Specific stimulation of T lymphocytes with erythropoietin for adoptive immunotherapy.

In adoptive T-cell immunotherapy of cancer, expansion and persistence of effector cells is a key determinant of response. We tested whether T lymphocytes could be rendered sensitive to erythropoietin (Epo) through ectopic expression of its wild-type receptor or a truncated form (EpoRm), which augments Epo signaling in erythrocyte progenitors. Both receptors could be expressed in human T lymphocytes; Epo ligation induced STAT5 phosphorylation, which was abrogated by nontoxic concentrations of the JAK1/2 inhibitor ruxolitinib. EpoRm had higher expression and triggered more potent stimulation than its wild-type counterpart, including superior T-cell survival and proliferation. Using a bicistronic vector, we expressed EpoRm together with an anti-CD19-41BB-CD3ζ chimeric antigen receptor (CAR), while maintaining the functions of each receptor. In the presence of Epo, EpoRm-CAR T cells had greater ex vivo expansion than CAR T cells and killed CD19+ leukemic cells more effectively in long-term cultures. In immunodeficient mice, physiologic levels of murine Epo were sufficient to preferentially expand EpoRm-CAR T cells, yielding a significantly higher antileukemic activity. Thus, outfitting adoptive T cells with EpoRm should yield greater effector-to-target ratios with a smaller number of infused cells; Epo or ruxolitinib administration could be used to adjust their levels postinfusion, maximizing antitumor activity and minimizing toxicity.

Bone Marrow Endothelial Cells Take Up Blood-Borne Immune Complexes via Fcγ Receptor IIb2 in an Erythropoietin-Dependent Manner.

Immune complexes (ICs) in blood are efficiently removed mainly by liver reticuloendothelial systems consisting of sinusoidal endothelial cells and Kupffer cells expressing FcγR. The bone marrow (BM) also has sinusoidal vasculatures, and sinusoidal BM endothelial cells (BMECs) bear unique function, including hematopoietic niches and traffic regulation of hematopoietic cells. In this study, we found that sinusoidal BMECs express FcγRIIb2, which is markedly increased in anemic conditions or by the administration of erythropoietin (Epo) in healthy mice. BMECs expressed Epo receptor (EpoR), and the Epo-induced increase in FcγRIIb2 expression was abolished in Epor-/- ::HG1-Epor transgenic mice, which lack EpoR in BMECs except for BM erythroblasts, suggesting the effect was directly mediated via EpoR on BMECs. Further, although BMECs hardly captured i.v.-injected soluble ICs in healthy mice, Epo administration induced a remarkable increase in the uptake of ICs in a FcγRIIb-dependent manner. Enhancement of the IC incorporation capacity by Epo was also observed in cultured BMECs in vitro, suggesting the direct effect of Epo on BMECs. Moreover, we found that i.v.-injected ICs in Epo-treated mice were more rapidly removed from the circulation than in PBS-treated mice. These results reveal a novel function of BMECs to efficiently remove circulating blood-borne ICs in an FcγRIIb2-mediated manner.

Antibody-mediated pure red cell aplasia related with epoetin-beta pegol (C.E.R.A.) as an erythropoietic agent: case report of a dialysis patient.

