Denis Cournoyer

Epoetin-associated pure red cell aplasia: past, present, and future considerations.

BACKGROUND: Since 1988, millions of patients have received epoetin products intravenously (IV) and subcutaneously. In 1998, epoetin‐associated pure red cell aplasia (PRCA) was first reported and causation was attributed to formulations without human serum albumin (HSA), subcutaneous administration, and uncoated rubber stoppers.

Avascular Necrosis of the Femoral Head: Vascular Hypotheses

Vascular hypotheses provide compelling pathogenic mechanisms for the etiology of avascular necrosis of the femoral head (ANFH). A decrease in local blood flow of the femoral head has been postulated to be the cause of the disease. Several studies in human and animal models of ANFH have shown microvascular thrombosis. Endothelial cell damage could be followed by abnormal blood coagulation and thrombus formation with any resulting degeneration distal to the site of vascular occlusion. Other studies suggest that thrombophilia, particularly impaired fibrinolysis, plays a potential role in thrombus formation in ANFH. Reduction in shear stress due to decreased blood flow could lead to apoptosis of endothelial cells, which can ultimately contribute to plaque erosion and thrombus formation. Dysregulation of endothelial cell activating factors and stimulators of angiogenesis or repair processes could also affect the progression and outcome of ANFH. Likewise, regional endothelium dysfunction (RED), referred to as a potential defect in endothelial cells located in the feeding vessels of the femoral head itself, may also have a crucial role in the pathogenesis of ANFH. Molecular gene analysis of regional endothelial cells could also help to determine potential pathways important in the pathogenesis of ANFH.

Anti-Erythropoietin Antibody-Mediated Pure Red Cell Aplasia after Treatment with Recombinant Erythropoietin Products: Recommendations for Minimization of Risk

Since 1998, there has been a marked increase in incidence of pure red cell aplasia secondary to development of anti-erythropoietin antibodies (Ab+ PRCA) in patients who have chronic kidney disease (CKD) and receive recombinant erythropoietin. The relationship between incidence of Ab+ PRCA and specific erythropoietin products has not been examined rigorously. Manufacturers provided data regarding exposure to erythropoietin products and incidence of Ab+ PRCA between January 1998 and March 2003 in patients with CKD. Assuming a Poisson distribution, a maximum likelihood estimate for the Poisson rate parameter was calculated for each product. A test for homogeneity of Poisson rates was conducted to compare likelihood estimates between products. Global incidence of Ab+ PRCA was relatively low. Likelihood estimates were not significantly different for Epogen, Procrit, and Aranesp, independent of their formulation or route of administration. Eprex lacking human serum albumin (HSA) and administered subcutaneously was associated with the greatest risk of Ab+ PRCA. HSA-containing Eprex administered subcutaneously was associated with a lower risk than HSA-free Eprex administered subcutaneously, but this risk exceeded that of intravenous Epogen and intravenous HSA-free Eprex. NeoRecormon administered subcutaneously was associated with less risk than subcutaneous HSA-free Eprex but more risk than intravenous Epogen. HSA-free Eprex should not be administered subcutaneously to patients with CKD due to increased risk of Ab+ PRCA. Although the subcutaneous administration of HSA-containing Eprex is riskier than intravenous Epogen and intravenous HSA-free Eprex, and the use of subcutaneous NeoRecormon is riskier than intravenous Epogen, there is currently no evidence that other products are safer.

Recommendations on haematological criteria for the diagnosis of epoetin-induced pure red cell aplasia

Abstract:  Pure red cell aplasia (PRCA) is a rare condition characterised by an arrest in red blood cell production, which may be congenital or acquired. Recombinant human erythropoietin (epoetin) was introduced in 1989 for the treatment of anaemia of chronic kidney disease patients and has maintained an excellent therapeutic and safety record while treating hundreds of thousands of patients. A very rare, but serious adverse event associated with epoetin administration is a condition in which patients develop neutralising anti‐erythropoietin antibodies and, consequently, PRCA. This condition is referred to as epoetin‐induced PRCA (epo‐PRCA). Since it is a rare condition, many haematologists and nephrologists around the world see the condition infrequently and may be uncertain about the diagnosis. For this reason, an ad hoc international working group of expert haematologists and nephrologists met together to derive new recommendations for the haematological diagnosis of epo‐PRCA. These recommendations, which represent the consensus opinions of the working group, address haematological approaches to monitor and investigate suspected epo‐PRCA and should help physicians differentiate between PRCA and other bone marrow diseases, as well as, between PRCA and epo‐PRCA.

