DNA hypermethylation of SFRP2 influences the pathology of rheumatoid arthritis through the canonical Wnt signaling in model rats

Abstract

In this work, the expression of secreted frizzled related protein 2 (SFRP2) in rheumatoid arthritis (RA) model rats and the mechanisms of SFRP2 on the RA pathogenesis were investigated. Data suggested that SFRP2 was significantly down-regulated in RA model rats compared with normal control, and overexpression of SFRP2 suppressed the RA pathogenesis and the canonical Wnt signaling in fibroblast-like synovial cells (FLS) from RA model rats, whereas knockdown of SFRP2 got an opposite observation. Interestingly, 5-azadC treatment up-regulated the SFRP2 expression, inhibited the FLS proliferation, suppressed the expression of IL-6 and IL-8 and the fibronectin production, suggesting that the decreased SFRP2 in RA model rats was due to the DNA methylation. Furthermore, DNMT1 knockdown up-regulated the SFRP2 expression, DNMT1 overexpression inhibited the SFRP2, and the quantitative methylation-specific PCR (qMSP) confirmed that the DNMT1 has direct methylation roles for the SFRP2 promoter, leading to a regulation of FLS proliferation and fibronectin expression in RA model rats. In addition, up-regulated MeCP2 was involved in the SFRP2 regulation and the pathogenesis of RA model rats, and MeCP2 and DNMT1 have synergistic inhibition roles in the SFRP2 expression. Combination of DNMT1 and DNA methylation may be a promising treatment strategy for individuals with RA in which SFRP2 is down-regulated.     

Immunization with plasmids encoding M2 acetylcholine muscarinic receptor epitopes impairs cardiac function in mice and induces autophagy in the myocardium

Abstract

Autoantibodies against the M2 subtype of muscarinic acetylcholine receptors with functional activities have been found in the sera of patients with dilated cardiomyopathy (DCM), and the second extracellular loop has been established as the predominant epitope. However, it has been shown that the third intracellular loop is recognized by Chagas disease patients with severe cardiac dysfunction. In this work, BALB/c mice were immunized with plasmids encoding these two epitopes, and a control group received the empty plasmid (pcDNA3 vector). Serum from these DNA-immunized animals had elevated and persistent titres of antibodies against respective antigens. Heart echocardiography indicated diminished left ventricular wall thickness and reduced ejection fraction for both epitope-immunized groups, and ergospirometry tests showed a significant decrease in the exercise time and oxygen consumption. Transfer of serum from these immunized mice into naïve recipients induced the same alterations in cardiac structure and function. Furthermore, electron microscopy analysis of donor-immunized animals revealed several ultrastructural alterations suggestive of autophagy and mitophagy, suggesting novel roles for these autoantibodies. Overall, greater functional and structural impairment was observed in the donor and recipient epitope groups, implicating the third intracellular loop epitope in the pathological effects for the first-time. Therefore, the corresponding peptides could be useful for autoimmune DCM diagnosis and targeted therapy.     

IMMUNIZATION OF MICE AGAINST ALMONELLA TYPHIMURIUM USING DIFFERENT DNA PREPARATIONS

Groups of female BALB/c mice were given primary and booster injections of whole genomic DNA extracted from S. typhimurium, P. aeruginosa, or S. aureus. Other groups of mice were immunized in a similar manner with the 1.57kb fragment of the mouse virulence gene (mviA), pTargeT vector (plasmid DNA)/1.57kb construct, pTargeT vector, or saline. Mice in all groups were challenged intraperitoneally with 100 LD50 of S. typhimurium. The bacterial genomic DNA was extracted using the Pure Gene extraction kit. Specific primers were used to amplify the 1.57kb fragment by PCR. The pTargeT Mammalian Expression Vector System was used to prepare the plasmid/1.57kb construct. Bacterial genomic DNA extracted from P. aeruginosa and S. aureus appeared to induce non-specific resistance in mice. Specific, in addition to non-specific resistance appeared to be induced when genomic DNA from S. typhimurium was used. There was a prolongation of survival in the groups of mice that received either the 1.57kb fragment or the pTargeT vector/1.57kb construct and 16.67% and 33.34% respectively, of mice in each group survived at 40 days post challenge. None of the mice in the saline control group survived by day 7 post challenge.

It is suggested that the non-specific resistance observed in this study might have been due to the adjuvant effect of the non-methylated CpG and other immunostimulatory motifs in bacterial DNA. Specific resistance obtained when genomic DNA from S. typhimurium was used might have been due to minute antigenic contamination, or virulence factor genes other than the mviA gene, might have been expressed in the host, which induced specific immunity.     

