In vitro response of T cells from aplastic anemia patients to antilymphocyte globulin and phytohemagglutinin: colony-stimulating activity and lymphokine production

The aim of the present study was to compare the response of bone marrow (BM) lymphocytes from patients with aplastic anemia (AA) or normal controls to increasing doses of antilymphocyte globulin (ALG) or phytohemagglutinin (PHA). For this purpose BM T-enriched cells from 11 AA patients and 9 normal individuals were incubated with ALG (0-1000 micrograms/ml) or PHA (0%-10%) for 1 day, and the supernatants were tested for suppression/enhancement of granulocyte-macrophage colony-forming unit (CFU-GM) growth and for release of granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) assayed with the enzyme-amplified sensitivity immunoassay (EASI). The production of colony-stimulating activity (CSA) by T cells primed with ALG and tested in the absence of exogenous GM-CSF correlated with the dose of ALG in priming cultures up to 14% EG (100% EG = CFU-GM growth with 30 ng/ml of GM-CSF). The amount of GM-CSF in the supernatants paralleled their capacity to sustain CFU-GM growth (up to 3.5 ng/ml of GM-CSF). Production of CSA or GM-CSF from T cells primed with PHA was significantly lower. Supernatants of PHA-primed T cells, when added to normal BM cells in the presence of exogenous GM-CSF, produced a dose-dependent inhibition of CFU-GM growth (down to 13% +/- 10% EG). The same supernatants contained detectable amounts of IFN-gamma and TNF-alpha (21 +/- 6.7 IU/ml and 4.6 +/- 2.9 ng/ml, respectively). IFN-gamma production from severe AA (SAA) T cells in response to PHA was significantly superior to the IFN-gamma production from normal T cells (21 +/- 6.7 IU/ml vs 9.5 +/- 7.1 IU/ml, p = 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)     

The two different receptors for tumor necrosis factor mediate distinct cellular responses.

The individual roles of the murine type 1 and type 2 tumor necrosis factor (TNF) receptors (TNF-R1 and TNF-R2) were investigated utilizing (i) the strong species specificity of TNF-R2 for murine TNF compared to human TNF and (ii) agonistic rabbit polyclonal antibodies directed against the individual TNF receptors. Proliferation of mouse thymocytes and the murine cytotoxic T-cell line CT-6 is stimulated by murine TNF but not by human TNF. Consistent with this observation, polyclonal antibodies directed against TNF-R2 induced proliferation in both of these cell types, whereas polyclonal antibodies directed against TNF-R1 had no effect. In contrast, cytotoxicity in murine LM cells (which are sensitive to murine and human TNF) was induced by antibodies against TNF-R1 but not by antibodies against TNF-R2. Also, the steady-state level of manganous superoxide dismutase mRNA in the murine NIH 3T3 cell line was induced by murine TNF, human TNF, and anti-TNF-R1 but not by anti-TNF-R2. These results suggest that TNF-R2 initiates signals for the proliferation of thymocytes and cytotoxic T cells, whereas TNF-R1 initiates signals for cytotoxicity and the induction of the protective activity, manganous superoxide dismutase. The nonredundant signaling observed for the two TNF receptors cannot be explained simply by the differential expression of the two TNF receptors in the various cell types, because LM cells express on their surface higher levels of TNF-R2 than TNF-R1, and LM cells, NIH 3T3 cells, and thymus cells all express mRNA corresponding to both receptor types. It is therefore likely that the two receptors initiate distinct signaling pathways that result in the induction of different cellular responses.     

Regulation of neutrophil migration and superoxide production by recombinant tumor necrosis factors-alpha and -beta: comparison to recombinant interferon-gamma and interleukin-1 alpha

We compared the ability of recombinant human tumor necrosis factor- alpha (rHuTNF-alpha) and tumor necrosis factor-beta (rHuTNF-beta) to stimulate polymorphonuclear neutrophil (PMN) migration and superoxide production. Significant PMN migration occurred across polycarbonate filters after stimulation with rHuTNF-alpha at concentrations ranging from 10(-7) to 10(-10) mol/L and at 10(-7) to 10(-8) mol/L for rHuTNF- beta and N-formylmethionyl-leucyl phenylalanine (FMLP), whereas recombinant human interferon-gamma was only minimally active at 10(-7) mol/L and recombinant human interleukin-1 alpha was inactive at the doses tested. In addition, antibodies to rHuTNF-alpha completely inhibited rHuTNF-alpha but not rHuTNF-beta or FMLP-induced PMN migration. Combinations of rHuTNF-alpha and rHuTNF-beta (at similar molar concentrations) stimulated PMN migration levels comparable to that obtained with rHuTNF-alpha alone. Checkerboard analyses performed by placing different concentrations of rHuTNF-alpha and rHuTNF-beta above and below polycarbonate filters of microchemotaxis chambers demonstrated that rHuTNF-alpha and rHuTNF-beta stimulated both chemotactic and chemokinetic responses by PMN. Additional studies demonstrated that 1 X 10(-8) mol/L rHuTNF-alpha and 3 X 10(-9) mol/L rHuTNF-beta (which represents 10(4) U/mL of each cytokine) were similar in their ability to induce superoxide production by PMNs; however, at ten- to 100-fold lower molar concentrations (10(3) and 10(2) units), rHuTNF-alpha was significantly more active than rHuTNF-beta. At the doses tested, both cytokines were less active than phorbol myristate acetate at stimulating O2- release. The results demonstrate that rHuTNF- alpha and rHuTNF-beta differ quantitatively but not qualitatively in their effects on PMN functions in vitro and suggest that rHuTNF-beta may be less toxic than rHuTNF-alpha in vivo.     