BACKGROUND: Erythropoietin-stimulating agents (ESAs) are used to treat anemia in patients with chronic kidney disease, enabling maintenance of stable hemoglobin levels and eliminating the need for multiple transfusions. Epoetin-beta pegol (C.E.R.A.) is a continuous erythropoietin receptor activator created by integrating a large methoxy-polyethylene-glycol-polymer chain into the erythropoietin molecule, which provides it with a longer half-life. On rare occasions, cases of antibody-mediated pure red cell aplasia (PRCA) related to ESAs are reported. They are characterized by abrupt onset of severe transfusion-dependent anemia, despite ESA therapy. We herein report a case of antibody-mediated PRCA in a dialysis patient receiving C.E.R.A. CASE PRESENTATION: A 44-year-old man with end-stage renal failure had been receiving continuous ambulatory peritoneal dialysis for 2 years. C.E.R.A. was administered subcutaneously as a sole ESA once a month at the hospital since 4 years ago for the treatment of renal anemia and his hemoglobin level was well controlled at 12 g/dl. From 10 months before diagnosis, however, his hemoglobin level suddenly declined, necessitating frequent transfusions. Based on the results of a bone marrow examination and detection of anti-C.E.R.A. antibodies, the patient was diagnosed with antibody-mediated PRCA. After successful elimination of the antibodies using oral prednisolone plus cyclosporine, the patient was re-administrated C.E.R.A. intravenously, as there are few reports of antibody-mediated PRCA related to ESA using that administration route. He responded to the C.E.R.A., and his anemia dramatically improved, eliminating the need for blood transfusions. CONCLUSIONS: This is the first reported case of recovery from an antibody-mediated PRCA with C.E.R.A. after its re-administration following a reversal of the antibody. It has been suggested that the additional large pegylation chain makes C.E.R.A. less likely to trigger antibody generation than other ESAs. Following successful treatment of antibody-mediated PRCA using immunosuppressive therapy, C.E.R.A. can be re-administered intravenously to treat renal anemia.

Antierythropoietin Antibody Production Is Not Associated with Malaria and Malaria-Related Anaemia in Humans.

INTRODUCTION: The pathophysiology of malaria-related anaemia is not fully understood although increased destruction of parasitized and nonparasitized erythrocytes, as well as inadequate erythropoiesis, has been proposed. Circulating antierythropoietin (anti-EPO) antibodies have also been implicated in malaria and malaria-related anaemia in mice. However, studies on this association have not been investigated in humans. This study therefore determined the prevalence of anti-EPO antibody production and assessed its association with malaria and malaria-related anaemia in humans. METHODS: A total of 86 children aged 1-10 years (57 children with malaria serving as the case group and 29 healthy children serving as control), all residents of Duayaw Nkwanta, Ghana, were recruited for this case-control study. Venous blood was collected for thick and thin films for malaria microscopy, full blood count by automated haematology analyzer, and antierythropoietin antibody and erythropoietin estimation by sandwich ELISA method. RESULTS: Out of the 86 participants recruited, only 3 (3.5%) were positive for anti-EPO antibody; 2.3% of the case group; and 1.2% of the control group. There was no association between the cases and the controls in the production of anti-EPO antibodies. Erythropoietin concentration was significantly higher in malaria-related anaemic subjects (p=0.032). CONCLUSION: Antierythropoietin antibodies are not associated with malaria infection and malaria-related anaemia in humans. Erythropoietin concentration is associated with malaria-related anaemia.

Development of a strategy and computational application to select candidate protein analogues with reduced HLA binding and immunogenicity.

Unwanted immune responses against protein therapeutics can reduce efficacy or lead to adverse reactions. T-cell responses are key in the development of such responses, and are directed against immunodominant regions within the protein sequence, often associated with binding to several allelic variants of HLA class II molecules (promiscuous binders). Herein, we report a novel computational strategy to predict 'de-immunized' peptides, based on previous studies of erythropoietin protein immunogenicity. This algorithm (or method) first predicts promiscuous binding regions within the target protein sequence and then identifies residue substitutions predicted to reduce HLA binding. Further, this method anticipates the effect of any given substitution on flanking peptides, thereby circumventing the creation of nascent HLA-binding regions. As a proof-of-principle, the algorithm was applied to Vatreptacog α, an engineered Factor VII molecule associated with unintended immunogenicity. The algorithm correctly predicted the two immunogenic peptides containing the engineered residues. As a further validation, we selected and evaluated the immunogenicity of seven substitutions predicted to simultaneously reduce HLA binding for both peptides, five control substitutions with no predicted reduction in HLA-binding capacity, and additional flanking region controls. In vitro immunogenicity was detected in 21·4% of the cultures of peptides predicted to have reduced HLA binding and 11·4% of the flanking regions, compared with 46% for the cultures of the peptides predicted to be immunogenic. This method has been implemented as an interactive application, freely available online at http://tools.iedb.org/deimmunization/.

