Odelia Katz

Erythropoietin enhances immune responses in mice

Erythropoietin (Epo) is the main erythropoietic hormone. Recombinant human Epo (rHuEpo) is thus used in clinical practice for the treatment of anemia. Accumulating data reveals that Epo exerts pleiotropic activities. We have previously shown an anti‐neoplastic activity of Epo in murine multiple myeloma (MM) models, and in MM patients. Our findings that this anti‐neoplastic effect operates via CD8+ T lymphocytes led us to hypothesize that Epo possesses a wider range of immunomodulatory functions. Here we demonstrate the effect of Epo on B lymphocyte responses, focusing on three experimental models: (i) tumor‐bearing mice, (5T2 MM mouse); (ii) antigen‐injected healthy mice; and (iii) antigen‐injected transgenic mice (tg6), overexpressing human Epo. In the MM model, despite bone marrow dysfunction, Epo‐treated mice retained higher levels of endogenous polyclonal immunoglobulins, compared to their untreated controls. In both Epo‐treated wild type and tg6 mice, Epo effect was manifested in the higher levels of splenocyte proliferative response induced in vitro by lipopolysaccharide. Furthermore, these mice had increased in vivo production of anti‐dinitrophenyl (DNP) antibodies following immunization with DNP‐keyhole limpet hemocyanin. Epo‐treated mice showed an enhanced immune response also to the clinically relevant hepatitis B surface antigen. These findings suggest a potential novel use of rHuEpo as an immunomodulator.

Erythropoietin treatment in advanced multiple myeloma is associated with improved immunological functions : could it be beneficial in early disease?

Erythropoietin (Epo) is the main growth regulator of red blood cells, and recombinant human erythropoietin (rHuEpo) is thus used in clinical practice for the treatment of anaemia, primarily in kidney disease and cancer. rHuEpo treatment was found to be associated with prolonged survival of multiple myeloma (MM) patients. This clinical observation was then supported by studies on murine myeloma models. It thus appeared that rHuEpo had an anti‐myeloma effect, causally related to an immunomodulatory function of rHuEpo. The present study investigated whether rHuEpo‐treated MM patients acquire improved immunological functions. Treatment with rHuEpo, prescribed for anaemia that occurs in advanced disease, was associated with effects on a variety of immunological parameters and functions. This was expressed in an actual normalisation of the CD4:CD8 cell ratio, enhanced T cell phytohaemagglutinin‐mediated activation and proliferation potential, T cell expression of the costimulatory CD28 and inhibitory CTLA‐4 molecules, as well as reduced interleukin‐6 serum values to normal levels. Furthermore, it was demonstrated that immunological abnormalities manifest in patients even in the early stages of MM. Our findings thus suggest that rHuEpo treatment might be effective in the early stages of MM, before anaemia develops. It is expected that this would boost the immune system, consequently achieving an anti‐myeloma function; affecting disease progression and improving the prognosis.

Erythropoietin has an anti-myeloma effect - a hypothesis based on a clinical observation supported by animal studies

Recombinant human erythropoietin (rHuEpo) was introduced into clinical practice more than a decade ago, and has been found to be effective in the treatment of several types of anemia, including anemia of end‐stage renal failure and cancer‐related anemia. No study has suggested that Epo might have an effect on the biology of the disease, nor any survival advantage to cancer patients treated with Epo for anemia has been reported. Here we report six patients with advanced multiple myeloma (MM) with very poor prognostic features, whose expected survival was <6 months. All six patients were treated with rHuEpo for their anemia, either without any chemotherapy or very mild chemotherapy for a short time. Yet, surprisingly they lived for 45–133 months totally from MM diagnosis and 38–94 months with rHuEpo (with a good quality of life). In fact, one patient, is still alive and well, more than 8 yr after chemotherapy was discontinued because of a resistant aggressive disease. The course in these six MM patients led us to hypothesize that Epo might have an antineoplastic or antimyeloma effect. We proceeded and tested that hypothesis in mouse models of myeloma (Mittelman M et al., Proc Natl Acad Sci USA 98:5181,2001). In these models we confirmed that rHuEpo induced tumor regression in about 50% of the BALB/c mice inoculated with MOPC‐315 myeloma cells. We then presented evidence that the mechanism is a new immune‐mediated phenomenon, via activation of CD8+ T cells. Furthermore, evidence from the literature supports the antineoplastic effect of Epo. Epo might be used as an adjunct immune treatment in various malignant diseases, in addition to the current regimens and chemotherapeutic protocols. Fututre trials should determine the role of Epo in myeloma and cancer treatment, besides clarifying concerns about the presence of Epo receptors on some tumor cells.

Erythropoietin treatment leads to reduced blood glucose levels and body mass: Insights from murine models

Erythropoietin (EPO) regulates proliferation and differentiation of erythroid precursor cells into erythrocytes. The last decade has revealed non-renal sites of EPO production and extrahematopoietic expression of the EPO receptor, thus suggesting that EPO has pleiotropic functions. Here, we addressed the interplay between EPO/glucose metabolism/body weight by employing a panel of relevant experimental murine models. The models focused on situations of increased EPO levels, including EPO-injected C57BL/6 and BALB/c mice, as well as transgenic mice (tg6) constitutively overexpressing human EPO, thus exposed to constantly high EPO serum levels. As experimental models for diabetes and obesity, we employed protein Tyr phosphatase 1B (PTP1B) knockout mice associated with resistance to diabetes (PTP1B(-/-)), and ob/ob mice susceptible to diabetes and obesity. The data presented herein demonstrate EPO-mediated decrease in blood glucose levels in all mice models tested. Moreover, in the ob/ob mice, we observed EPO-mediated attenuation of body weight gain and reduction of hemoglobin A1c. Taken together, our data bear significant clinical implications of EPO treatment in the management of a wide range of metabolic diseases, thus adding an important novel therapeutic potential to this pleiotropic hormone.