High-dose cyclophosphamide followed by autografting can improve the outcome of relapsed or resistant non-Hodgkin's lymphomas with involved or hypoplastic bone marrow

We report our experience of high-dose cyclophosphamide (HDCY) followed by high-dose therapy (HDT) and peripheral blood progenitor cell (PBPC) autografting in patients with diffuse, intermediate and high-grade non-Hodgkin's lymphomas who have failed conventional treatment. From 1991 to 1996, 54 consecutive patients pre-treated with a median of two chemotherapy lines entered the study. Eighteen patients (33%) were still responders to conventional chemotherapy (sensitive relapse), and 20 patients (37%) were in partial response (PR) after chemotherapy (CT). Sixteen patients (30%) were resistant to conventional CT either at presentation (non responder) or in relapse (resistant relapse). Thirty-nine patients had bone marrow involved by disease and fifteen had an hypoplastic marrow following conventional treatment. Patients received HDCY (7gr/m2) and G-CSF or GM-CSF in order to collect PBPC. Median collected CD34+ cells was 12.3 x 10(6)/Kg (range 0.7-197). After HDT (BEAM or Melphalan + TBI) 50 patients underwent PBPC autografting. According to intention to treat, 44 (81%) of 54 patients achieved complete remission (CR) (50% after HDCY and 31% after HDT). Procedure related death occurred in 6 patients (11%), one after HDCY and 5 after autografting. Twenty-nine (66%) of 44 patients are still in CR, 7 to 63 months (median 27 months) after the procedure. Three-year probability of survival, disease-free survival and progression-free survival are 63%, 64% and 52% respectively. In conclusion, HDCY is an effective procedure not only in mobilizing PBPC, but also in reducing tumour burden. HDT with PBPC support may further improve the outcome in this category of high-risk non-Hodgkin's lymphomas.     

Bone marrow transplantation for paroxysmal nocturnal hemoglobinuria

BACKGROUND AND OBJECTIVE: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disease of the hemopoietic stem cell (HSC) characterized by intravascular hemolysis and increased risk of venous thrombosis. There are different therapeutic approaches for PNH which do not cure the disease, but can decrease its complications. Allogeneic bone marrow transplantation (BMT) may cure PNH. We reports here our experience of seven PNH patients who underwent allogeneic BMT. DESIGN AND METHODS: Between January 1991 and January 1999 seven patients with PNH, aged 23 to 37, were transplanted with unmanipulated bone marrow from HLA identical siblings. Median time from diagnosis to BMT was 2.5 years (range: 1-16). All patients were transfusion-dependent and had received various treatments before BMT: steroids, vitamins, cyclosporin A (CyA), growth factors. One patient had also been treated with anti-thymocyte globulin. One patient was HbsAg positive and one anti-HCV positive. At the time of BMT the median value of hemoglobin (Hb) was 9 g/dL (range 6.5-11), white blood cells 5&10(9)/L (range: 2.9-7.7), platelets 97&10(9)/L (range: 31-355), LDH: 2726 U/L. The conditioning regimen was cyclophosphamide (160 mg/kg) and busulfan (10-14 mg/kg), followed by unmanipulated bone marrow (median of 5&10(8) cells/kg) and CyA (+MTX in two patients) for prophylaxis of graft-versus-host disease (GvHD). RESULTS: All seven patients are alive, full chimeras, with complete hematologic recovery and no evidence of PNH, at a median follow up of 51 months post-BMT (6-103). Time to achieve a granulocyte count of 0.5&10(9)/L, platelets 30&10(9)/L and Hb 10 g/dL was respectively 16, 19 and 22 days. Acute GvHD was limited or mild in six patients, and severe in one. Chronic GvHD was extensive in two patients. INTERPRETATION AND CONCLUSIONS: This study confirms that HLA identical sibling BMT is an effective therapeutic option for PNH, also in the hemolytic phase of the disease: it also suggests that HBV and HCV infections are not an absolute contraindication.     

