A simple qualitive procedure for the detection of chloroquine in urine for use in clinical analytical toxicology in resource poor settings.

Objective: To develop and validate a simple procedure for the qualitative determination of chloroquine in urine with potential for use in developing countries lacking sophisticated analytical equipment and expensive reagents. Design: This was a laboratory based study making use of which combines a colorimetric test, Dill-Glazko's test, and UV/Visible absorbance spectrometry to confirm the presence of chloroquine. The spectrophotometric method was cross validated with the standard Baselt's method for quantification of chloroquine in biological fluids. Setting: Pharmacology laboratory at the Department of Clinical Pharmacology, College of Health Sciences, University of Zimbabwe. Main Outcome Measures: Recovery of the methods was assessed by comparing the peak absorbances and the resolution of the peaks at 329nm and 343nm. Sensitivity and specificity was determined by analysing in a blinded manner. The limits of detection of both the Dill-Glazko's test and the confirmatory test was determined. Results: In the prevalidation procedures increasing the volume of the ethylacetate and the volume of the lower aqueous layer extracted was found to increase the recovery of the confirmatory test. There was a significant difference between both the peak absorbances and the peak resolution for the two methods (p<0.0001). The confirmatory test had a sensitivity of 90% and a specificity of 100%, whereas the Baselt's method had a sensitivity of 83.3% and a specificity of 96.7%. The limit of detection of the Dill-Glazko's test was 15mg/Land that of the confirmatory test was 5mg/L. Conclusions: The confirmatory test had better recovery and was more sensitivity compared with the Baselt's method. The limit of detection of the combination method (Dill-Glazko's plus confirmatory test) was 15mg/L. The combination test showed appreciable sensitivity to be suitable for application to clinical toxicology.

Ovarian recovery after stem cell transplantation.

Autologous or allogeneic SCT with conventional conditioning (chemotherapy with or without irradiation) has emerged as an effective and potentially curative therapy in patients with hematologic malignancies and in other selected solid tumors; however, several patients experience significant early and delayed side effects, including long-term endocrine imbalance and infertility. In spite of several reproductive recovery and pregnancy reports published in the oncology literature, review of medical literature reveals a paucity of comparable information in the SCT field. We report here four cases of ovarian recovery in patients who received hormonal replacement therapy after diagnosis of primary ovarian failure due to high-dose chemotherapy and SCT.

Heterogeneity of clonogenic cells in acute myeloblastic leukemia.

The expression of differentiation-associated surface antigens by the clonogenic leukemic cells from 20 patients with acute myeloblastic leukemia (AML) was studied with a panel of seven cytotoxic monoclonal antibodies (anti-Ia, -MY9, -PM-81, -AML-2-23, -Mol, -Mo2, and -MY3). The surface antigen phenotypes of the clonogenic cells were compared with the phenotypes of the whole leukemic cell population, and with the phenotypes of normal hematopoietic progenitor cells. In each case the clonogenic leukemic cells were found within a distinct subpopulation that was less "differentiated" than the total cell population. Clonogenic leukemic cells from different patients could be divided into three phenotype groups. In the first group (7 of 20 cases), the clonogenic cells expressed surface antigens characteristic of the normal multipotent colony-forming cell (Ia, MY9). These cases tended to have "undifferentiated" (FAB M1) morphology, and the total cell population generally lacked expression of "late" monocyte antigens such as MY3 and Mo2. A second group (seven cases) of clonogenic cells expressed surface antigens characteristic of an "early" (day 14) colony-forming unit granulocyte-monocyte (CFU-GM), and a third group (six cases) was characteristic of a "late" (day 7) CFU-GM. The cases in these latter two groups tended to have myelomonocytic (FAB M4) morphology and to express monocyte surface antigens. These results suggest that the clonogenic cells are a distinct subpopulation in all cases of AML, and may be derived from normal hematopoietic progenitor cells at multiple points in the differentiation pathway. The results further support the possibility that selected monoclonal antibodies have the potential to purge leukemic clonogenic cells from bone marrow in some AML patients without eliminating critical normal progenitor cells.

Gamma interferon induces monocytoid differentiation in the HL-60 cell line.

