in rabbit. The inhibition of bone-marrow adipogenesis and concomitant enhancement of osteogenesis with RNAi may provide a novel approach to the prevention of steroid-induced osteonecrosis.     

Evaluation of five DNA extraction methods for detection of H. pylori in formalin-fixed paraffin-embedded (FFPE) liver tissue from patients with hepatocellular carcinoma

Since Helicobacter spp. DNA was identified in liver tissue resected from patients with hepatocelullar carcinoma (HCC), researchers have suggested a role of this bacterium in hepatic carcinogenesis. Archives of formalin-fixed, paraffin-embedded (FFPE) tissues represent an extraordinary source for clinical studies providing many advantages. However, DNA extraction from FFPE tissues is laborious, time-consuming and still remains a challenge. The aim of this study was to evaluate five protocols for DNA extraction from FFPE liver obtained from patients with HCC in order to detect Helicobacter pylori DNA. These methods were: (1) QIAamp FFPE Tissue Kit, (2) QIAamp DNA Mini Kit, (3) Wizard SV Genomic DNA Purification System, (4) RealiaPrep FFPE gDNA Miniprep System and (5) phenol–chloroform. H. pylori detection was performed using 16S rRNA gene amplification by PCR. The highest total amount of DNA was obtained using the phenol–chloroform method. Analyses of 16S rRNA gene amplification did not show statistically significant differences among the methods (p = 0.466), although the highest percentage of positive cases (70%) was found in samples extracted with phenol-chloroform. We suggest that of the five methods evaluated, phenol/chloroform is the most suitable for detection of H. pylori in FFPE liver from patients with HCC.     

Novel and atypical splicing mutation in a compound heterozygous UNC13D defect presenting in Familial Hemophagocytic Lymphohistiocytosis triggered by EBV infection

Familial Hemophagocytic Lymphohistiocytosis type 3 (FHL3) is a genetic disorder caused by mutations in UNC13D gene, coding the granule priming factor Munc13-4 that intervenes in NK and T cell cytotoxic function. Here we report the case of a 17-month-old girl with prolonged symptomatic EBV infectious mononucleosis and clinical symptoms of hemophagocytic syndrome. In vitro functional analysis pointed to a degranulation defect. The genetic analysis of UNC13D gene identified initially a heterozygous mutation (c.753 + 1G > T) in the donor splice-site that resulted in exon 9 skipping (maternal allele). Mutations in other genes were considered, but additional analysis of UNC13D cDNA revealed in the paternal allele a heterozygous transition from G to A (c.2448 − 13G > A) at the 3′ acceptor splice-site in intron 25, generating a new acceptor splice-site that leads to a frameshift and a premature STOP codon. Allele specific amplification of the cDNA confirmed the absence of a functional mRNA from the paternal allele. This case illustrates an atypical compound heterozygous UNC13D mutation affecting the RNA splicing that generates a typical FHL3 phenotype.     

Patients co-infected with hepatitis C virus (HCV) and human immunodeficiency virus recover genotype cross-reactive neutralising antibodies to HCV during antiretroviral therapy

When severely immunodeficient HIV/HCV co-infected patients are treated with antiretroviral therapy, it is important to know whether HCV-specific antibody responses recover and whether antibody profiles predict the occurrence of HCV-associated immune restoration disease (IRD). In 50 HIV/HCV co-infected patients, we found that antibody reactivity and titres of neutralising antibodies (nAb) to JFH-1 (HCV genotype 2a virus) increased over 48 weeks of therapy. Development of HCV IRD was associated with elevated reactivity to JFH-1 before and during the first 12 weeks of therapy. Individual analyses of HCV IRD and non-HCV IRD patients revealed a lack of an association between nAb responses and HCV viral loads. These results showed that increased HCV-specific antibody levels during therapy were associated with CD4⁺ T-cell recovery. Whilst genotype cross-reactive antibody responses may identify co-infected patients at risk of developing HCV IRD, neutralising antibodies to JFH-1 were not involved in suppression of HCV replication during therapy.     

