Novel antibodies directed against the human erythropoietin receptor: creating a basis for clinical implementation

Recombinant human erythropoietin (rHuEPO) is an effective treatment for anaemia but concerns that it causes disease progression in cancer patients by activation of EPO receptors (EPOR) in tumour tissue have been controversial and have restricted its clinical use. Initial clinical studies were flawed because they used polyclonal antibodies, later shown to lack specificity for EPOR. Moreover, multiple isoforms of EPOR caused by differential splicing have been reported in cancer cell lines at the mRNA level but investigations of these variants and their potential impact on tumour progression, have been hampered by lack of suitable antibodies. The EpoCan consortium seeks to promote improved pathological testing of EPOR, leading to safer clinical use of rHuEPO, by producing well characterized EPOR antibodies. Using novel genetic and traditional peptide immunization protocols, we have produced mouse and rat monoclonal antibodies, and show that several of these specifically recognize EPOR by Western blot, immunoprecipitation, immunofluorescence, flow cytometry and immunohistochemistry in cell lines and clinical material. Widespread availability of these antibodies should enable the research community to gain a better understanding of the role of EPOR in cancer, and eventually to distinguish patients who can be treated safely by rHuEPO from those at increased risk from treatment.     

GABAB receptor deficiency causes failure of neuronal homeostasis in hippocampal networks

Stabilization of neuronal activity by homeostatic control systems is fundamental for proper functioning of neural circuits. Failure in neuronal homeostasis has been hypothesized to underlie common pathophysiological mechanisms in a variety of brain disorders. However, the key molecules regulating homeostasis in central mammalian neural circuits remain obscure. Here, we show that selective inactivation of GABA_B, but not GABA_A, receptors impairs firing rate homeostasis by disrupting synaptic homeostatic plasticity in hippocampal networks. Pharmacological GABA_B receptor (GABA_BR) blockade or genetic deletion of the GB_1a receptor subunit disrupts homeostatic regulation of synaptic vesicle release. GABA_BRs mediate adaptive presynaptic enhancement to neuronal inactivity by two principle mechanisms: First, neuronal silencing promotes syntaxin-1 switch from a closed to an open conformation to accelerate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly, and second, it boosts spike-evoked presynaptic calcium flux. In both cases, neuronal inactivity removes tonic block imposed by the presynaptic, GB_1a-containing receptors on syntaxin-1 opening and calcium entry to enhance probability of vesicle fusion. We identified the GB_1a intracellular domain essential for the presynaptic homeostatic response by tuning intermolecular interactions among the receptor, syntaxin-1, and the Ca_V2.2 channel. The presynaptic adaptations were accompanied by scaling of excitatory quantal amplitude via the postsynaptic, GB_1b-containing receptors. Thus, GABA_BRs sense chronic perturbations in GABA levels and transduce it to homeostatic changes in synaptic strength. Our results reveal a novel role for GABA_BR as a key regulator of population firing stability and propose that disruption of homeostatic synaptic plasticity may underlie seizure''s persistence in the absence of functional GABA_BRs.Neural circuits achieve an ongoing balance between plasticity and stability to enable adaptations to constantly changing environments while maintaining neuronal activity within a stable regime. Hebbian-like plasticity, reflected by persistent changes in synaptic and intrinsic properties, is crucial for refinement of neural circuits and information storage; however, alone it is unlikely to account for the stable functioning of neural networks (1). In the last 2 decades, major progress has been made toward understanding the homeostatic negative feedback systems underlying restoration of a baseline neuronal function after prolonged activity perturbations (2–4). Homeostatic processes may counteract the instability by adjusting intrinsic neuronal excitability, inhibition-to-excitation balance, and synaptic strength via postsynaptic or presynaptic modifications (5, 6) through a profound molecular reorganization of synaptic proteins (7, 8). These stabilizing mechanisms have been collectively termed homeostatic plasticity. Homeostatic mechanisms enable invariant firing rates and patterns of neural networks composed from intrinsically unstable activity patterns of individual neurons (9).However, nervous systems are not always capable of maintaining constant output. Although some mutations, genetic knockouts, or pharmacologic perturbations induce a compensatory response that restores network firing properties around a predefined “set point” (10), the others remain uncompensated, or their compensation leads to pathological function (11). The inability of neural networks to compensate for a perturbation may result in epilepsy and various types of psychiatric disorders (12). Therefore, determining under which conditions activity-dependent regulation fails to compensate for a perturbation and identifying the key regulatory molecules of neuronal homeostasis is critical for understanding the function and malfunction of central neural circuits.In this work, we explored the mechanisms underlying the failure in stabilizing hippocampal network activity by combining long-term extracellular spike recordings by multielectrode arrays (MEAs), intracellular patch-clamp recordings of synaptic responses, imaging of synaptic vesicle exocytosis, and calcium dynamics, together with FRET-based analysis of intermolecular interactions at individual synapses. Our results demonstrate that metabotropic, G protein-coupled receptors for GABA, GABA_BRs, are essential for firing rate homeostasis in hippocampal networks. We explored the mechanisms by which GABA_BRs gate homeostatic synaptic plasticity. Our study raises the possibility that persistence of epileptic seizures in GABA_BR-deficient mice (13–15) is directly linked to impairments in a homeostatic control system.     