Will there still be a role for the originator erythropoiesis-simulating agents after the biosimilars and the hypoxia-inducible factor stabilizers approval?

PURPOSE OF REVIEW: To discuss if there will still be a role for the originator ESAs after the already available biosimilars and the approval of HIF stabilizers in the near future. RECENT FINDINGS: Current treatment with erythropoiesis-simulating agents (ESAs) is effective and generally well tolerated, but requires parenteral injections. It is also surrounded by safety concerns and is still expensive. Functional iron deficiency is the major obstacle for efficient ESA therapy. ESA resistance may develop, calling for high ESA doses, further increasing the side effects associated with ESA use. Biosimilars were introduced for reducing costs. In searching for an ideal antianemic drug, new investigational strategies have been proposed including the attractive alternative hypoxia-inducible factor (HIF) stabilizers, which stimulate endogenous EPO production. However, we should caution in translating the historical results referring to the side effects of ESAs to current clinical practice, considering that hemoglobin targets and ESAs doses are now much lower. We could anticipate that side effects will be much less. SUMMARY: According to preliminary data, orally administered HIF stabilizers could provide pharmacological advantages over the existing ESAs. These will need confirmation by the findings of large, phase-3, clinical trials. Finally, cost will be an important issue determining their future use.

Effects of experimental manipulation of hematocrit on avian flight performance in high- and low-altitude conditions.

Despite widely held assumptions that hematocrit (Hct) is a key determinant of aerobic capacity and exercise performance, this relationship has not often been tested rigorously in birds and results to date are mixed. Migration in birds involves high-intensity exercise for long durations at various altitudes. Therefore, it provides a good model system to examine the effect of Hct on flight performance and physiological responses of exercise at high altitude. We treated yellow-rumped warblers (Setophaga coronata) with avian erythropoietin (EPO) and anti-EPO to experimentally manipulate Hct and assessed flight performance at low and high altitudes using a hypobaric wind tunnel. We showed that anti-EPO-treated birds had lower Hct than vehicle- and EPO--treated birds post-treatment. Anti-EPO-treated birds also had marginally lower exercise performance at low altitude, committing a higher number of strikes (mistakes) in the first 30 min of flight. However, anti-EPO-treated birds performed significantly better at high altitude, attaining a higher altitude in a ramped altitude challenge to 3000 m equivalent altitude, and with a longer duration of flight at high altitude. Birds exercising at high altitude showed decreased Hct, increased glucose mobilization and decreased antioxidant capacity, regardless of treatment. In summary, we provide experimental evidence that the relationship between Hct and exercise performance is dependent on altitude. Future studies should investigate whether free-living birds adaptively modulate their Hct, potentially through a combination of erythropoiesis and plasma volume regulation (i.e. hemodilution), based on the altitude they fly at during migratory flight.

Phenotype, proliferation and apoptosis of B lymphocytes in hemodialysis patients treated with recombinant human erythropoietin.

One of the major causes of disorders of the immune response in patients undergoing hemodialysis (HD) is weaker activity of their helper T lymphocytes (Th cells), mainly reduced proliferative capacity associated with decreased expression of key surface antigens. Since cooperation between Th and B lymphocytes is essential for B cell function, changes in Th cell phenotype and ability to proliferate or produce cytokines could directly translate into an impaired humoral response. Therefore, we investigated the T cell-dependent activity of B cells in HD patients focusing mainly on their proliferative kinetics, susceptibility to apoptosis and the ability to produce antibodies. Since our previous studies have shown the beneficial effects of recombinant human erythropoietin (rhEPO) on T lymphocytes, we also investigated the in vivo and in vitro influence of rhEPO on B cells. Our results show that B lymphocytes of HD patients, especially of those who are not treated with rhEPO, have reduced proliferative capacity in vitro, reflected in low number of cell divisions, decreased percentage of proliferating cells and an increased susceptibility to apoptosis. They are also characterized by impaired ability to produce immunoglobulins. We have found no significant changes in the expression of key antigens of B lymphocytes with the exception of IL-10R. Furthermore, we demonstrated a time- and health status-dependent impact of rhEPO on patient's B cells. Our results show possible mechanisms responsible for the deficiency of humoral responses in HD patients which, at least partially, can be modulated through the supplementation with rhEPO.