Should IFN-γ, IL-17 and IL-2 be considered predictive biomarkers of acute rejection in liver and kidney transplant? Results of a multicentric study

Acute rejection (AR) remains a major challenge in organ transplantation, and there is a need for predictive biomarkers. In the present multicenter study, we prospectively examined a series of biomarkers in liver and kidney recipients. Intracellular expression of IFN-γ, IL-17 and IL-2 and IL-17 soluble production were evaluated both pre-transplantation and post-transplantation (1st and 2nd week, 1st, 2nd and 3rd month). 142 transplant patients (63 liver/79 kidney) were included in the study. Twenty-eight recipients (14 liver/14 kidney) developed AR. Pre- and post-transplantation intracellular expression of %IFN-γ⁺ in CD4⁺CD69⁺ and in CD8⁺CD69⁺ and soluble IL17 identified liver and kidney transplant patients at high risk of AR. Pre-transplantation, %IL-2⁺ in CD8⁺CD69⁺ also identified kidney patients at high risk. We constructed pre- and post-transplantation risk prediction models, based on a composite panel of biomarkers, which could provide the basis for future studies and will be a useful tool for the selection and adjustment of immunosuppressive treatments.     

A genetic program theory of aging using an RNA population model

Aging is a common characteristic of multicellular eukaryotes. Copious hypotheses have been proposed to explain the mechanisms of aging, but no single theory is generally acceptable. In this article, we refine the RNA population gene activating model (Lv et al., 2003) based on existing reports as well as on our own latest findings. We propose the RNA population model as a genetic theory of aging. The new model can also be applied to differentiation and tumorigenesis and could explain the biological significance of non-coding DNA, RNA, and repetitive sequence DNA. We provide evidence from the literature as well as from our own findings for the roles of repetitive sequences in gene activation. In addition, we predict several phenomena related to aging and differentiation based on this model.     

New advances in the pathophysiologic and radiologic basis of the exstrophy spectrum

The exstrophy–epispadias complex is a rare spectrum of anomalies affecting the genitourinary system, anterior abdominal wall, and pelvis. Recent advances in the repair of classic bladder exstrophy (CBE) and cloacal exstrophy (CE) have resulted in significant changes in outcomes of surgical management (including higher continence rate, fewer surgical complications, and better cosmesis) and health-related quality of life in these patients. These noteworthy changes resulted from advances in the pathophysiological and genetic backgrounds of this disease and better radiologic assessment of the three-dimensional anatomy of the bony pelvis and its musculature. A PubMed search was performed with the keyword exstrophy. The resulting literature pertaining to genetics, stem cells, imaging, tissue engineering, epidemiology, and endocrinology was reviewed. The following represents an overview of the advances in basic science understanding and imaging of the exstrophy–epispadias spectrum and discusses their possible and future effects on the management of CBE and CE.     

The Effects of Osterix on the Proliferation and Odontoblastic Differentiation of Human Dental Papilla Cells

Introduction

Dental papilla cells (DPCs) are precursors of odontoblasts and have the potential to differentiate into odontoblasts. Osteoblasts and odontoblasts have many common characteristics. Osterix (Osx) is essential for osteoblast differentiation. However, no information is available for the effects of Osx on the odontoblastic differentiation of DPCs. The purpose of this study was to investigate the effects of Osx on the proliferation and odontoblastic differentiation of DPCs.

Methods

An immortalized human dental papilla cell (hDPC) line was used. Osx was stably overexpressed or knocked down in hDPCs with infection of lentiviral particles to determine its biological effects on hDPCs. The proliferation of cells was measured by the 5-ethynyl-2′-deoxyuridine incorporation assay and direct cell counting. Expressions of dentin sialophosphoprotein, nestin, dentin matrix protein 1, and alkaline phosphatase were detected by real-time polymerase chain reaction to determine the odontoblastic differentiation of cells. The mineralization ability of cells was evaluated by von Kossa staining and alkaline phosphatase activity assay.

Results

Overexpression of Osx retarded the proliferation of hDPCs, whereas knockdown of Osx increased the cell proliferation. Overexpression of Osx promoted the odontoblastic differentiation of hDPCs by up-regulating odontoblastic differentiation genes and increased the mineralization ability of hDPCs. Knockdown of Osx down-regulated odontoblastic differentiation genes and decreased the mineralization ability of hDPCs.

Conclusions

Osx might function as a potential regulator for the proliferation and odontoblastic differentiation of hDPCs.