Increased susceptibility to dextran sulfate sodium induced colitis in the T cell protein tyrosine phosphatase heterozygous mouse.

T cell protein tyrosine phosphatase (TC-PTP / PTPN2) is an enzyme that is essential for the proper functioning of the immune system and that participates in the control of cell proliferation, and inflammation. We previously observed that TC-PTP−/− mice display various immunodeficiencies, hypersensitivity to LPS and die within three weeks of birth due to anemia and widespread inflammation. A recent analysis of the Wellcome Trust Case Control Consortium (WTCC) genome wide scan data, reported in 2007, indicated a potential role for TC-PTP in inflammatory bowel disease (IBD). To further investigate the potential role of TC-PTP in IBD, we studied heterozygous TC-PTP mutant mice challenged with dextran sulfate sodium (DSS) in their drinking water. In comparison to control animals, we observed significant changes in the colon mucosa of DSS-treated TC-PTP+/− mice, in the ratio of colon to body weight, as well as an up-regulation of mRNA transcripts for IL-6, IL-23, 1L-12β, IFN-γ, TNF-α. Moreover, up-regulation of serum IL-6 levels in DSS-treated TC-PTP+/− mice confirms that mice with a single copy of the TC-PTP gene display increased susceptibility to systemic inflammation due to bowel epithelial erosion resulting from DSS challenge. Our findings support the lack of modulation of Janus kinases 1 and 3 (Jak1, Jak3), and the downstream signal transducer and activator of transcription 1,3 and 5 (Stat1, Stat3, Stat 5) by PTPN2 in the development of IBD like condition. Pathological and molecular analysis reveal that the deficiency of TC-PTP results in pro-inflammatory condition in the bowel of heterozygous TC-PTP+/− mice. These novel findings in TC-PTP hemi-deficiency support the hypothesis that TC-PTP is an important regulator of inflammatory cytokine signaling and that it may be implicated in the pathophysiology of IBD.

Transfection of murine fibroblast cells with human cytidine deaminase cDNA confers resistance to cytosine arabinoside.

One of the major limitations in the use of cytosine arabinoside (Ara-C) in cancer chemotherapy is the hematopoietic toxicity produced by this nucleoside analog. One approach to overcome this problem would be to Insert a gene for drug resistance to Ara-C in normal hematopoietic cells to protect them from drug toxicity. An interesting candidate gene for this aim is cytidine deaminase which catalyzes the deamination of Arac-C, resulting in a significant loss of its antineoplastic activity. We have llgated the human cDNA for cytidine deaminase into the plasmid vector pMFG. Transfection of NIH 3T3− derlved GP + E86 murine fibroblasts cells with this vector resulted In a marked increase (> 50-fold) in the expression of cytidine deaminase. In addition, the transfected cells showed resistance to the cytotoxic action and to the Inhibition of DNA synthesis produced by Ara-C. Northern and Western blot analysis of the transfected cells showed increased expression of mRNA for cytidine deaminase and increased immunologically detectable enzyme. The ability to confer drug resistance to Ara-C through gene transfer of cytidine deaminase may have the potential as a selectable marker and for the protection of the bone marrow from the toxicity produced by this analog so as to increase its effectiveness in cancer chemotherapy.

New insights into the pathogenesis of glucocorticoid-induced avascular necrosis: microarray analysis of gene expression in a rat model

IntroductionAvascular necrosis of the femoral head (ANFH) occurs variably after exposure to corticosteroids. Microvascular thrombosis is a common pathological finding. Since systemic thrombophilia is only weakly linked with ANFH, we propose that microvascular vessel pathology may be more related to local endothelial dysfunction and femoral head apoptosis. Corticosteroid effects on the endothelium and resultant apoptosis have been reported. We hypothesize that corticosteroids contribute to a differential gene expression in the femoral head in rats with early ANFH.MethodsBesides bone marrow necrosis, which is a common sign in ANFH and reported in the early stages, we include the presence of apoptosis in this study as a criterion for diagnosing early disease. Forty Wistar Kyoto (WKY) rats were randomized to either a corticosteroid-treated group or an age-matched control group for six months. After sacrifice, the femoral heads were examined for ANFH. Total mRNA was extracted from femoral heads. Affymetrix exon array (Santa Clara, CA, USA) was performed on 15 selected RNA samples. Validation methods included RT-PCR and immunohistochemistry (IHC).ResultsAlthough rat exon array demonstrated a significant upregulation of 51 genes (corticosteroid(+)/ANFH(+) VS control), alpha-2-macroglobulin (A2M) gene was particularly over-expressed. Results were validated by RT-PCR and IHC. Importantly, A2M is known to share vascular, osteogenic and cartilage functions relevant for ANFH.ConclusionsThe findings suggest that corticosteroid-induced ANFH in rats might be mediated by A2M. Investigation of A2M as a potential marker, and a treatment target, for early ANFH should be carried out.