In vitro inhibition and reversal of Plasmodium falciparum cytoadherence to endothelium by monoclonal antibodies to ICAM-1 and CD36.

BACKGROUND: Sequestration of parasitized red blood cells from the peripheral circulation during an infection with Plasmodium falciparum is caused by an interaction between the parasite protein PfEMP1 and receptors on the surface of host endothelial cells, known as cytoadherence. Several lines of evidence point to a link between the pathology of severe malaria and cytoadherence, therefore blocking adhesion receptors involved in this process could be a good target to inhibit pRBC sequestration and prevent disease. In a malaria endemic setting this is likely to be used as an adjunct therapy by reversing existing cytoadherence. Two well-characterized parasite lines plus three recently derived patient isolates were tested for their cytoadherence to purified receptors (CD36 and ICAM-1) as well as endothelial cells. Monoclonal antibodies against human CD36 and ICAM-1 were used to inhibit and reverse infected erythrocyte binding in static and flow-based adhesion assays. RESULTS: Anti-ICAM-1 and CD36 monoclonal antibodies were able to inhibit and reverse P. falciparum binding of lab and recently adapted patient isolates in vitro. However, reversal of binding was incomplete and varied in its efficiency between parasite isolates. CONCLUSIONS: The results show that, as a proof of concept, disturbing existing ligand-receptor interactions is possible and could have potential therapeutic value for severe malaria. The variation seen in the degree of reversing existing binding with different parasite isolates and the incomplete nature of reversal, despite the use of high affinity inhibitors, suggest that anti-adhesion approaches as adjunct therapies for severe malaria may not be effective, and the focus may need to be on inhibitory approaches such as vaccines.

Circulating autoantibodies to endogenous erythropoietin are associated with chronic hepatitis C virus infection-related anemia.

BACKGROUND: Chronic hepatitis C virus (HCV) infection is associated with autoimmune phenomena and is often complicated by anemia. Circulating autoantibodies to endogenous erythropoietin (anti-EPO) have been detected in patients with chronic viral infections and were correlated to anemia. The present study aimed to determine anti-EPO prevalence in patients with chronic HCV infection and investigate its possible association with anemia. METHODS: Ninety-three consecutive patients (62 males and 31 females) with chronic HCV infection, who had never received antiviral therapy or recombinant EPO, were enrolled in the study. Circulating anti-EPO were detected in the serum by using an ELISA assay. Quantitative determination of serum EPO levels was done by radioimmunoassay. HCV RNA viral load measurement and genotype sequencing were also performed. RESULTS: Circulating anti-EPO were detected in 10.8% of HCV-infected patients and the prevalence of anti-EPO was significantly higher in patients with anemia (19.4% vs 5.3%, P=0.040) compared to that in those without anemia. Compared to anti-EPO negative cases, anti-EPO positive patients had higher frequency of anemia (70.0% vs 34.9%, P=0.030), lower EPO concentrations (median 16.35 vs 30.65 mU/mL, P=0.005), and higher HCV RNA viral load (median 891.5X103 vs 367.5X103 IU/mL, P=0.016). In multivariate regression analysis the presence of anti-EPO remained an independent predictor of anemia (adjusted OR: 14.303, 95% CI: 1.417-36.580, P=0.024). EPO response to anemia was less prominent among anti-EPO positive patients (P=0.001). CONCLUSIONS: Circulating anti-EPO are detected in a significant proportion of treatment-naive HCV-infected patients and are independently associated with anemia, suggesting a further implication of autoimmunity in the pathophysiology of HCV-related anemia.

Detection of erythroblast antibodies in mitogen-stimulated bone marrow cultures from patients with myelodysplastic syndromes.

