Modified recombinant human erythropoietin with potentially reduced immunogenicity.

Recombinant human erythropoietin (rHuEPO) is a biopharmaceutical drug given to patients who have a low hemoglobin related to chronic kidney disease, cancer or anemia. However, some patients repeatedly receiving rHuEPO develop anti-rHuEPO neutralizing antibodies leading to the development of pure red cell aplasia (PRCA). The immunogenic antibody response activated by rHuEPO is believed to be triggered by T-cells recognizing EPO epitopes bound to MHC molecules displayed on the cell surface of APCs. Previous studies have reported an association between the development of anti-rHuEpo-associated PRCA and the HLA-DRB1*09 gene, which is reported to be entrenched in the Thai population. In this study, we used computational design to screen for immunogenic hotspots recognized by HLA-DRB1*09, and predicted seventeen mutants having anywhere between one through four mutations that reduce affinity for the allele, without disrupting the structural integrity and bioactivity. Five out of seventeen mutants were less immunogenic in vitro while retaining similar or slightly reduced bioactivity than rHuEPO. These engineered proteins could be the potential candidates to treat patients who are rHuEpo-dependent and express the HLA-DRB1*09 allele.

Unusual Anemias.

Many processes lead to anemia. This review covers anemias that are less commonly encountered in the United States. These anemias include hemoglobin defects like thalassemia, bone marrow failure syndromes like aplastic anemia and pure red cell aplasia, and hemolytic processes such as paroxysmal nocturnal hemoglobinuria. The pathogenesis, diagnostic workup, and treatment of these rare anemias are reviewed.

Overlooked non-motor symptoms in myasthenia gravis.

Patients with myasthenia gravis (MG) may have various non-motor symptoms in addition to fatigability and weakness of skeletal muscles. Thymomas contain abundant immature thymocytes and developing CD4 and CD8 T cells. Thymomas are found in 15-25% of patients with MG and are associated with severe symptoms. We suggest that non-motor symptoms are based on the autoimmune disorders probably owing to an abnormal T cell repertoire from thymomas. Using previously reported cases and cases from our multicentre cooperative study, we review the clinical characteristics of patients with thymoma-associated MG who have non-motor symptoms. CD8 T cell cytotoxicity against haematopoietic precursor cells in bone marrow and unidentified autoantigens in hair follicles lead to the development of pure red cell aplasia, immunodeficiency and alopecia areata. In contrast, neuromyotonia, limbic encephalitis, myocarditis and taste disorders are autoantibody-mediated disorders, as is MG. Autoantibodies to several types of voltage-gated potassium channels and the related molecules can evoke various neurological and cardiac disorders. About 25% of patients with thymoma-associated MG have at least one non-motor symptom. Non-motor symptoms affect many target organs and result in a broad spectrum of disease, ranging from the impairment of quality of life to lethal conditions. Since relatively little attention is paid to non-motor symptoms in patients with thymoma-associated MG, the symptoms may be overlooked by many physicians. Early diagnosis is important, since non-motor symptoms can be treatable. A complete understanding of non-motor symptoms is necessary for the management of patients with thymoma-associated MG.

Response of pure red cell aplasia to cyclophosphamide after failure of mycofenolate mofetil in a patient with polyglandular syndrome type I.

A 26-year-old female with the classic major and minor components of autoimmune polyglandular syndrome type 1 was diagnosed as having pure red cell aplasia. Treatment with 1.5 g/d mycofenolate mofetil for 3 months failed to restore erythroid production. Treatment with cyclosporine A produced a good partial response but led to renal toxicity and was therefore substituted with cyclophosphamide, which had a good partial effect and lasted for 18 months. The relapse of anemia was not observed during the 6-month follow-up period after the cessation of treatment.

Linking drugs to obscure illnesses: lessons from pure red cell aplasia, nephrogenic systemic fibrosis, and Reye's syndrome. a report from the Southern Network on Adverse Reactions (SONAR).