Effect of high-dose dexamethasone on endothelial haemostatic gene expression and neutrophil adhesion

Glucocorticoid usage especially at high doses is complicated by adverse outcomes such as thrombotic events or acceleration of inflammatory response in conditions like myeloma and osteonecrosis. The mechanism(s) through which high-dose dexamethasone (HDDEXA) causes vascular injury remains unclear. We hypothesized that HDDEXA sensitizes endothelial cells (EC) to the effect of inflammatory mediators and modulates endothelial haemostatic gene expression and leukocyte adhesion. Human umbilical vein endothelial cells (HUVECs) were grown in the absence or presence of HDDEXA and were also tested in the presence or absence of tumor necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS) or thrombin. mRNA and protein expression were measured and the functional consequences of HDDEXA preconditioning on cell adhesion molecules (CAM) were determined by agonist-mediated leukocyte adhesion assay. Treatment with HDDEXA resulted in an increased induction of CAM, tissue factor and von Willebrand factor, while down-regulating thrombomodulin and urokinase. HDDEXA alone had no effect on adhesion but resulted in enhanced TNF-alpha- and LPS-mediated adhesion of neutrophils. Together, these findings suggest that HDDEXA sensitizes HUVEC to the effect of inflammatory mediators and induces a pro-adhesive environment in primary EC. This finding is of importance when glucocorticoid usage is required at therapeutic high doses in patients with or without thrombotic risk factors.

Targeting DNA Repair Proteins: A Promising Avenue for Cancer Gene Therapy

Enhanced DNA repair in many cancer cells can be correlated to the resistance to cancer treatment, and thus contributes to a poor prognosis. Ionizing radiation and many anti-cancer drugs induce DNA double-strand breaks (DSBs), which are usually regarded as the most toxic types of DNA damages. Repair of DNA DSBs is vital for maintaining genomic stability and hence crucial for survival and propagation of all cellular organisms. Therefore, reducing the capacity of cancer cells to repair DSBs could sensitize tumors to radio/chemotherapy. Many investigators have used gene therapy strategies to down-regulate or inactivate proteins involved in the repair of DSBs in order to reduce the survival of cancer cells. Herein, are reviewed several protein candidates that have been targeted by different gene therapy approaches. Results obtained from in vitro and in vivo experiments are presented and discussed in the perspective of potential gene therapy clinical trials.

A rat model of early stage osteonecrosis induced by glucocorticoids

BackgroundGlucocorticoid (GC)-induced osteonecrosis (ON) is an important complication of medical therapy. The exact pathomechanisms of ON has not been clearly elucidated. There is a need for a reproducible animal model that better approximates the clinical scenario.MethodsTo determine the genetic susceptibility of rats to develop GC-induced femoral head ON, we evaluated 5 different inbred strains of rats (Spontaneous Hypertensive Rat, Wistar Kyoto, Wistar Furth, SASCO Fisher and Lewis). Prednisone pellets (dosage of 1.82-2.56 mg/kg/day) were implanted subcutaneously for 90. After 90 days, the femurs were resected and examined histologically and radiographically. Pathological and histological examination was performed. Hematoxylin and eosin (H & E) staining was used to delineate the femoral head osteonecrosis lesions as well as abnormalities of articular cartilage and growth plate.ResultsThe greatest differences in H & E staining were seen in the Wistar Kyoto and Wistar Furth groups. In these groups 4 out of 5 and 3 out of 5, respectively, steroid-induced rats revealed growth plate disruption with acellular areas. The TUNEL apoptosis staining assay for apoptosis revealed that 4 out of 5 of Wistar Kyoto rats, 5 out of 5 of Wistar Furth, 2 out of 4 of surviving Lewis and 2 out of 2 of the surviving spontaneous hypertensive rats had apoptotic osteocytes in trabeculae, whereas none of the Fisher rats showed apoptotic osteocytes.ConclusionsWe postulate that Wistar Kyoto, Wistar Furth and spontaneous hypertensive rats may be strains of rats more susceptible to develop ON of the femoral head while Fisher rats were the most resistant.