BACKGROUND: Low-risk myelodysplastic syndromes (MDS) show several immunologic abnormalities, including increased frequency of autoimmune manifestations and/or overt autoimmune diseases, whose prognostic significance still remains controversial. STUDY DESIGN AND METHODS: We studied the presence of erythroblast antibodies in mitogen-stimulated bone marrow (BM) cultures of 70 patients with early-stage MDS (refractory anemia and refractory anemia with ringed sideroblasts). RESULTS: Sixty-six percent of patients showed positive erythroblast antibodies, along with BM erythroid hyperplasia and a hemolytic picture in the peripheral blood. Supernatants from positive cultures induced an increase of overall cellularity, the appearance of erythroblastic clustering, and dyserythropoietic signs in normal BM. We identified CD45(dim) Gly-A(dim) CD71(bright) cells (red blood cell precursors at different maturation stage) as the target of the antibodies. Erythropoietin (EPO) levels were reduced and EPO receptors (EPO-R) increased in BM culture supernatants from positive patients. However, flow cytometric analysis showed that neither EPO nor EPO-R was involved in an abnormal stimulation driven by these autoantibodies. Values of the proapoptotic protein Bax were increased in positive patients and Bcl-2 levels were decreased, although not significantly. CONCLUSION: MDS patients with anti-erythroblast autoimmunity showed increased BM apoptosis, suggesting that the autoimmune reaction may contribute to an unfavorable BM microenvironment for optimal erythropoiesis.

Incidence of erythropoietin antibody-mediated pure red cell aplasia: the Prospective Immunogenicity Surveillance Registry (PRIMS).

BACKGROUND: Subcutaneous administration of Eprex(®) (epoetin alfa) in patients with chronic kidney disease (CKD) was contraindicated in the European Union between 2002 and 2006 after increased reports of anti-erythropoietin antibody-mediated pure red cell aplasia (PRCA). The Prospective Immunogenicity Surveillance Registry (PRIMS) was conducted to estimate the incidence of antibody-mediated PRCA with subcutaneous administration of a new coated-stopper syringe presentation of Eprex(®) and to compare this with the PRCA incidence with subcutaneous NeoRecormon(®) (epoetin beta) and Aranesp(®) (darbepoetin alfa). METHODS: PRIMS was a multicentre, multinational, non-interventional, parallel-group, immunogenicity surveillance registry. Adults with CKD receiving or about to initiate subcutaneous Eprex(®), NeoRecormon(®) or Aranesp(®) for anaemia were enrolled and followed for up to 3 years. Unexplained loss or lack of effect (LOE), including suspected PRCA, was reported, with antibody testing for confirmation of PRCA. RESULTS: Of the 15 333 patients enrolled, 5948 received Eprex(®) (8377 patient-years) and 9356 received NeoRecormon(®)/Aranesp(®) (14 286 patient-years). No treatment data were available for 29 patients. Among 23 patients with LOE, five cases of PRCA were confirmed (Eprex(®), n = 3; NeoRecormon(®), n = 1; Aranesp(®), n = 1). Based on exposed time, PRCA incidence was 35.8/100 000 patient-years (95% CI 7.4-104.7) for Eprex(®) versus 14.0/100 000 patient-years (95% CI 1.7-50.6) for NeoRecormon(®)/Aranesp(®). The incidence of PRCA with Eprex(®) was not significantly different versus comparator ESAs (rate ratio: 2.56; 95% CI 0.43-15.31). An analysis based on observed time produced similar findings. CONCLUSION: This large, prospective registry demonstrates that PRCA is rare with subcutaneous administration of either the new coated-stopper syringe presentation of Eprex(®), or NeoRecormon(®) or Aranesp(®).

Erythropoietin attenuates loss of potassium chloride co-transporters following prenatal brain injury.