Prognostic factors in COPP-treated patients with Hodgkin's disease

Summary In a recently published review of the literature [40] we came to the conclusion that the Ann-Arbor staging classification is of limited prognostic value for chemotherapy of Hodgkin's disease (Table 2). Four risk factors accounted for impaired complete remission rates: stage IVB, lymphocyte depletion or not classifiable histologic type, previous chemotherapy, and older age.Fifty-eight evaluable patients were treated with COPP; 23 reached a complete remission (40%). Disease-free survival was 31%, overall survival 49% after five years [33].Besides the known risk factors, impaired bone marrow function (leucocyte counts <4×10⁹/1, platelet counts <100×10⁹/1) at the start of therapy was associated with poor treatment results: none of six patients achieved a complete remission [41].Eleven of 16 patients with no and 11 of 23 patients with one risk factor achieved a complete remission, as did only one patient with more than one risk factor. Survival rates after 30 months were; 87% with no, 66% with one, 36% with two, and 13% with more than two risk factors. We can conclude from our results that the prognosis of patients undergoing chemotherapy for Hodgkin's disease depends on the number of risk factors.     

Improved outcome in adult B-cell acute lymphoblastic leukemia

A total of 68 adult patients with B-cell acute lymphoblastic leukemia (B-ALL) were treated in three consecutive adult multicenter ALL studies. The diagnosis of B-ALL was confirmed by L3 morphology and/or by surface immunoglobulin (Slg) expression with > 25% blast cell infiltration in the bone marrow (BM). They were characterized by male predominance (78%) and a median age of 34 years (15 to 65 y) with only 9% adolescents (15 to 20 y), but 28% elderly patients (50 to 65 y). The patients received either a conventional (N = 9) ALL treatment regimen (ALL study 01/81) or protocols adapted from childhood B-ALL with six short, intensive 5-day cycles, alternately A and B. In study B-NHL83 (N = 24) cycle A consisted of fractionated doses of cyclophosphamide 200 mg/m2 for 5 days, intermediate-dose methotrexate (IdM) 500 mg/m2 (24 hours), in addition to cytarabine (AraC), teniposide (VM26) and prednisone. Cycle B was similar except that AraC and VM26 were replaced by doxorubicin. Major changes in study B-NHL86 (N = 35) were replacement of cyclophosphamide by ifosphamide 800 mg/m2 for 5 days, an increase of IdM to high-dose, 1,500 mg/m2 (HdM) and the addition of vincristine. A cytoreductive pretreatment with cyclophosphamide 200 mg/m2, and prednisone 60 mg/m2, each for 5 days was recommended in study B-NHL83 for patients with high white blood cell (WBC) count (> 2,500/m2) or large tumor burden and was obligatory for all patients in study B-NHL86. Central nervous system (CNS) prophylaxis/treatment consisted of intrathecal methotrexate (MTX) therapy, later extended to the triple combination of MTX, AraC, and dexamethasone, and a CNS irradiation (24 Gy) after the second cycle. Compared with the ALL 01/81 study where all the patients died, results obtained with the pediatric protocols B-NHL83 and B-NHL86 were greatly improved. The complete remission (CR) rates increased from 44% to 63% and 74%, the probability of leukemia free survival (LFS) from 0% to 50% and 71% (P = .04), and the overall survival rates from 0% to 49% and 51% (P = .001). Toxicity, mostly hematotoxicity and mucositis, was severe but manageable. In both studies B-NHL83 and B-NHL86, almost all relapses occurred within 1 year. The time to relapse was different for BM, 92 days, and for isolated CNS and combined BM and CNS relapses, 190 days (P = .08). The overall CNS relapses changed from 50% to 57% and 17%, most probably attributable to the high-dose MTX and the triple intrathecal therapy. LFS in studies B-NHL83 and B-NHL86 was significantly influenced by the initial WBC count < or > 50,000/microL, LFS 71% versus 29% (P = .003) and hemoglobin value > or < 8 g/dL, LFS 67% versus 27% (P = .02). Initial CNS involvement had no adverse impact on the outcome. Elderly B- ALL patients (> 50 years) also benefited from this treatment with a CR rate of 56% and a LFS of 56%. It is concluded that this short intensive therapy with six cycles is effective in adult B-ALL. HdM and fractionated higher doses of cyclophosphamide or ifosphamide seem the two major components of treatment.     

