Intraoperative antegrade lavage and anastomotic healing in acute colonic obstruction

The management of acute distal colonic obstruction remains controversial. The advent of intraoperative colonic irrigation has allowed primary anastomosis to be performed in obstructed bowel. Fifteen patients, with acute distal obstruction due to carcinoma, diverticulitis or sigmoid volvulus were managed by primary resection and anastomosis. There were two leaks (14 percent) and the duration of operation was prolonged. In an experimental study of anastomotic healing after acute obstruction, intraoperative irrigation improved early anastomotic colonic collagen content (P<0.02) and perianastomotic proximal and distal collagen content (P<0.002,P<0.05). Intraoperative lavage is a useful technique to permit safer primary resection and anastomosis in obstructed colon.     

Inhibition of receptor binding and neutralization of bioactivity by anti-erythropoietin monoclonal antibodies

We have generated four high affinity monoclonal antibodies (MoAbs) to recombinant human erythropoietin (EPO). All four MoAbs immunoprecipitate radioiodinated native EPO, and the concentrations of MoAbs required for maximum binding range from 10 nmol/L to 100 nmol/L. Two MoAbs, designated Group I MoAbs, bind to an epitope within the N- terminal 20 amino acids of EPO and also immunoprecipitate sodium dodecyl sulfate (SDS)-denatured EPO. Two other MoAbs (Group II MoAbs) do not immunoprecipitate SDS-denatured EPO and do not bind to any of the eight endo C fragments of EPO. We first used murine erythroleukemia (MEL) cells to test the MoAbs for inhibition of EPO-receptor binding. MEL cells, although unresponsive to EPO, express 760 high affinity receptors for EPO per cell (Kd = 0.24 nmol/L). To assay our MoAbs, MEL cells were grown as monolayers on fibronectin-coated Petri dishes and incubated at 4 degrees C with radioiodinated EPO. Group I MoAbs do not inhibit binding of radioiodinated EPO to the MEL EPO-receptor, but Group II MoAbs do inhibit binding in a dose-dependent manner. We next examined the neutralization of EPO bioactivity by our MoAbs, using EPO- dependent cell line. Only Group II MoAbs inhibit a newly developed EPO- dependent cell growth, demonstrating that inhibition of EPO-receptor binding correlates with neutralization of EPO bioactivity.     

Distinctive gene expression profiles of CD34 cells from patients with myelodysplastic syndrome characterized by specific chromosomal abnormalities

Aneuploidy, especially monosomy 7 and trisomy 8, is a frequent cytogenetic abnormality in the myelodysplastic syndromes (MDSs). Patients with monosomy 7 and trisomy 8 have distinctly different clinical courses, responses to therapy, and survival probabilities. To determine disease-specific molecular characteristics, we analyzed the gene expression pattern in purified CD34 hematopoietic progenitor cells obtained from MDS patients with monosomy 7 and trisomy 8 using Affymetrix GeneChips. Two methods were employed: standard hybridization and a small-sample RNA amplification protocol for the limited amounts of RNA available from individual cases; results were comparable between these 2 techniques. Microarray data were confirmed by gene amplification and flow cytometry using individual patient samples. Genes related to hematopoietic progenitor cell proliferation and blood cell function were dysregulated in CD34 cells of both monosomy 7 and trisomy 8 MDS. In trisomy 8, up-regulated genes were primarily involved in immune and inflammatory responses, and down-regulated genes have been implicated in apoptosis inhibition. CD34 cells in monosomy 7 showed up-regulation of genes inducing leukemia transformation and tumorigenesis and apoptosis and down-regulation of genes controlling cell growth and differentiation. These results imply distinct molecular mechanisms for monosomy 7 and trisomy 8 MDS and implicate specific pathogenic pathways.     

Persistence of host-type hematopoiesis after allogeneic bone marrow transplantation for leukemia is significantly related to the recipient's age and/or the conditioning regimen, but it is not associated with an increased risk of relapse

We investigated the chimerism pattern within flow-sorted peripheral blood- or bone marrow-derived cell populations after allogeneic bone marrow transplantation (BMT) for the treatment of leukemia in children. This study was performed to define the identity of persistent host-type cells, to identify prognostic variables for the persistence of host- type hematopoiesis, and to determine the prognostic significance of the chimerism pattern on the duration of the leukemia-free interval, the overall survival, and the leukemia-free survival. The patients received either HLA-identical non-T-cell-depleted (n = 46) or HLA nonidentical T- cell-depleted (n = 7) BMT. In the peripheral blood, the children showed either stable mixed chimerism (SMC; ie, persistent host-type hematopoiesis; n = 14), (transient) mixed T-lymphoid chimerism (MTLC; n = 9), or complete chimerism (CC; n = 30). In the bone marrow, only donor-type cells were found in children with either CC (n = 8) or MTLC (n = 2), and a mixture of donor- and recipient-type cells was found in children with SMC (n = 7). The persistence of host-type hematopoiesis (SMC) was significantly related to a lower age of the recipient, the type of conditioning regimen, a lower total body irradiation dose, T- cell depletion of the bone marrow graft, and the use of cyclosporine A for acute graft-versus-host disease prophylaxis. No significant differences were found between patients with (SMC) or without (CC/MTLC) persistent host-type hematopoiesis with respect to the duration of the leukemia-free interval, the overall survival, or the leukemia-free survival. We conclude that ablation of host-type hematopoiesis is not compulsory for long-term leukemia-free survival after allogeneic BMT for various hematologic malignancies.     