Hyaluronan-grafted particle clusters loaded with Mitomycin C as selective nanovectors for primary head and neck cancers.

CD44, a well-documented cell surface receptor, is involved in cell proliferation, migration, signaling, adhesion, differentiation and angiogenesis, which are important properties for normal and cancerous cell function. We recently developed particle clusters coated with hyaluronan (termed gagomers; GAG), and showed that they can deliver the insoluble drug paclitaxel directly into CD44-over-expressing tumors in a mouse tumor model. Here, we tested primary head and neck cancers (HNC) and normal cells taken from the same patient, and found that although CD44 expression in both types of cells was high, GAGs bind only to the cancerous cells in a selective manner. We next formulated the anti cancer agent mitomycin C (MMC) in the GAGs. MMC-based chemoradiation is a potential treatment for HNC, however, due to patients toxicity, MMC is not part of the standard treatment of HNC. MMC encapsulation efficiency was about 70% with a half-life drug efflux of 1.2 ± 0.3 days. The Ex vivo study of the targeted MMC-GAG showed significant increase in the therapeutic effect on HNC cells (compared to free MMC), while it had no effect on normal cells taken from the same patient. These results demonstrate the specificity of the nanovectors towards head and neck cancers, which might be applicable as future therapy to many CD44-expressing tumors.

Erythropoietin Induced Tumour Mass Reduction in Murine Lymphoproliferative Models

leukocyte (PBL) counts begin to increase a few weeks following injection, exceeding 20 ! 10 6 cells/ml. Mice were injected (subcutaneously) with 10 4 MOPC-315 tumour cells in the abdomen area as described [7] . The rHuEPO (epoetin alfa, Eprex®; Janssen-Cilag, Baar, Switzerland) treatment (rHuEPO 30 U) was administered daily for 10 consecutive days, followed by three times per week for an additional 2–3 weeks to the MOPC-315-bearing mice as described [7] . The linear mixed effect (repeated measures) model was used to determine the effect of EPO on tumour size. Variance among mice was taken into account in the analysis by considering the mice to be randomly selected from a larger population. Starting 9 days after tumour cell injection, 28 MOPC315-bearing mice were injected with rHuEPO or albumin (control). The survival curve, shown in fi gure 1 d, demonstrates approximately 50 and 15% survival of the EPOtreated and control mice, respectively, which is in line with our previously documented observations [7] . Tumour growth kinetics in each of the EPO-treated and albumin-treated mice are presented in fi gures 1 a and b, respectively. Statistical analysis of the tumour size of 7 EPO-treated and 11 control progressor mice, which displayed only one localized tumour, using the linear mixed effect (repeated measures) model, yielded an R value of 0.875 ( fi g. 1 c). The predicted mean rate of tumour growth was 0.539 and 1.238 mm/day for EPO and albumin-treatRecombinant erythropoietin (rHuEPO) is widely used in clinical practice in the treatment of several types of anaemia [1–4] . We observed that patients with end-stage multiple myeloma (MM) treated with EPO live longer than expected, despite their original poor prognostic features [5, 6] . BALB/c mice in which MM was induced by transplantation of mineral oil-induced plasmacytoma cells (MOPC-315) showed complete T cell-mediated tumour regression in 30–60% of the animals after treatment with EPO [7] , suggesting that EPO may also act as an antitumour immunotherapeutic agent. Here, we raised a question regarding the effect of EPO on tumour load, rather than on the ultimate survival, by studying two lymphoproliferative murine models, MOPC-315 MM [8] and B cell leukaemia/lymphoma (BCL1) [9] . BCL1 is a B-cell leukaemia/lymphoma that developed spontaneously in a 2-year-old female BALB/c mouse [10] ; its advantage as a model is due to its analogies to human chronic lymphocytic leukaemia/lymphoma. In both models, we focused on tumour-bearing mice that did not achieve complete tumour regression after EPO treatment (‘progressors’). Female inbred BALB/c mice, aged 6–8 weeks, were obtained from the Tel-Aviv University Breeding Center. BCL1 tumour cells (10 4 cells), derived from spleens of tumour-bearing BALB/c mice, were injected intra-peritoneally into syngeneic mice. Typically, peripheral blood Received: January 18, 2005 Accepted after revision: May 9, 2005

Non-erythroid effects of erythropoietin: Are neutrophils a target?

We have previously shown that erythropoietin (EPO) potentiates the immune response. Analysis of various possible cellular mediators was performed on EPO-injected mice and transgenic mice overexpressing human EPO (tg6). Here we present our studies on neutrophils, peritoneal (casein induced), and from the peripheral blood, spleen and bone marrow. Neutrophil counts were elevated in peripheral blood and spleens of the tg6 mice, yet, no other EPO-associated effects were detected in the count and function of the different neutrophil populations. Hence, neutrophils are probably not mediators of the EPO immunological effects, although their counts may be affected by extreme EPO levels.