Cell-free DNA release during programmed cell death in ischemia reperfusion injury

Transplantation is invariably associated with acute allograft injury caused by ischemia reperfusion injury (IRI). This injury causes cells of the allograft to undergo various forms of programmed cell death including apoptosis and necroptosis. During programmed cell death, immunogenic molecules are released from cells, one of which is cell-free DNA (cfDNA). We hypothesize that cfDNA is released by microvascular endothelial cells (MVECs) during programmed cell death of IRI and that cfDNA acts as both a biomarker for cellular injury as well as a biologically active molecule capable of amplifying inflammation and organ injury. Our results indicate that cfDNA is released by MVECs under both apoptotic and necroptotic conditions in vitro, as well as during IRI in an in vivo mouse model. We have also shown that cfDNA release is ameliorated by blocking necroptosis in vivo with the use of RIPK3-/-mice that are incapable of undergoing necroptosis. Lastly, we have shown that cfDNA is capable of activating immune cells, showing that NK cell activation markers are upregulated when purified NK cells are subjected to cfDNA in vitro. Our results indicate that cfDNA is a potential biomarker of allograft injury in a renal transplant setting. Donor-derived cfDNA from blood or urine may give rise to novel non-invasive tests to diagnose graft damage. cfDNA also appears to exacerbate inflammation by activating immune cells to produce pro-inflammatory cytokines which further escalates inflammation. It may be prudent to inhibit the release of cfDNA in a transplant scenario, a goal our lab is currently working towards

The relationship between predicted peptide–MHC class II affinity and T-cell activation in a HLA-DRβ1*0401 transgenic mouse model

The HLA-DRB1*0401 MHC class II molecule (DR4) is genetically associated with rheumatoid arthritis. It has been proposed that this MHC class II molecule participates in disease pathogenesis by presenting arthritogenic endogenous or exogenous peptides to CD4(+) T cells, leading to their activation and resulting in an inflammatory response within the synovium. In order to better understand DR4 restricted T cell activation, we analyzed the candidate arthritogenic antigens type II collagen, human aggrecan, and the hepatitis B surface antigen for T-cell epitopes using a predictive model for determining peptide–DR4 affinity. We also applied this model to determine whether cross-reactive T-cell epitopes can be predicted based on known MHC–peptide–TCR interactions. Using the HLA-DR4-IE transgenic mouse, we showed that both T-cell proliferation and Th1 cytokine production (IFN-γ) correlate with the predicted affinity of a peptide for DR4. In addition, we provide evidence that TCR recognition of a peptide–DR4 complex is highly specific in that similar antigenic peptide sequences, containing identical amino acids at TCR contact positions, do not activate the same population of T cells

A novel expression platform for the production of diabetes-associated autoantigen human glutamic acid decarboxylase (hGAD65)

<p>Abstract</p> <p>Background</p> <p>Human glutamic acid decarboxylase 65 (hGAD65) is a key autoantigen in type 1 diabetes, having much potential as an important marker for the prediction and diagnosis of type 1 diabetes, and for the development of novel antigen-specific therapies for the treatment of type 1 diabetes. However, recombinant production of hGAD65 using conventional bacterial or mammalian cell culture-based expression systems or nuclear transformed plants is limited by low yield and low efficiency. Chloroplast transformation of the unicellular eukaryotic alga <it>Chlamydomonas reinhardtii </it>may offer a potential solution.</p> <p>Results</p> <p>A DNA cassette encoding full-length <it>hGAD65</it>, under the control of the <it>C. reinhardtii </it>chloroplast <it>rbc</it>L promoter and 5'- and 3'-UTRs, was constructed and introduced into the chloroplast genome of <it>C. reinhardtii </it>by particle bombardment. Integration of <it>hGAD65 </it>DNA into the algal chloroplast genome was confirmed by PCR. Transcriptional expression of <it>hGAD65 </it>was demonstrated by RT-PCR. Immunoblotting verified the expression and accumulation of the recombinant protein. The antigenicity of algal-derived hGAD65 was demonstrated with its immunoreactivity to diabetic sera by ELISA and by its ability to induce proliferation of spleen cells from NOD mice. Recombinant hGAD65 accumulated in transgenic algae, accounts for approximately 0.25–0.3% of its total soluble protein.</p> <p>Conclusion</p> <p>Our results demonstrate the potential value of <it>C. reinhardtii </it>chloroplasts as a novel platform for rapid mass production of immunologically active hGAD65. This demonstration opens the future possibility for using algal chloroplasts as novel bioreactors for the production of many other biologically active mammalian therapeutic proteins.</p

Arthritis induced by posttranslationally modified (citrullinated) fibrinogen in DR4-IE transgenic mice