Complement proteins C5b-9 stimulate procoagulant activity through platelet prothrombinase

The capacity of platelets treated with nonlytic concentrations of the C5b-9 proteins to catalyze prothrombin activation and thereby trigger clot formation has been investigated. When suspended in the presence of exogenous factors Xa and Va, gel-filtered platelets treated with purified C5b-9 proteins catalyzed prothrombin to thrombin conversion at rates up to tenfold above controls, and exceeded by up to fourfold the prothrombinase activity observed for thrombin-stimulated platelets. In the absence of added factor Va, C5b-9 assembly on the platelet surface significantly shortened the lag period before prothrombinase expression that was observed for untreated platelets and increased the maximum catalytic rate of thrombin formation. A comparison with other platelet stimuli revealed that the C5b-9-induced activation of platelet prothrombinase closely paralleled the effects mediated by calcium ionophore A23187. Our data suggest that the C5b-9 proteins promote the release of platelet factor V and the assembly of the prothrombinase complex, thereby potentiating the effects of thrombin on the activation of prothrombinase. Membrane assembly of the C5b-9 proteins was also observed to markedly accelerate the rate of platelet-catalyzed plasma clotting, suggesting a direct link between C5b-9-mediated prothrombinase activation and procoagulant activity accompanying immunologic damage to the platelet.     

Timed sequential chemotherapy of cytoxan-refractory multiple myeloma with cytoxan and adriamycin based on induced tumor proliferation

Malignant plasma cell proliferation and induced humoral stimulatory activity (HSA) occur in vivo at a predictable time following drug administration. Sequential sera from 11 patients with poor-risk multiple myeloma (MM) undergoing treatment with Cytoxan (CY) 2400 mq/sq m were assayed for their in vitro effects on malignant bone marrow plasma cell tritiated thymidine (3HTdR) incorporation. Peak HSA was detected day 9 following CY. Sequential changes in marrow malignant plasma cell 3HTdR-labeling indices (LI) paralleled changes in serum activity, with peak LI occurring at the time of peak HS. An in vitro model of chemotherapy demonstrated that malignant plasma cell proliferation was enhanced by HSA, as determined by 3HTdR incorporation assay, 3HTdR LI, and tumor cells counts, and that stimulated plasma cells were more sensitive to cytotoxic effects of adriamycin (ADR) than were cells cultured in autologous pretreatment serum. Based on these studies, we designed a clinical trial to treat 12 CY-refractory poor- risk patients with MM in which ADR (60 mg/sq m) was administered at the time of peak HSA and residual tumor cell LI (day 9) following initial CY, 2400 mg/m (CY1ADR9). Eight of 12 (67%) responded to timed sequential chemotherapy with a greater than 50% decrement in monoclonal protein marker and a median survival projected to be greater than 8 mo duration (range 4-21+ mo). These clinical results using timed sequential CY1ADR9 compare favorably with results obtained using ADR in nonsequential chemotherapeutic regimens.     

Pharmacologic marrow purging in murine T cell leukemia

Deoxycoformycin in combination with deoxyadenosine was used to purge 6C3HED malignant T cells from murine marrow in vitro. Adenosine deaminase activity of 6C3HED cells was ablated by incubation with 10(- 6) mol/L deoxycoformycin (dCF). During a 12-hour incubation with 10(-6) mol/L dCF and 10(-4) mol/L deoxyadenosine, tumor cells sequentially accumulated dATP, became depleted of NAD followed by ATP, then died. More than 5 logs of 6C3HED cells were killed as measured by survival of mice injected with treated tumor cells. Identical incubation of 5 x 10(6) marrow cells did not interfere with rescue of syngeneic lethally irradiated mice. Long-term survival was demonstrated in 12 of 14 mice that received marrow that had been contaminated with 5% 6C3HED cells, incubated with deoxycoformycin and deoxyadenosine, then used to rescue lethally irradiated mice. This murine model provides information not available from in vitro assays and may be useful in the development of strategies to purge malignant T cells from marrow.     

Evidence for a multistep pathogenesis of chronic myelogenous leukemia

To study the relationship of the Philadelphia chromosome (Ph1) to the pathogenesis of chronic myelogenous leukemia, multiple B-lymphoid cell lines were established from a patient with Ph1-positive leukemia who was heterozygous for the X-chromosome-linked enzyme glucose-6-phosphate dehydrogenase. Both A and B types of enzyme were found in a 1:1 proportion in normal tissues, but 45 of 63 (71%) Ph1-negative B- lymphoid cells lines derived from this patient showed only the single glucose-6-phosphate dehydrogenase (type B) found in the Ph1-positive leukemic clone. Furthermore, 8 of 33 analyzable lines with B-type enzyme had chromosomal aberrations compared to 0 of 14 lines with A- type glucose-6-phosphate dehydrogenase. These results provide evidence for the suggestion that some cells of the abnormal clone do not express the Ph1 abnormality. Thus, acquisition of Ph1 may not be a sufficient cause for the disease. It is possible that at least two steps are involved in the pathogenesis of Ph1-positive chronic myelogenous leukemia, one causing abnormal proliferation of a clone of pluripotent hematopoietic stem cells and the other inducing Ph1 in descendants of these progenitors.