We investigated the ability of purified, recombinant DNA-derived interferons (IFN) to induce phenotypic changes in cells of the HL-60 promyelocytic leukemia cell line. Changes in cell surface markers detected by monoclonal antibodies as well as morphologic, histochemical, and functional changes were monitored. We found that gamma-IFN, but not alpha- or beta-IFN, induced the expression of antigens characteristic of monocytes and granulocytes (AML-2-23, 63D3, and 61D3), as well as changes in morphology consistent with monocytoid differentiation. These included induction of alpha-naphthyl acetate esterase, increased cell size, and a decrease in azurophilic granules. The gamma-IFN dose dependency and time course of the effect on antigen expression suggest that de novo protein synthesis was induced by gamma-IFN. The activity of gamma-IFN and of mixed-lymphocyte culture supernatant was blocked by a monoclonal antibody to gamma-IFN. Significant augmentation in the ability of the HL-60 cells to mediate antibody-dependent cellular cytotoxicity was induced by gamma-IFN. These findings suggest that gamma-IFN plays a role in the regulation of hematopoiesis.

Cytotoxic activity of gemtuzumab ozogamicin (Mylotarg) in acute myeloid leukemia correlates with the expression of protein kinase Syk.

Acute myeloid leukemia (AML) cells express the cell surface antigen CD33 that, upon ligation with a monoclonal antibody (mAb), is a downregulator of cell growth in a Syk-dependent manner. An anti-CD33 mAb coupled to a toxin, gemtuzumab ozogamicin (GO), is used for the treatment of AML (Mylotarg). Therefore, we investigated whether the response of AML cells to GO treatment also depends on Syk expression. Forty primary AML samples (25 Syk-positive and 15 Syk-negative) were tested for their response to the anti-proliferative effects of GO and unmodified anti-CD33 mAb. A correlation between Syk expression and the response of leukemia cells to GO and anti-CD33 mAb was found. 'Blocking' of Syk by small interfering RNA resulted in unresponsiveness of AML cells to both GO and anti-CD33 mAb-mediated cytotoxicity. Syk upregulation by the de-methylating agent 5-azacytidine (5-aza) induced re-expression of Syk in some cases, resulting in enhanced GO and anti-CD33-mediated inhibition of leukemia cell growth. Thus, the cytotoxicity of both GO and anti-CD33 in primary AML samples was associated with Syk expression. 5-Aza restored Syk and increased the sensitivity of originally Syk-negative, non-responsive cells to CD33 ligation to levels of Syk-positive cells. These data have clinical significance for predicting response to GO and designing clinical trials.

Monoclonal antibodies reactive with small cell carcinoma of the lung.

Murine monoclonal antibodies (MoAb) reactive with antigens associated with small cell carcinoma of the lung (SCCL) were prepared and partially characterized. Four were selected for further study on the basis of their lack of reactivity with normal leukocytes and erythrocytes. These MoAb, designated SCCL-41, SCCL-114, SCCL-124, and SCCL-175, are all IgM immunoglobulins. The binding of these MoAb to patient-derived SCCL tumor cells, SCCL cell lines, and non-SCCL cell lines was studied by indirect immunofluorescence and flow cytometry. Considerable heterogeneity in the expression of these cell surface antigens was noted among both the patient-derived tumor cells and the SCCL cell lines. One of the MoAb, SCCL-175, reacted with 7 of 7 patient-derived tumor cell samples and 9 of 10 SCCL cell lines. None of the antigens defined by these MoAb were expressed on non-SCCL lung tumor cell lines. SCCL-175 reacted with cells from both a choriocarcinoma and a colon carcinoma cell line, whereas the other 3 MoAb were unreactive with these and several other tumor cell lines. These MoAb may be useful in the diagnosis and subclassification of SCCL tumors.

ras oncogene activation and occupational exposures in acute myeloid leukemia.