Identification of serious adverse drug reactions (sADRS) associated with commonly used drugs can elude detection for years. Reye's syndrome (RS), nephrogenic systemic fibrosis (NSF), and pure red cell aplasia (PRCA) among chronic kidney disease (CKD) patients were recognized in 1951, 2000, and 1998, respectively. Reports associating these syndromes with aspirin, gadodiamide, and epoetin, were published 29, 6, and 4 years later, respectively. We obtained primary information from clinicians who identified causes of these sADRs and reviewed factors contributing to delayed identification of these toxicities. Overall, 3,500 aspirin-associated RS cases in the United States, 1,605 gadolinium-associated NSF cases, and 181 epoetin-associated PRCA cases were reported. Delays in FDA regulation of over-the- counter medications and administration of aspirin to children contributed to development of RS. For NSF, in 1996, the Danish Medicine Agency approved high-dose gadodiamide administration to chronic kidney disease (CKD) patients undergoing MR scans. Overall, 88 % of Danish NSF cases were from two hospitals and 97 % of United States' NSF cases were from 60 hospitals. These hospitals frequently administered high-doses of gadodiamide to CKD patients. Another factor was the decision to administer linear chelated contrast agents versus lower risk macrocyclic chelated agents. For PRCA, increased use of subcutaneous epoetin formulations to CKD patients, in part due to convenience and cost-savings considerations, and a European regulatory requirement requiring removal of albumin as a stabilizer, led to toxicity. Overall, 81, 13, and 17 years elapsed between drug introduction into practice and identification of a causal relationship for aspirin, erythropoietin, and gadodiamide, respectively. A substantial decline in new cases of these sADRs occurred within two years of identification of the offending drug. Clinicians should be vigilant for sADRs, even for frequently-prescribed pharmaceuticals, particularly in settings where formulation or regulatory changes have occurred, or when over-the-counter, off-label, or pediatric use is common.

The patterns of MHC association in aplastic and non-aplastic paroxysmal nocturnal hemoglobinuria.

The deficiency of glycosyl-phosphatidylinositol (GPI)-anchored proteins in plasma membranes of PIG-A gene mutated hematopoietic stem cells (HSCs) is so far insufficient to explain the domination of paroxysmal nocturnal hemoglobinuria (PNH) clone over the normal HSC. We attempted to elucidate possible link between MHC and initial severe aplastic anemia (ISAA/PNH) type and non-aplastic (n/PNH) outcome of PNH. In 50 PNH patients assigned as ISAA/PNH (n = 13), n/PNH (n = 33) or nonassigned (n = 4) and 200 ethnically matched controls we analyzed MHC associations. Our data confirmed strong associations of DRB1*15:01 (RR = 3.51, p = 0.0011) and DQB1*06:02 (RR = 7.09, p = 0.000026) alleles, especially with n/PNH subtype. B*18:01 allele was associated with increased risk of ISAA/PNH subtype (RR = 5.25, p = 0.0028). We conclude that both class II and class I MHC alleles are associated with different subsets of PNH. Clonal selection of PIG-A mutated cells with cognate metabolic block is associated with MHC class II alleles DRB1*15:01 and DQB1*06:02 independent from initial severe AA clone selection. MHC class I molecule B*18:01 can additionally influence the domination of PNH clone in PNH subjects with initial severe aplastic anemia.

[Myelodysplastic syndrome with erythroblastopenia]. / Syndromes myélodysplasiques érythroblastopéniques.

Myelodysplastic syndrome with erythroid hypoplasia or erythroblastopenia has not yet been clearly defined, and in most patients it is mistaken for acquired pure red cell aplasia. Including one additional patient reported in this article, a literature review revealed only 50 cases over the last 20 years. These patients were predominantly elderly males, all required regular packed red cell transfusions, and they had a poor prognosis, mainly because of acute transformation. The mechanisms of erythroid aplasia remain unclear. However, recent data suggest the association of an intrinsic stem cell defect with immunological implication.

Anti-erythropoietin antibody-mediated pure red cell aplasia in a living donor liver transplant recipient treated for hepatitis C virus.

After liver transplantation, reinfection of the newly engrafted liver with hepatitis C virus is essentially universal in patients who are viremic at the time of transplantation. Treatment with interferon preparations with or without ribavirin is recommended in patients with marked histologic injury; however, hematologic toxicity associated with therapy has been reported, which is usually treated with growth factor support, including erythropoietin analogues. We present the first reported case of anti-erythropoietin antibody-mediated pure red cell aplasia arising in the setting of hepatitis C virus therapy in a patient who underwent living donor liver transplantation.

Prevalence of anti-erythropoietin antibodies in hemodialysis patients without clinical signs of pure red cell aplasia. Comparison between hypo- and normoresponsive patients treated with epoetins for renal anemia.