Cytotoxicity by Tumor Necrosis Factor is Linked with the Cell Cycle But Does Not Require DNA Synthesis

Abstract

The relationship between the kinetics of cell death induced by TNF and the cell cycle in L929.10 target cells was investigated by comparison of growing, asynchronous cells with target cells synchronized at G₁/S using a double thymidine block. The induction phase of lysis, the time following TNF addition but before loss of cell viability, was shortened in asynchronous cells by increasing the level of saturation of the TNF receptor. However, in synchronized target cells, the length of the induction phase showed no dependence on receptor occupancy. Almost all cell death occurred within a 3 hr period 4-7 hr after the addition of TNF regardless of the concentration of TNF. Target cell lysis in Synchronized cells was concomitant with mitosis as verified by flow cytometry and DNA staining with propidium iodide. The narrow window of cytotoxicity was not due to cell cycle-related changes in the expression of the TNF receptor as measured by [¹²⁵I]TNF binding. Treatment with TNF did not accelerate or retard the progression of cells through S and G₂/M nor did target cells accumulate at G₂/M. When the kinetic experiments were repeated in the presence of 2 MM thymidine, TNF-treated cells died with identical dose and kinetic responses as those in which the thymidine block had been removed. Under these conditions, flow cytometric analysis revealed that DNA synthesis remained inhibited. These results suggest that TNF-induced cytotoxicity is linked to cell cycle-associated processes and that TNF is capable of overriding the normal cellular controls that coordinately link the DNA replicative cycle with the mitotic cycle. In the L929.10 target cell, TNF may induce a fatal mitosis-linked event.     

RNF43 is a tumour suppressor gene mutated in mucinous tumours of the ovary

Mucinous carcinomas represent a distinct morphological subtype which can arise from several organ sites, including the ovary, and their genetic characteristics are largely under‐described. Exome sequencing of 12 primary mucinous ovarian tumours identified RNF43 as the most frequently somatically mutated novel gene, secondary to KRAS and mutated at a frequency equal to that of TP53 and BRAF. Further screening of RNF43 in a larger cohort of ovarian tumours identified additional mutations, with a total frequency of 2/22 (9%) in mucinous ovarian borderline tumours and 6/29 (21%) in mucinous ovarian carcinomas. Seven mutations were predicted to truncate the protein and one missense mutation was predicted to be deleterious by in silico analysis. Six tumours had allelic imbalance at the RNF43 locus, with loss of the wild‐type allele. The mutation spectrum strongly suggests that RNF43 is an important tumour suppressor gene in mucinous ovarian tumours, similar to its reported role in mucinous pancreatic precancerous cysts. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

VAGINAL IMMUNITY IN THE HSV-2 MOUSE MODEL

Herpes simplex virus type 2 (HSV-2) is a sexually transmitted pathogen that infects the genital tract. Efforts to develop vaccines to protect women against this and other sexually transmitted pathogens would be facilitated by a better understanding of the immune mechanisms that protect the female reproductive tract against such infections. Such information would be invaluable in developing vaccine strategies to promote the type and magnitude of immune responses in the genital tract that would effectively protect against infection. This review focuses on recent studies using a progestin-treated adult mouse model to explore mucosal immunity to HSV-2 in the vagina. Evidence indicating a major role for both humoral and T cell immunity is presented.     

A Snapshot of Immunotherapy for Rheumatoid Arthritis

Apoptosis represents the cell's innate ability to self-destruct and is an important cellular response mechanism against virus infection. The purpose of this series of articles is to critically review significant advances in important areas of virology with respect to virus infection and its relationship with apoptosis. In the Introduction to the first issue (Intern. Rev. Imm., 22: 321–326, 2003), we dedicated these entire two issues to the memory of Dr. Lois K. Miller. Dr. Miller's Ph.D. advisor, Robert D. Wells, and two of her students, Rollie Clem and Lorena Passarelli, contributed touching remembrances of their associations with Dr. Miller. In the Introduction to this issue, an overview of key points of discussion detailed in each chapter of the two issues is provided. The future of the emerging field of viral apoptosis remains bright, indeed.     

From Plasmids to Protection: A Review of DNA Vaccines Against Infectious Diseases

The field of DNA vaccine development began over 16 years ago with the observation that plasmid DNA could be injected into and expressed in vivo and drive adaptive immune responses. Since then, there has been great interest in developing this technology to create a new generation of vaccines with the ability to elicit both humoral and cellular immune responses from an inherently innocuous injection. However, DNA vaccines have yet to proceed past phase I/II clinical trials in humans – primarily due to a desire to induce more potent immune responses. This review will examine how DNA vaccines function to induce an immune response and how this information might be useful in future vaccine design.     

The FHA and BRCT domains recognize ADP-ribosylation during DNA damage response

Poly-ADP-ribosylation is a unique post-translational modification participating in many biological processes, such as DNA damage response. Here, we demonstrate that a set of Forkhead-associated (FHA) and BRCA1 C-terminal (BRCT) domains recognizes poly(ADP-ribose) (PAR) both in vitro and in vivo. Among these FHA and BRCT domains, the FHA domains of APTX and PNKP interact with iso-ADP-ribose, the linkage of PAR, whereas the BRCT domains of Ligase4, XRCC1, and NBS1 recognize ADP-ribose, the basic unit of PAR. The interactions between PAR and the FHA or BRCT domains mediate the relocation of these domain-containing proteins to DNA damage sites and facilitate the DNA damage response. Moreover, the interaction between PAR and the NBS1 BRCT domain is important for the early activation of ATM during DNA damage response and ATM-dependent cell cycle checkpoint activation. Taken together, our results demonstrate two novel PAR-binding modules that play important roles in DNA damage response.