Therapeutic agents that restore the inhibitory actions of γ-amino butyric acid (GABA) by modulating intracellular chloride concentrations will provide novel avenues to treat stroke, chronic pain, epilepsy, autism, and neurodegenerative and cognitive disorders. During development, upregulation of the potassium-chloride co-transporter KCC2, and the resultant switch from excitatory to inhibitory responses to GABA guide the formation of essential inhibitory circuits. Importantly, maturation of inhibitory mechanisms is also central to the development of excitatory circuits and proper balance between excitatory and inhibitory networks in the developing brain. Loss of KCC2 expression occurs in postmortem samples from human preterm infant brains with white matter lesions. Here we show that late gestation brain injury in a rat model of extreme prematurity impairs the developmental upregulation of potassium chloride co-transporters during a critical postnatal period of circuit maturation in CA3 hippocampus by inducing a sustained loss of oligomeric KCC2 via a calpain-dependent mechanism. Further, administration of erythropoietin (EPO) in a clinically relevant postnatal dosing regimen following the prenatal injury protects the developing brain by reducing calpain activity, restoring oligomeric KCC2 expression and attenuating KCC2 fragmentation, thus providing the first report of a safe therapy to address deficits in KCC2 expression. Together, these data indicate it is possible to reverse abnormalities in KCC2 expression during the postnatal period, and potentially reverse deficits in inhibitory circuit formation central to cognitive impairment and epileptogenesis.

Selection of antibodies that regulate phenotype from intracellular combinatorial antibody libraries.

A method is presented that uses combinatorial antibody libraries to endow cells with new binding energy landscapes for the purpose of regulating their phenotypes. Antibodies that are expressed in cells infected with a lentiviral combinatorial antibody library are selected directly for function rather than only for binding. The potential diversity space can be very large because more than one lentivirus can infect a single cell. Thus, the initial combinatorial diversity of ~1.0 × 10(11) members generated by the random association of antibody heavy and light chains is greatly increased by the reassortment of the antibody Fv domains themselves inside cells. The power of the system is illustrated by its ability to select unusual antibodies. Here, the selected antibodies are potent erythropoietin agonists whose ontogeny depends on recombination at the protein level of pairs of antibodies expressed in the same cell to generate heterodimeric bispecific antibodies. The obligate synergy between the different binding specificities of the antibody's monomeric subunits appears to replicate the asymmetric binding mechanism of authentic erythropoietin.

Resistance to Recombinant Human Erythropoietin Therapy in a Rat Model of Chronic Kidney Disease Associated Anemia.

This study aimed to elucidate the mechanisms explaining the persistence of anemia and resistance to recombinant human erythropoietin (rHuEPO) therapy in a rat model of chronic kidney disease (CKD)-associated anemia with formation of anti-rHuEPO antibodies. The remnant kidney rat model of CKD induced by 5/6 nephrectomy was used to test a long-term (nine weeks) high dose of rHuEPO (200 UI/kg bw/week) treatment. Hematological and biochemical parameters were evaluated as well as serum and tissue (kidney, liver and/or duodenum) protein and/or gene expression of mediators of erythropoiesis, iron metabolism and tissue hypoxia, inflammation, and fibrosis. Long-term treatment with a high rHuEPO dose is associated with development of resistance to therapy as a result of antibodies formation. In this condition, serum EPO levels are not deficient and iron availability is recovered by increased duodenal absorption. However, erythropoiesis is not stimulated, and the resistance to endogenous EPO effect and to rHuEPO therapy results from the development of a hypoxic, inflammatory and fibrotic milieu in the kidney tissue. This study provides new insights that could be important to ameliorate the current therapeutic strategies used to treat patients with CKD-associated anemia, in particular those that become resistant to rHuEPO therapy.

Erythropoietin is a hypoxia inducible factor-induced protective molecule in experimental autoimmune neuritis.