BACKGROUND: Epidemiologic studies of acute myeloid leukemias (AMLs) show small increases in risk of disease associated with certain occupations and chemical exposures. PURPOSE: This study was designed to determine whether the presence of mutationally activated ras oncogenes in AML are associated with occupational and chemical exposures. METHODS: We interviewed 62 patients with newly diagnosed AML (or their next-of-kin), all of whom were enrolled in a national multicenter clinical trial, and 630 healthy control subjects. DNA extracted from patients' pretreatment bone marrow samples was amplified by using the polymerase chain reaction and probed with allele-specific oligonucleotides for activating point mutations at the 12th, 13th, and 61st codons of three protooncogenes: H-ras (also known as HRAS), K-ras (also known as KRAS2), and N-ras (also known as NRAS). RESULTS: Patients with ras mutation-positive AML had a higher frequency (six of 10 patients) of working 5 or more years in an a priori high-risk occupation than did patients with ras mutation-negative AML (eight of 52; odds ratio [OR] = 6.8; 95% confidence interval [CI] = 1.3-36). Patients with ras mutation-positive AML were more likely than patients with ras mutation-negative AML to have breathed chemical vapor on the job (OR = 9.1; 95% CI = 1.3-64) or to have had skin contact with chemicals (OR = 6.9; 95% CI = 1.3-37). When ras-positive patients were compared with healthy control subjects, the ORs for occupation and occupational exposures remained elevated, while patients with ras mutation-negative AML showed no increased risk when compared with control subjects. CONCLUSION: Activation of ras proto-oncogenes may identify an etiologic subgroup of AML caused by occupation and chemical exposure. IMPLICATION: Disease etiology may be better understood if epidemiologic measures of exposure are integrated with molecular assays of the genetic defects responsible for cancer initiation and promotion.

Elimination of small cell carcinoma of the lung from human bone marrow by monoclonal antibodies and immunomagnetic beads.

The majority of patients with small cell carcinoma of the lung (SCCL) have bone marrow involvement detected by monoclonal antibodies (mAb). High dose chemotherapy followed by autologous bone marrow transplantation may improve treatment results for patients with SCCL, but the bone marrow may need to be purged of contaminating tumor cells. This study investigates the reactivity of a panel of mAb with two SCCL cell lines and normal bone marrow and the ability of the mAb and immunomagnetic beads to eliminate the SCCL cells from a mixture of 90% normal bone marrow cells and 10% SCCL cells. The mAb and immunomagnetic beads removed 4 to 5 log of SCCL cells in the model system. The immunomagnetic separation did not significantly adversely affect normal hematopoietic progenitor cells, as determined by bone marrow colony-forming units. The results suggest that the mAb and immunomagnetic beads could safely and effectively separate SCCL cells from the bone marrow for autologous bone marrow transplantation following high dose chemotherapy.

Expression of antigens associated with small cell carcinoma of the lung on hematopoietic progenitor cells.

We have previously described a panel of four monoclonal antibodies (MoAbs) reactive with antigens expressed on tumor cells from small cell carcinoma of the lung (SCCL). These IgM MoAbs are cytotoxic to SCCL cells in the presence of complement and thus have potential as reagents for the removal of SCCL cells contaminating bone marrow autografts. Therefore, we examined the cytotoxicity of these MoAbs against normal hematopoietic progenitor cells as a preliminary step toward their use in clinical trials. In this paper we report the results of treating normal bone marrow and peripheral blood mononuclear cells with the four IgM MoAbs, as well as an IgG2a MoAb that we have recently prepared against an SCCL cell line, DMS 406. Peripheral blood and bone marrow mononuclear cells were treated with the MoAbs alone or in combination, in the presence of rabbit complement, and then plated into colony-forming assays. Notably, only one MoAb, SCCL-1, had any demonstrable cytotoxicity against progenitor cells. This toxicity was limited to bone marrow burst-forming unit-erythroid and all classes of blood progenitor cells. A MoAb cocktail containing a combination of either four or five MoAbs + complement spared most marrow progenitor cells. These studies extend the base of information regarding the expression of SCCL-associated antigens on hematopoietic cells and indicate that selected MoAbs may be used safely for the removal of SCCL cells from autografts by complement-dependent lysis or other means.

A novel monoclonal antibody, SCCL 175, with specificity for small cell neuroendocrine carcinoma of the lung.