Foscarnet prophylaxis of cytomegalovirus infections in patients undergoing allogeneic bone marrow transplantation (BMT): a dose-finding study

This is a dose-finding study using foscarnet for CMV prophylaxis after allogeneic bone marrow transplantation (BMT) in 20 high risk patients (unrelated donors, or T cell depleted, and/or advanced disease). Foscarnet was started on day +1 after BMT and continued until day +100. We explored four different dose levels, patients being entered at the lowest dose level until one patient experiences CMV-reactivation, identified as two consecutive positive CMV antigenemias (CMVAg-emia). The four dose levels expressed as mg/kg/day between days 1 and 30 (induction) and between days 31 and 100 (maintenance) were respectively: dose level I = 60/30 (n = 5); dose level II = 120/60 (n = 4); dose level III = 120/90 (n = 5) and dose level IV = 120/120 (n = 6). All patients showed engraftment: PMN > or =0.5 x 109/l at a median interval of 16, 21, 17, 15 days after BMT, and Plt > or =30x10(9)/l on days 19, 16, 17, 17 respectively. CMVAg-emia was seen in 10 patients at a median interval of 53 days post-BMT (range 33-89) with a median of 10 CMV antigen+ cells (range 1-16). There was a dose effect of foscarnet on CMVAg-emia: respectively 4/5 patients (80%), 2/4 (50%), 3/5 (60%) and 1/6 (18%) at dose levels I, II, III, IV (P = 0.1). CMV disease was seen in 3/9 (33%) at dose levels I, II and 0/11 at dose levels III, IV (P = 0. 07). The median number of CMV antigen-positive cells at diagnosis of CMV infection was different: 13 in dose levels I-II and two in dose levels III-IV (P = 0.01). Increased creatininine was seen in 15 patients with a mean of 1.8 mg% (range 1.5-5.7) and was the cause of discontinuation in nine patients (45%). Renal toxicity was reversible in all nine patients. Overall actuarial TRM at 2 years was 31%: 47% for patients at dose levels I-II and 19% for patients at dose levels III-IV. In conclusion, foscarnet exhibits a dose-dependent prophylactic effect on CMVAg-emia, CMV disease and transplant-related mortality with acceptable and reversible renal toxicity.     

Philadelphia-chromosome-negative peripheral blood stem cells can be mobilized in the early phase of recovery after a myelosuppressive chemotherapy in Philadelphia-chromosome-positive acute lymphoblastic leukaemia

Ten patients in first or second relapse with Philadelphia chromosome acute lymphoblastic leukaemia, ineligible for allogeneic sibling marrow transplantation, were treated with an intensive chemotherapy regimen including idarubicin, intermediate-dose arabinosylcytosine, etoposide and G-CSF. Peripheral blood stem cells were collected by leukapheresis during initial early WBC recovery from chemotherapy-Induced aplasia. In 5/10 patients all metaphases in leukapheresis products were found to be Philadelphia-chromosome-negative and they have been used as autotransplants after conditioning with TBI/etoposide/cyclophosphamide (or idarubicin) and G-CSF. All five patients showed sustained engraftment and one of them is alive and well Philadelphia-chromosome-negative 18 months after transplant. These preliminary results suggest that it is possible to recover Philadelphia-chromosome-negative blood stem cells after intensive chemotherapy, even in advanced patients, and to perform autografting with these cells.     

Monoclonal antibody therapy of human cancer: Taking the HER2 protooncogene to the clinic

The HER2 protooncogene encodes a 185-kDa transmembrane protein (p185^HER2) with extensive homology to the epidermal growth factor (EGF) receptor. Clinical and experimental evidence supports a role for overexpression of the HER2 protooncogene in the progression of human breast, ovarian, and non-small cell lung carcinoma. These data also support the hypothesis that p185^HER2 present on the surface of overexpressing tumor cells may be a good target for receptor-targeted therapeutics. The anti-p185^HER2 murine monoclonal antibody (muMAb) 4D5 is one of over 100 monoclonals that was derived following immunization of mice with cells overexpressing p185^HER2. The monoclonal antibody is directed at the extracellular (ligand binding) domain of this receptor tyrosine kinase and presumably has its effect as a result of modulating receptor function.In vitro assays have shown that muMAb 4D5 can specifically inhibit the growth of tumor cells only when they overexpress the HER2 protooncogene. MuMAb 4D5 has also been shown to enhance the TNF- sensitivity of breast tumor cells that overexpress this protooncogene. Relevant to its clinical application, muMAb 4D5 may enhance the sensitivity of p185^HER2-overexpressing tumor cells to cisplatin, a chemotherapeutic drug often used in the treatment of ovarian cancer.In vivo assays with a nude mouse model have shown that the monoclonal antibody can localize at the tumor site and can inhibit the growth of human tumor xenografts which overexpress p185^HER2. Modulation of p185^HER2 activity by muMAb 4D5 can therefore reverse many of the properties associated with tumor progression mediated by this putative growth factor receptor. Together with the demonstrated activity of muMAb 4D5 in nude mouse models, these results support the clinical application of muMAb 4D5 for therapy of human cancers characterized by the overexpression of p185^HER2.