Experimental amebiasis: immunohistochemical study of immune cell populations.

Distribution of immune cell populations was studied in a C3H/HeJ mouse model of intestinal amebiasis from 5 to 60 days post-inoculation with Entamoeba histolytica, using immunoperoxidase techniques. At various time intervals, the ceca from mice were fixed in 10% formalin, dehydrated, embedded and sectioned at 5 microm. Sections were incubated with conjugated peroxidase-labelled antibodies to mouse IgA, IgM, and IgG. Color was developed with 3, 3'-diaminobenzidine tetrahydrochloride (DAB)/H2O2 solution. CD3, CD4, and CD8 cells, as well as neutrophils were detected by reacting with biotin-conjugated anti-mouse CD3, CD4, CD8, and CD11 monoclonal antibodies, followed by their incubation with avidin-peroxidase and color development with DAB/H2O2 solution. Erythrocin B and toluidine blue were used to stain eosinophils and Mast cells, respectively. It was observed that the IgA+ plasma cell was the dominating immune cell present in the mucosa, although eosinophils, neutrophils, CD3+, CD4+, CD8+, IgM+, IgG+ cells and Mast cells were also seen. Results of this study suggest that infiltration of immune cells at the mucosal surface during intestinal amebiasis might be important in the defense against this parasite.

Studies of the function and structure of in vitro propagated human granulocytes.

In our study, hematopoietic progenitor cells isolated from human umbilical cord blood were grown in an in vitro liquid culture system using the recombinant colony stimulating factors IL-3 plus granulocyte-macrophage colony-stimulatory factor (GM-CSF) or IL-3 plus granulocyte colony stimulating factor (G-CSF). The morphology and function of the cells produced were then studied, and it was demonstrated that continuous exposure to IL-3 plus GM-CSF produced predominantly eosinophilic granulocytes, whereas IL-3 plus G-CSF produced neutrophilic granulocytes. Cells from IL-3/GM-CSF cultures showed progressively increasing oxygen metabolism and locomotive capabilities over time, which became equivalent to peripheral blood neutrophils at wk 4 and 3, respectively. Phagocytic activity of these cells was poor. IL-3/G-CSF cultures produced cells with progressive increases in oxygen metabolism, locomotion, and phagocytosis. These functions never became equivalent to those of peripheral blood neutrophils. Flow cytometric analysis of IL-3/G grown cells showed that they expressed CD11b on their surfaces and that surface expression increased 2-fold after secondary granule secretagogue exposure. Ultrastructurally, the eosinophilic granulocyte nature of the IL-3/GM grown cells was confirmed by immunogold-lectin staining and IL-3/G grown cells were shown to contain antigenic myeloperoxidase. The data demonstrate that human umbilical cord blood mononuclear cells can be used to propagate granulocytes in vitro, that the types of granulocytes produced in this culture system depend on the growth factors used, that the cells produced in vitro develop several of the functional characteristics of peripheral blood granulocytes, and that ultrastructural details of developing human granulocytes can be carefully examined in this model system.

Hematopoiesis in limiting dilution cultures: influence of cytokines on human hematopoietic progenitor cells.

We have modified a limiting dilution liquid culture assay, used to quantify hematopoietic progenitor frequency, to simultaneously assess cellular proliferation and differentiation. The frequency of colonies from cord blood obtained with recombinant human interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in combination was comparable to cultures with IL-3 alone. However, IL-3 and GM-CSF in combination were synergistic for higher granulocyte proliferation than IL-3 alone, indicating that enhanced granulocyte production occurred via action of GM-CSF on progenitor cell populations already stimulated into proliferation by IL-3. Peak proliferation was evident at 4 weeks in culture, with metamyelocytes predominating; at 6 weeks, mostly neutrophils and eosinophils were present, and eosinophils were more numerous in cultures with IL-3. Increasing concentrations of erythropoietin (epo) in liquid culture with IL-3 or GM-CSF decreased absolute granulocyte yield while stimulating erythroid proliferation. The influence of epo on lineage morphology for cells plated in methylcellulose was markedly less evident by comparison, arguing against an inductive effect of epo to shift progenitor cell lineage. This liquid culture methodology may be a useful tool for preclinical screening of cytokines on human hematopoietic progenitor cells.

High dose cyclophosphamide, BCNU and VP-16 with autologous blood stem cell support for refractory multiple myeloma.