The murine monoclonal antibody SCCL 175, which is one of several monoclonal antibodies directed against small cell neuroendocrine carcinoma developed by one of us (E.D.B.), was studied for its immunohistochemical reactivity against normal human tissues and a spectrum of bronchopulmonary and metastatic carcinomas using the avidin-biotin complex technique. SCCL 175 reacted with 40 of 44 small cell carcinomas including both primary and metastatic sites and was distributed both on the cell surface and intracytoplasmically. Staining was seen in fresh frozen tissues, cytology preparations, and in a limited number of paraffin-embedded tissue sections after trypsin pretreatment. It was nonreactive with all non-small cell lung carcinomas, neuroendocrine carcinomas from other primary sites, and nonpulmonary carcinomata studied to date. Its distribution in normal adult human tissues was limited to some hypothalamic neurons and the apical membranes of renal proximal tubular epithelium. Cytotrophoblastic and syncytotrophoblastic cells from placental tissue demonstrated variable SCCL 175 immunoreactivity. Of choriocarcinomas studied, one of three demonstrated focal staining. These findings demonstrate the diagnostic utility of SCCL 175 in phenotyping small cell carcinoma of lung, and its specificity suggests a potential role in the therapy of this disease.

Expression of myeloid and major histocompatibility antigens on small cell carcinoma of the lung cell lines analyzed by cytofluorography: modulation by gamma-interferon.

Recent reports have described the expression of myeloid cell surface antigens on cells of small cell carcinoma of the lung (SCCL). In order to confirm and extend these findings, we have examined by cytofluorography a large panel of well-characterized cell lines derived from SCCL tumors for the expression of several myeloid cell-associated antigens, some of which were not examined in previous reports. In addition, we have studied the expression of Classes I and II HLA antigens on these SCCL cell lines. Finally, we examined the effect of gamma-interferon on the expression of several cell surface markers and on proliferation of SCCL cells. We have found that several SCCL cell lines expressed a Mr 55,000 polypeptide antigen, My23, previously found only on monocytes and monocytic leukemia cells. In addition, the cell lines studied expressed another antigen, defined by monoclonal antibody AML-1-99, which is associated with monocytes and hematopoietic stem cells. We confirmed previous studies that the Leu-7 antigenic determinant is expressed on SCCL cells but observed only minimal or absent binding of monoclonal antibody OKM1 to most cell lines. Class I HLA antigens were present on eight of nine lines examined while Class II HLA was expressed on three of nine lines. Gamma-Interferon decreased the proliferative rate of all lines examined. However, this lymphokine was capable of inducing Class I HLA on several lines. The effect of gamma-interferon on other cell surface antigens was variable. These studies confirm that some myeloid cell-associated antigens are expressed on cultured SCCL lines and, additionally, show that their expression can be modulated by immune interferon. Determining the significance of finding myeloid cell-associated antigens on SCCL cells will require further study.

Reactivity of anti-CD15 monoclonal antibody PM-81 with breast cancer and elimination of breast cancer cells from human bone marrow by PM-81 and immunomagnetic beads.

The CD15 carbohydrate antigen, lacto-N-fucopentaose III is expressed on a variety of human cancer cells including acute myeloid leukemia, small cell carcinoma of the lung, and colorectal carcinomas. We have found that cells from breast cancer cell lines and patient-derived tissue are strongly CD15 positive, as seen by binding to the PM-81 monoclonal antibody. In this report we show that monoclonal antibody PM-81 and immunomagnetic beads can remove breast cancer cells from bone marrow and thus be used as "purging" agents for autologous bone marrow transplantation. PM-81 and immunomagnetic beads removed up to 3 log of SK-BR-3 and MCF7 breast carcinoma cell line cells while minimally affecting normal hematopoietic progenitor cells. This technique may be useful for purging marrow for autologous bone marrow transplantation in breast cancer.

Human dendritic cells genetically engineered to express high levels of the human epithelial tumor antigen mucin (MUC-1).