Experimental autoimmune neuritis (EAN), an autoantigen-specific T-cell-mediated disease model for human demyelinating inflammatory disease of the peripheral nervous system, is characterized by self-limitation. Here we investigated the regulation and contribution of erythropoietin (EPO) in EAN self-limitation. In EAN sciatic nerves, hypoxia, and protein and mRNA levels of hypoxia-inducible factor 1α (HIF-1α), HIF-2α, EPO and EPO receptor (EPOR) were induced in parallel at disease peak phase but reduced at recovery periods. Further, the deactivation of HIF reduced EAN-induced EPO/EPOR upregulation in EAN, suggesting the central contribution of HIF to EPO/EPOR induction. The deactivation of EPOR signalling exacerbated EAN progression, implying that endogenous EPO contributed to EAN recovery. Exogenous EPO treatment greatly improved EAN recovery. In addition, EPO was shown to promote Schwann cell survival and myelin production. In EAN, EPO treatment inhibited lymphocyte proliferation and altered helper T cell differentiation by inducing increase of Foxp3(+)/CD4(+) regulatory T cells and decrease of IFN-γ(+)/CD4(+) Th1 cells. Furthermore, EPO inhibited inflammatory macrophage activation and promoted its phagocytic activity. In summary, our data demonstrated that EPO was induced in EAN by HIF and contributed to EAN recovery, and endogenous and exogenous EPO could effectively suppress EAN by attenuating inflammation and exerting direct cell protection, indicating that EPO contributes to the self-recovery of EAN and could be a potent candidate for treatment of autoimmune neuropathies.

Risk of pure red cell aplasia in patients with hepatitis C receiving antiviral therapy and an erythropoiesis-stimulating agent.

Antibody-mediated pure red cell aplasia (PRCA) has been primarily observed in patients with chronic kidney disease treated with an erythropoiesis-stimulating agent (ESA); only a few anecdotal cases have been reported in other patient populations. We searched the Amgen Global Safety Adverse Event Database and identified 14 patients with hepatitis C who developed severe anemia, anti-erythropoietin antibodies, and bone marrow biopsy-proven PRCA, while receiving interferon therapy (with or without ribavirin) and an ESA. During the follow-up period and after ESA treatment stopped, 11 patients no longer required transfusions and 3 did. Analysis of antibody isotypes showed that, contrary to reports of patients with chronic kidney disease, immunoglobulin G1 was the predominant isotype rather than immunoglobulin G4 (immunoglobulin G4 was detected in only 1 of 6 patients). Epitope mapping showed the anti-erythropoietin antibodies bound domains required for receptor binding. Therefore, the potential benefits of ESA therapy must be weighed against the risk for PRCA in patients with hepatitis C who are receiving treatment with interferon and ribavirin.

Rechallenge with intravenous recombinant human erythropoietin can be successful following the treatment of anti-recombinant erythropoietin associated pure red cell aplasia.

Anti recombinant human erythropoietin (r-HuEpo) associated pure red cell aplasia (PRCA) is an immunologic adverse effect of using subcutaneous r-HuEpo. Immunosuppressive agents have been suggested as treatment of this serious complication. After the reversal of anti-r-HuEpo antibody, the patients continue to have renal anemia and require long-term blood transfusion, albeit less frequently than when the antibody is positive. It is controversial whether re-challenging the patients with r-HuEpo is appropriate because re-challenging may cause the reappearance of the antibody. To balance the risk of antir-HuEpo antibody reappearance and longterm blood transfusion complications, we re-challenged r-HuEpo in five anti-r-HuEpo associated PRCA cases after a successful reversal of antibody using prednisolone in combination with cyclophosphamide. The rechallenge was performed intravenously since there were no reports of anti-r-HuEpo associated PRCA cases using this administration route. The duration after the reversal of antibody was 2.4 months before the re-challenge. Two patients were immediately re-challenged as soon as the antibodies reversed. After rechallenge with intravenous r-HuEpo, all patients responded to r-HuEpo: target level of Hb was maintained, blood transfusion was not required, and anti-r-HuEpo was consistently negative. All patients were followed for at least 6 months after re-challenge. Our data suggest that re-challenge with intravenous r-HuEpo can successfully treat anti- r-HuEpo associated PRCA.

A simple screening test for the detection of erythropoietin antibodies.

Antibody-mediated pure red cell aplasia (PRCA) is a rare complication of erythropoietin (EPO) therapy. Identification and demonstration of functional activity of EPO antibodies required to diagnose this condition is difficult and only performed in selected laboratories worldwide. In this article we report a recent cluster of three cases of antibody-mediated PRCA over a 16-month period in a single center associated with EPREX use. We also describe the use of a simple low-cost inhibitor assay that can be used to screen for PRCA in local laboratories.