Rheumatoid arthritis (RA) is a common autoimmune disease that afflicts the synovium of diarthrodial joints. The pathogenic mechanisms inciting this disease are not fully characterized, but may involve the loss of tolerance to posttranslationally modified (citrullinated) antigens. We have demonstrated that this modification leads to a selective increase in antigenic peptide affinity for major histocompatibility complex (MHC) class II molecules that carry the RA-associated shared epitope, such as HLA-DRB1*0401 (DR4). We describe the induction of arthritis in DR4-IE transgenic (tg) mice with citrullinated fibrinogen, a protein commonly found in inflamed synovial tissue and a frequent target of autoantibodies in RA patients. The disease induced in these mice was characterized by synovial hyperplasia followed by ankylosis, but lacked a conspicuous polymorphonuclear cell infiltrate. Immunological analysis of these mice through T cell epitope scanning and antibody microarray analysis identified a unique profile of citrulline-specific reactivity that was not found in DR4-IE tg mice immunized with unmodified fibrinogen or in wild-type C57BL/6 mice immunized with citrullinated fibrinogen, two conditions where arthritis was not observed. These observations directly implicate citrullinated fibrinogen as arthritogenic in the context of RA-associated MHC class II molecules

Acute rejection is associated with antibodies to non-Gal antigens in baboons using Gal-knockout pig kidneys

We transplanted kidneys from α1,3-galactosyltransferase knockout (GalT-KO) pigs into six baboons using two different immunosuppressive regimens, but most of the baboons died from severe acute humoral xenograft rejection. Circulating induced antibodies to non-Gal antigens were markedly elevated at rejection, which mediated strong complement-dependent cytotoxicity against GalT-KO porcine target cells. These data suggest that antibodies to non-Gal antigens will present an additional barrier to transplantation of organs from GalT-KO pigs to humans. © 2005 Nature Publishing Group

Late Kidney Allograft Loss: What We Know about It, and What We Can Do about It

Despite dramatic improvements in immunosuppression, late graft loss after kidney transplantation remains a common and difficult problem. Histologic evaluation may reveal changes related to BK polyomavirus infection, hypertension, or calcineurin inhibitor toxicity, which can help to guide therapy. The designation chronic allograft nephropathy should thus be reserved for biopsies with tubular atrophy and interstitial fibrosis without an apparent cause. Although the cause clearly includes both antigen-dependent and antigen-independent events, the approach remains largely to exclude immune mechanisms. Although this review discusses the potential contribution of antibody to chronic injury, it focuses on the basic elements of kidney injury, the role of parenchymal cells in promoting injury, and the proliferative and inflammatory responses that accompanying injury. Strategies to manage these recipients include close attention to accompanying hypertension, diabetes, and hyperlipidemia, as well as consideration for altering immunosuppression; however, therapies that limit epithelial-to-mesenchymal transition or directly block fibrosis pathways may reduce chronic allograft fibrosis and may prove to be useful. Understanding the basic pathogenesis sufficiently to allow early intervention may finally benefit patients who are at high risk for tubular atrophy and interstitial fibrosis and promote their long-term graft function

Use of a Pelvic Kidney for Living Transplantation: Case Report and Review of the Literature

The world-wide shortage of cadaveric kidneys for transplantation has resulted in a tendency to extend the indications for procurement from cadaveric donors and increased our reliance on living donors. Individuals with ectopic kidneys located in the bony pelvis (pelvic kidneys) have up to now been excluded from being renal donors, because pelvic kidneys are usually small, and may have anomalous vascular supplies, as well as complex urinary drainage systems. We report the use of a pelvic kidney for living-related renal transplantation. Transplantation of the pelvic kidney is technically feasible, and therefore individuals with pelvic kidneys should be considered as extended renal donors

Emerging technologies to achieve oral delivery of GLP-1 and GLP-1 analogs for treatment of type 2 diabetes mellitus (T2DM)

Glucagon-like peptide-1 (GLP-1) is a gastrointestinal (GI) peptide hormone that stimulates insulin secretion, gene expression and &#x3b2;-cell proliferation, representing a potentially novel and promising therapeutic agent for the treatment of T2DM. DPP-IV-resistant, long-acting GLP-1 analogs have already been approved by FDA as injectable drugs for treating patients with T2DM. Oral delivery of therapeutic peptides and proteins would be preferred owing to advantages of lower cost, ease of administration and greater patient adherence. However, oral delivery of proteins can be affected by rapid enzymatic degradation in the GI tract and poor penetration across the intestinal membrane, which may require amounts that exceed practical consideration. Various production strategies have been explored to overcome challenges associated with the oral delivery of therapeutic peptides and proteins. The goal of this review is to provide an overview of the current state of progress made towards the oral delivery of GLP-1 and its analogs in the treatment of T2DM, with special emphasis on the development of plant and food-grade bacterial delivery systems. Recently, genetically engineered plants and food-grade bacteria have been increasingly explored as novel carrier systems for the oral delivery of peptide and protein drugs. These have a largely unexplored potential to serve both as an expression system and as a delivery vehicle for clinically relevant, cost effective therapeutics. As such, they hold great promise for human biopharmaceuticals and novel therapies against various diseases