We have achieved stable high-level expression of a human tumor antigen, epithelial cell mucin (MUC-1), on human dendritic cells (DCs) by retroviral transduction of CD34+ progenitor cells and their subsequent cytokine-induced differentiation into DCs. The process of retroviral transduction did not alter the growth or differentiation of DCs from CD34+ progenitor cells. Immunofluorescence and electron microscopy studies revealed that the expression of mucin was limited to the body of the DCs and was excluded from the cytoplasmic veils of the DCs. Furthermore, the expression of mucin on DCs was similar, if not identical, to the nonpolarized expression of mucin found on carcinoma cells. In functional studies, the MUC-1(+)-transduced DCs were potent stimulators of allogeneic CD4+ T cells and, in fact, were superior to MUC-1- DCs. Thus, MUC-1+ DCs are expected to be a valuable tool in the immunotherapeutic treatment of patients with tumors that express MUC-1.

Increased expression of the differentiation-defective granulocyte colony-stimulating factor receptor mRNA isoform in acute myelogenous leukemia.

Granulocyte colony-stimulating factor (G-CSF) critically affects all stages of granulopoiesis by activating a signaling cascade initiated by dimerization of its receptor (G-CSFR). Five human G-CSFR isoforms have been identified (classes I-V). A quantitative polymerase chain reaction (Q-PCR) technique was used to examine the expression of these five isoforms in normal and leukemic myeloid cells. We demonstrated that neutrophils expressed predominantly the class I isoform and low levels of class IV isoform (IV/I = 0.037 +/- 0.005). No expression of the class II, class III, or class V isoform was detected. In contrast, all AML cell lines and acute myelogenous leukemia (AML) patient samples expressed increased relative amounts of the class IV isoform (IV/I = 0.047-0.350). When compared to normal immature myeloid cells, as represented by the CD34+ fraction of adult bone marrow (ABM) cells, three of eight AML cell lines and three of six AML patient samples expressed significantly increased levels of the class IV isoform relative to class I. This suggests that the increase in the relative expression of the class IV isoform seen in a considerable portion of AML cell samples is related to their leukemic phenotype. Given the inability of the class IV G-CSFR to drive myeloid maturation, the relative increase in class IV expression in AML cells may contribute to their aberrant response to G-CSF.

Monocyte-mediated lysis of acute myeloid leukemia cells in the presence of the bispecific antibody 251 x 22 (anti-CD33 x anti-CD64).

Immunotherapy using bispecific antibodies (BsAb) to direct immune effector cells toward target tumor cells has been shown to be effective in a number of studies. Several immune trigger molecules have been characterized. Among them, FcgammaRI appears to play an important role in antibody-dependent cellular cytotoxicity. It is expressed mainly on monocytes, macrophages, and neutrophils under certain clinical situations. The expression of FcgammaRI can be regulated by a variety of cytokines, primarily by IFN-gamma. Recent studies have shown that granulocyte-colony-stimulating factor (G-CSF) and granulocyte-macrophage-colony stimulating factor (GM-CSF) can increase the number of the FcgammaRI-positive monocytes, increase the expression of FcgammaRI on circulating neutrophils after in vivo infusion, and greatly enhance the cytotoxic activity of circulating neutrophils. CD33 is a glycoprotein expressed on the cell surface of mature monocytes, myeloid progenitor cells, and myeloid leukemic blasts, but not on the earliest hematopoietic progenitor cells and other normal tissues. Herein, we report the construction of a BsAb, 251 x 22, by conjugating an anti-CD33 mAb (mAb 251) to an anti-FcgammaRI mAb (mAb 22). The BsAb 251 x 22 is capable of enhancing the cytotoxicity of several leukemia cell lines by cytokine-activated monocytes. Our data also show that G-CSF- and GM-CSF-stimulated monocytes can mediate cytotoxicity of target leukemia cells comparable to that of IFN-gamma-stimulated monocytes. The expression of FcgammaRI on monocytes after 24-h in vitro incubation with G-CSF and GM-CSF was increased, although not significantly. Prolonged incubation of monocytes with G-CSF for 48 h significantly increased the FcgammaRI expression. Because humanized anti-CD33 and anti-FcgammaRI mAb are available, and because GM-CSF and G-CSF have been used widely for patients after chemotherapy to stimulate the recovery of myeloid hematopoiesis, additional clinical development of this project is feasible. A BsAb comprised of humanized anti-CD33 and anti-FcgammaRI could have clinical application in the treatment of myeloid leukemia, especially in the management of minimal residual disease.