The development of cellular immunity to Epstein-Barr virus after allogeneic bone marrow transplantation

Epstein-Barr virus-induced lymphoproliferative disease (EBV-LPD) is a potentially lethal complication during the first 6 months after allogeneic bone marrow transplantation (BMT). To determine whether deficiencies of EBV-specific cellular immunity contribute to EBV-LPD susceptibility and distinguish patients at risk, we performed limiting dilution analysis to quantify anti-EBV cytotoxic T-lymphocyte precursor (CTLp) frequencies in 26 recipients of unmodified or T-cell-depleted (TCD) grafts from EBV-seropositive donors. At 3 months post-BMT (n = 26), only five patients had EBV CTLp frequencies in the range of seropositive normal controls, irrespective of the type of transplant administered. By 6 months post-BMT, 9 of 13 patients tested had EBV CTLp frequencies within the normal range. The time period in which these patients had deficient cellular immunity to EBV corresponds to the period in which we have observed EBV-LPD in most prior patients. One patient with a low EBV CTLp frequency at 4 months post-BMT developed an EBV-LPD. Within 2 weeks of receiving an infusion of donor peripheral blood mononuclear cells (PBMC) providing less than 1,200 EBV- specific cytotoxic T-cell precursors, populations of EBV-specific CTL in the circulation were restored to levels detected in normal seropositive adults. Concurrently, the patient achieved a regression of the EBV-LPD, which has been sustained without further therapy. These studies indicate that recipients of both unmodified and TCD marrow grafts have profound deficiencies of EBV-specific T cell-mediated immunity early posttransplant, and that the period of risk for EBV-LPD closely corresponds to this interval of severe deficiency. Treatment of one patient with EBV-LPD with marrow donor-derived PBMC induced a rapid expansion of EBV-specific cytotoxic T-cell populations that occurred contemporaneously with the clinical regression of disease.     

Factor V Quebec revisited

Factor V Quebec has been described as a bleeding disorder that exhibits an autosomal dominant inheritance pattern and presents severe bleeding after trauma. Two members of a fourth-generation (IV.13 and IV.15) Canadian family have been studied in detail and are the subject of this report. Their clinical presentations and histories have been described previously (Tracy et al: J Clin Invest 74:1221, 1984). Persistent abnormalities include mild thrombocytopenia and defective platelet factor V. Plasma factor V is present at near normal concentration and is fully functional. Thus, the bleeding diathesis appears to reflect the absence of platelet factor V activity. The recent report (Hayward et al: Blood 84:110a, 1994 [suppl, abstr]) of multimerin deficiency in these individuals led us to reevaluate these patients. Western blot analyses of platelet lysates developed with a variety of monoclonal antibodies show that the alpha-granule proteins, fibrinogen, von Willebrand factor, factor V and osteonectin are decreased in concentration and significantly degraded in the platelets of these patients. Thrombospondin, while not degraded, is substantially decreased. In contrast, platelet factor 4 and beta-thromboglobulin do not appear to be affected. These observations suggest that the alpha- granules are correctly assembled but the contents are subsequently subjected to proteolytic degradation. The results indicate that factor V Quebec disorder is probably associated with a generalized defect that leads to degradation of most proteins of the alpha-granules.     