BACKGROUND/AIMS: The prevalence of anti-erythropoietin antibodies in renal patients without clinical evidence of pure red cell aplasia (PRCA) who respond poorly to epoetin is unknown. This study tested for anti-erythropoietin antibodies in hemodialysis patients who were either hypo- or normoresponsive to epoetin treatment. METHODS: Epoetin hyporesponsiveness (hemoglobin < or =10.5 g/dl and epoetin > or =9,000 IU/week) and normoresponsiveness (hemoglobin >10.5 g/dl and epoetin <7,000 IU/week) were arbitrarily defined. Prevalence of anti-erythropoietin antibodies in hemodialysis patients without symptoms of PRCA was determined by screening sera of 536 patients from 35 German KfH dialysis units, using enzyme-linked immunosorbent assay (ELISA). Positive results were verified by radioimmunoprecipitation assay (RIP) and neutralizing activity was determined by bioassay. RESULTS: Anti-erythropoietin antibodies were detected in 3 hyporesponsive and 3 normoresponsive patients using ELISA. One patient per group was verified as borderline by RIP testing; the other 4 were negative. The bioassay was negative for 1 patient; the other died unrelated to PRCA before testing. Follow-up with RIP testing after 15 months under continuous epoetin treatment was negative (4 patients, 2 deceased). CONCLUSION: This survey did not identify anti-erythropoietin antibodies in hemodialysis patient's hyporesponsive to epoetin and does not support presumptive antibody screening as a routine work-up in these patients.

Diamond blackfan anemia: New paradigms for a "not so pure" inherited red cell aplasia.

Diamond Blackfan anemia (DBA) is a genetically and clinically heterogeneous disorder characterized by erythroid failure, congenital anomalies, and a predisposition to cancer. Faulty ribosome biogenesis is hypothesized to be the underlying defect, leading to erythroid failure due to accelerated apoptosis in affected erythroid progenitors/precursors. Since first observed in DBA, pro-apoptotic hematopoiesis has been recognized as a common mechanism for hematopoietic failure in virtually all of the inherited bone marrow failure syndromes. Inherited as an autosomal dominant trait, one of what appears to be multiple DBA genes, coding for ribosomal protein RPS19, has been cloned. The discovery of additional genes will no doubt clarify the molecular pathophysiology of this disorder. Even within families, individuals may vary dramatically as to the degree of anemia, treatment response, and the presence of congenital anomalies. The study of DBA has been facilitated by the creation of international patient registries that provide more reliable information regarding clinical presentation, genetics, and outcome, as well as descriptions of congenital malformations and cancer predisposition, than can be culled from the literature. Analysis of registry data has led to improvements in clinical care and provides patients and research specimens for clinical and laboratory investigations.

Resistant pure red cell aplasia after allogeneic stem cell transplantation with major ABO mismatch treated by escalating dose donor leukocyte infusion.

We report a case of pure red cell aplasia (PRCA) following allogeneic stem cell transplantation (SCT) with major ABO mismatch which proved resistant to all standard treatment options such as change in immunosuppressive treatment, high-dose erythropoietin (EPO) or plasma exchange. We therefore proceeded to administer five cycles of Rituximab therapy, without success. Finally, escalating doses of donor-derived leukocyte infusion (DLI) resolved the PRCA of our patient 415 d after bone-marrow transplantation (BMT) and 140 d after the first infusion of donor leukocytes. A review of the literature shows the efficacy of various treatments; the role of DLI and other treatment options are discussed. Furthermore, the underlying pathophysiological mechanisms especially with regard to the role of NK cells in alloreactivity after allogeneic SCT are explained.

Defect in the lymphoid compartment might account for CD8+-mediated effects in the pathophysiology of pure red cell aplasia.

Pure red cell aplasia (PRCA) is a rare hematological syndrome characterized by the lack of red cell progenitors in an otherwise normocellular bone marrow. Many agents and mechanisms have been implicated in the pathophysiology of PRCA, including immune-mediated dysfunctions. This report describes three patients with PRCA with unknown underlying cause and showed that for each, increases in CD8+ cells blunted the maturation of early erythroid (BFU-E). Each patient subsequently responded to immunosuppressive therapy. Peripheral blood mononuclear cells from age- and sex-matched healthy controls showed comparable distribution of CD3, CD4 and CD16, but significant increase in CD8 and decreased CD19. The distribution of lymphocyte subsets correlated with mitogen responses, but showed no difference in allogeneic responses when compared to controls. The adherent population in PRCA is important for mediating the hyper-immune state of patients, when IL-2 levels were used as readout. There was a trend for decreased BFU-E in patients, but marked reduction for late erythroid progenitors (CFU-E). CD8+ cells from PRCA blunted the maturation of BFU-E, despite increasing erythropoietin concentrations. These results strongly suggest that there are defects in the lymphoid compartment that feedback on the erythroid lineage of PRCA.