Eleven patients with advanced multiple myeloma refractory to standard doses of alkylating agents and salvage therapy with vincristine, adriamycin and dexamethasone (VAD) were treated with high dose cyclophosphamide, BCNU and VP-16 (CBV) with autologous blood stem cell support. Seven patients had marked marrow plasmacytosis (greater than 30%) and four had extensive pelvic bone disease precluding autologous marrow harvest. Four patients responded with a median remission duration of 7 months. Recovery of granulocytes and platelets occurred promptly in 10 evaluable patients with complete hematologic recovery. Autologous blood stem cells can provide safe and effective support for high dose CBV treatment of myeloma patients with extensive marrow plasmacytosis. The short remissions call for better cytoreductive regimens with consideration for earlier use when the myeloma may be more responsive to therapy.

Circulating myeloid progenitor cell kinetics during hematologic recovery from chemotherapy and subsequent recombinant human granulocyte-macrophage colony-stimulating factor administration.

Hematopoietic recovery from chemotherapy may be associated with an increase in circulating myeloid progenitor cell concentration (CFU-GM); these cells may be harvested by apheresis and used for autologous transplantation after high-dose cytoreductive therapy. Not all patients will demonstrate this increase, possibly due to damage to the stem cell compartment from prior chemoradiotherapy. Elevated circulating CFU-GM has also been reported in patients after short-term administration of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF); whether elevation would persist during longer duration is unknown. We measured circulating CFU-GM (by both limiting dilution in liquid culture and colony formation in semisolid media) in patients with sarcoma who began infusion of rhGM-CSF during recovery from chemotherapy. Patients with elevated circulating CFU-GM did not sustain these levels during subsequent rhGM-CSF infusion. By contrast, patients without rebound elevation of circulating CFU-GM following chemotherapy recovery did increase CFU-GM levels with rhGM-CSF administration. The proportion of marrow CFU-GM in cell cycle during chemotherapy recovery was elevated in both patient groups and remained elevated with rhGM-CSF administration. Both marrow and peripheral blood limiting dilution assays demonstrated linear growth kinetics, indicating a direct effect of the in vitro growth factor (also rhGM-CSF) on progenitor cells without excessive influence or dependence on accessory cells in culture. The use of rhGM-CSF to restore circulating CFU-GM for apheresis during recovery in patients lacking such elevation merits further study.

Analysis of peripheral blood granulocyte-macrophage colony growth by limiting dilution assay.

Analysis of myeloid progenitor cells in the peripheral blood (peripheral blood colony-forming unit granulocyte-macrophage; PBCFU-GM) is limited by their low frequency and by the presence of inhibitory cell populations. These factors limit the study of cytokines and cellular influences on PBCFU-GM in semisolid media assays and complicate the interpretation of data. We have developed a limiting dilution assay (LDA) in liquid culture for PBCFU-GM that allows evaluation of inhibitory or accessory effects of other cell populations and estimation of progenitor cell frequency. Using this system we have examined the inhibitory effect of autologous monocytes on in vitro colony growth. After monocyte depletion by counterflow centrifugal elutriation and adherence, colony growth with recombinant human granulocyte-macrophage colony-stimulating factor was linear over a wide range of cell densities, indicating a direct proliferative effect on circulating myeloid progenitor cells. Simultaneous PBCFU-GM assays in agar demonstrated monocyte inhibition but did not afford reliable interpretation of either progenitor frequency or linear growth kinetics in a statistically verifiable fashion. LDA in liquid culture may be a useful tool to study the effects of various cytokines and cell populations on PBCFU-GM in vitro and in vivo.

Analysis of risk factors for fatal hemorrhage during induction therapy of patients with acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL), an uncommon subtype of acute myelogenous leukemia (AML), has a high incidence of early hemorrhagic death during induction therapy; however, patients with APL surviving induction and obtaining complete remission have a good prognosis, with a longer remission duration than other subtypes of AML. We sought to determine if changes in selected clinical and laboratory coagulation parameters during induction therapy were significantly associated with fatal hemorrhage in that week, or were predictive of fatal hemorrhage in the following week. The first six weeks of induction therapy were studied in 65 patients, during which time 23 patients (35%) died from hemorrhagic complications. Time intervals analyzed were week 1, week 2, weeks 3 and 4, and weeks 5 and 6. There was no significant difference in the prevalence of severe hypofibrinogenemia (less than 150 mg%), prolongation of the prothrombin time (greater than 14 s), or presence of infection between patients who sustained fatal hemorrhage versus those who did not, for any given time period. The most significant parameter was the inability to obtain a posttransfusion platelet count of over 40,000/microliters in at least one half of the platelet transfusions administered in that week (p less than 0.001). This last parameter was the only variable that was also significantly associated with an increased risk of fatal hemorrhage for the following week (p less than 0.005). Clinical trials investigating management of coagulopathy in APL should evaluate the effect of therapy on response to platelet transfusion as well as on other laboratory parameters.