Secondary acute leukaemias with 11q23 rearrangement: clinical, cytogenetic, FISH and FICTION studies

Three patients with secondary acute leukaemia after treatment with topoisomerase II inhibitor agents are described. Two patients had acute myeloid leukaemia (AML), FAB M5a, one had pro-B-acute lymphoblastic leukaemia (ALL). The interval between initiation of chemotherapy and the onset of secondary acute leukaemia was 19–20 months. 11q23 rearrangements were detected in all cases. They were due to translocations t(11;19) (q23;p13.3), t(11;16)(q23;p13) and t(4;11)(q21;q23), respectively. Fluorescence in situ hybridization (FISH) with Yeast Artificial Chromosome (YAC) probe 13HH4 spanning the ALL-1 gene on 11q23 confirmed that in each case the ALL-1 gene had been disrupted by the translocations. The study underlined the relationship between the development of secondary acute leukaemias with 11q23 rearrangement and previous chemotherapy with topoisomerase II inhibitor agents. So far, however, only six adult patients with secondary ALL with t(4;11) after treatment with topoisomerase II inhibitor agents have been reported. ALL with t(4;11) mostly occurs in infants or young children. Our patient received epirubicin continuously for >19 months. This indicates that both myeloid and lymphoid leukaemias with involvement of the ALL-1 gene can be induced by exogenous agents, especially topoisomerase II inhibitors. Thus they may have a common biological background. This hypothesis was substantiated by means of combined immunophenotyping and FISH (FICTION). In the case of AML M5a with t(11;19), the tumour cells with ALL-1 rearrangement expressed CD34. Moreover, the pro-B-ALL with t(4;11) was CD34 positive. These findings suggest that the cell of origin of secondary AML and ALL with 11q23 rearrangement is an immature haemopoietic progenitor cell.     

An inhibitory monoclonal antibody to factor X that blocks prothrombin activation but not prothrombinase enzyme assembly

A monoclonal antibody (designated alpha BFX-2b) prepared against bovine factor X inhibited factor X activity in human, bovine, porcine, rabbit, and canine plasma. In assays using purified prothrombinase components, factor Xa, factor Va, phospholipid vesicles, and calcium ion with the fluorescent active site thrombin inhibitor dansylarginyl-N-(3-ethyl-1,5- pentanediyl)amide, the antibody inhibited the conversion of prothrombin to thrombin. Antibody alpha BFX-2b also blocked prothrombinase cleavage of the macromolecular substrates prethrombin 1 and prethrombin 2 but did not inhibit factor Xa hydrolysis of the synthetic substrate benzoyl- Ile-Glu-Gly-Arg-p-nitroanilide. The antibody also prevented the inactivation of factor Xa by antithrombin III but did not prevent the inactivation by soybean trypsin inhibitor. Antibody alpha BFX-2b bound factor Xa with a stoichiometry of 1:1 and an apparent dissociation constant of 9.0 x 10(-11) mol/L as estimated from its inhibition of prothrombinase activity. Antibody alpha BFX-2b did not prevent binding of factor Xa to factor Va-phospholipid as measured by using fluorescence polarization or high-pressure liquid gel chromatography with the fluorescent Factor Xa analogue dansyl-glutamyl-glycyl-arginyl- Xa. Immunoblotting of factor X following electrophoresis on sodium dodecyl sulphate-polyacrylamide gels and transfer to nitrocellulose indicated that the antigenic determinant recognized by antibody alpha BFX-2b was found on the heavy chain of factors X and Xa. From these observations it can be concluded that antibody alpha BFX-2b recognizes a highly conserved epitope on the factor X heavy chain that is remote from the topographic sites required for prothrombinase complex assembly and substrate hydrolysis but may be located at or near a portion of the macromolecular substrate binding site.     