Acute myeloblastic leukemia with hyperleukocytosis: risk factors for early mortality in induction.

Eighty-five patients with acute myeloblastic leukemia (AML) presenting with hyperleukocytosis (HL) were analyzed to assess morbidity and mortality in early induction. Patients who failed to achieve remission were older and more often had pulmonary leukostasis (62% vs 23%, p = .01) and hepatomegaly (54% vs 31%, p = .06) at presentation. Thirty-seven patients (44%) did not achieve complete remission (CR); 17 (54%) died early in induction therapy, 11 directly as a result of pulmonary hemorrhage with respiratory failure, while 5 had both pulmonary hemorrhage with respiratory failure and CNS hemorrhage. Early death patients were older and more often had pulmonary leukostasis (88% vs 29%, p less than .0001), hepatomegaly (71% vs 34%, p = .01), hyperbilirubinemia (60% vs 16%, p = .01) and hypofibrinogenemia (47% vs 12%, p less than .01) at presentation. Primarily for technical reasons, preinduction leukapheresis was not employed as often in this very-high-risk group as in other patients (56% vs 82%, respectively). Thus, sufficient heterogeneity exists in patients presenting with HL to define a subset of patients at particularly high risk for early mortality. Preinduction leukapheresis applied in a prospective controlled fashion should be evaluated to assess if such treatment may decrease early mortality in this group.

Clinical trial of plasma perfusion over immobilized staphylococcal protein A in metastatic breast cancer.

Nineteen patients with metastatic breast cancer refractory to conventional therapy were treated with plasma perfusion over 200 mg of staphylococcal Protein A immobilized on a silica matrix. Fever and chills (33%), pain at the site of tumor (18%), and dyspnea (16%) were the most frequent toxic effects encountered. Four patients (21%) developed a disseminated rash which necessitated cessation of treatment. Of 16 patients evaluable for response, one achieved a minor response of chest wall disease and two had no change in hepatic metastases for 4 and 5 months' duration. Potential mechanisms of antitumor effect are discussed.

Correlative patterns of neutrophil and platelet counts during very early remission of acute myeloblastic leukemia.

Patterns of recovery for peripheral blood neutrophils and platelets were analyzed in 65 patients with acute myeloblastic leukemia (AML) undergoing their first course of induction chemotherapy. Specifically, for every patient linear correlation coefficients (r values) were computed for neutrophils versus platelet counts every three days from Day 12 to 27 of therapy. Correlations were designated as "significant" if r greater than or equal to 0.82 (p less than or equal to 0.05). Patients who would enter complete remission with the first course of therapy were significantly more likely to have significant neutrophil-platelet correlations in the recovery phase than patients who did not achieve remission with the first course or who would have resistant disease (76% versus 20% and 76% versus 15%, respectively, both p less than 0.001). Although these data are preliminary and retrospective they nonetheless demonstrate a trend that may prove useful for patient monitoring if proven correct by formal studies. A prospective investigation examining neutrophil and platelet correlations within a defined period of early hematologic recovery is indicated to assess if such parameters have early prognostic value for the prediction of remission in individual patients receiving induction therapy for AML.

Pneumonia with Cunninghamella species in patients with hematologic malignancies. A case report and review of the literature.

A patient with chronic myelogenous leukemia in blastic crisis who developed pulmonary and systemic infection with Cunninghamella species is described. The emerging role of this newly recognized pathogen in immunocompromised patients is discussed.

Reversible bilateral lateral rectus muscle palsy associated with high-dose cytosine arabinoside and mitoxantrone therapy.

A patient with acute myelogenous leukemia in relapse developed reversible bilateral lateral rectus muscle palsy and cerebellar dysfunction after receiving chemotherapy with high-dose cytosine arabinoside and mitoxantrone. The differential diagnosis of this syndrome is reviewed.