Early blast clearance by remission induction therapy is a major independent prognostic factor for both achievement of complete remission and long-term outcome in acute myeloid leukemia: data from the German AML Cooperative Group (AMLCG) 1992 Trial

Risk assessment in acute myeloid leukemia (AML) using pretreatment characteristics may be improved by incorporating parameters of early response to therapy. In the 1992 trial of the German AML Cooperative Group (AMLCG), the amount of residual leukemic blasts in bone marrow was assessed one week after the first induction course (day 16 blasts). A total of 449 patients 16 to 76 years of age (median, 53 years) with de novo AML entered the trial and were evaluable. Treatment included TAD/HAM (thioguanine, cytosine arabinoside, and daunorubicin/high-dose cytosine arabinoside and mitoxantrone) double induction, TAD consolidation, and randomly either maintenance therapy or S-HAM consolidation. Cytogenetics were favorable, intermediate, unfavorable and not available in 10.0%, 48.3%, 13.1%, and 28.5%, respectively. Day 16 blasts ranged from 0% to 100% (median, 5%, mean +/- SD, 18.6 +/- 28.5%). Complete remission (CR) rate was 72.6%, 17.6% had persistent leukemia (PL), and 9.8% succumbed to hypoplastic death. Median overall survival (OS), event-free survival (EFS), and relapse-free survival (RFS) were 18, 9, and 15 months with 28.4%, 21.6%, and 30.1% at 5 years, respectively. As a continuous variable, day 16 blasts were related to CR rate (P < 0.0001), PL rate (P < 0.0001), OS (P < 0.0001), EFS (P < 0.0001), and RFS (P = 0.0049). Multivariate analyses identified the following parameters to be associated with the respective end points. CR rate: day 16 blasts (P <.0001), age (P =.0036), and LDH (P =.0072); OS: unfavorable cytogenetics (P <.0001), day 16 blasts (P <.0001), age (P <.0001), and LDH (P =.0040); EFS: unfavorable cytogenetics (P <.0001), LDH (P <.0001), day 16 blasts (P <.0001), and age (P =.0061); RFS: unfavorable cytogenetics (P <.0001), LDH (P <.0001), and day 16 blasts (P =.0359). The prognostic significance of day 16 blasts is independent of pretherapeutic parameters and predicts outcome even in patients achieving a CR.     

Surgical treatment strategies for giant inguinoscrotal hernia – a case report with review of the literature

Background

An inguinoscrotal hernia is defined as “giant” if descending below the midpoint of the inner thigh of a patient in upright position. In developed countries this is a rare entity. In the literature different surgical techniques have been reported so far to achieve a successful treatment.

Case presentation

We present the case of a 63 year-old man suffering from a giant inguinoscrotal hernia, whom we treated using a combined open transabdominal and inguinal approach following an unsuccessful laparoscopic attempt. Meshes were placed in a premuscular position (Lichtenstein’s procedure) and in a preperitoneal position. In addition, a reconstruction of the abdominal wall by modified components separation technique was performed. During the early postoperative period no complications were registered. Intensive care treatment was not necessary. The patient was discharged on postoperative day 8 in an excellent condition. Six months after surgery a scrotal hematocele was diagnosed and operatively removed. After a follow-up of 1.5 years neither hernia recurrence, nor chronic groin pain were recorded. The patient reported to be sexually active. His quality of life improved notably.Additionally, a Medline and PubMed database research was performed to create an overall picture of the existing surgical treatment strategies. Included were patients with diagnosis of primary giant inguinoscrotal hernia according to the given definition. Emergency interventions and cases without details of the surgical approach were excluded.

Conclusions

Firstly, this report describes a novel, successful surgical treatment of a giant inguinoscrotal hernia without administering preoperative progressive pneumoperitoneum therapy or visceral resection. Secondly, we summarize cases previously reported as a practical guide for possible surgical therapy approaches.