Autologous peripheral blood stem cell transplantation and adoptive immunotherapy with activated natural killer cells in the immediate posttransplant period.

Relapse after high-dose chemotherapy supported by peripheral blood stem cell transplantation (HDC-PBSCT) is the main cause of therapeutic failure in patients with lymphoma and breast cancer. Adoptive immunotherapy with activated natural killer (A-NK) cells and interleukin 2 might eliminate surviving residual tumor without adding to toxicity. Eleven patients with relapsed lymphoma and one with metastatic breast cancer were entered on a pilot clinical trial of HDC-PBSCT followed on day 2 after transplant by infusion of cultured autologous A-NK cells. Simultaneously, recombinant human interleukin 2 (rhIL-2) was initiated as a 4-day continuous i.v. infusion at 2 x 10(6) IU/m2/day, referred to as high-dose rhIL-2. Therapy with high-dose rhIL-2 was followed by a 90-day continuous i. v. infusion at 3 x 10(5) IU/m2/day, referred to as low-dose rhIL-2. All patients engrafted and nine completed treatment. Posttransplant days to a neutrophil count of 500/microliter and to a platelet count of 50,000/microliter were similar to comparable patients treated with HDC-PBSCT alone. Generation of A-NK cells for therapy was feasible in all patients except the three patients with Hodgkin's disease, whose cells did not proliferate in culture. Overall toxicity associated with early posttransplant transfer of A-NK cells and interleukin 2 did not differ from that observed with peripheral blood stem cell transplantation alone in comparable patients. There was early amplification of natural killer cell activity in the peripheral blood of four patients that appeared to result from the transfused A-NK cells. Adoptive transfer of A-NK cells and rhIL-2 during the pancytopenic phase after HDC-PBSCT was feasible and well tolerated, did not adversely affect engraftment, and resulted in amplified natural killer activity in the peripheral blood during the immediate posttransplantation period.

Phase I clinical trial of serotherapy in patients with acute myeloid leukemia with an immunoglobulin M monoclonal antibody to CD15.

Sixteen patients with acute myeloid leukemia (AML) were treated with a continuous i.v. infusion of mAb PM-81, an IgM mAb directed against the cellular differentiation antigen CD15, which is expressed on leukemia cells of >95% of patients with AML. MAb PM-81, also referred to as MDX-11, is capable of activating human and rabbit complement and lysing CD15-positive AML cells. In this Phase I study, patients were treated with 0.5, 1.0, or 1.5 mg/kg MDX-11 delivered over a 24-h period followed by conventional chemotherapy. Transient decreases in circulating blast cells postinfusion (prior to chemotherapy) were observed at all doses. We were able to show MDX-11 binding to bone marrow blasts in those patients who achieved stable serum levels of MDX-11. Serum MDX-11 was detectable at the 1. 0- and 1.5-mg/kg doses. Doses of 0.5 and 1.0 mg/kg were generally well tolerated, with no toxicities greater than grade II (Eastern Cooperative Oncology Group) reported. However, two of five patients receiving the 1.5-mg/kg dose experienced grade IV toxicities that resolved with treatment (one of these patients completed the infusion). Common toxicities reported included fever, chills, and hypotension. Only one patient developed human antimouse antibodies at 4 weeks posttreatment. This study determined that 1.0 mg/kg is a biologically effective dose that can be administered safely with little toxicity. Based on these results, we are pursuing a Phase I/II study of MDX-11 infusion following chemotherapy for patients with relapsed AML.

Expression of the CD15 antigen on normal and leukemic myeloid cells: effects of neuraminidase and variable detection with a panel of monoclonal antibodies.

Normal and malignant myeloids cells are known to express cell surface molecules having in common the carbohydrate antigen lacto-N-fucopentaose-III (LNF-III--termed CD15). We used flow cytometry to examine the variability of CD15 expression in normal cells and acute myeloid leukemia (AML) cells as detected by 24 murine monoclonal antibodies (mAb). Important differences in the levels of binding were observed with the various mAb. Titrations of each mAb were performed to confirm that these differences in binding were due to increased antigen detection and not differences in concn. In studies of CD15 expression on AML cells selected from a large prospective study, anti-CD15-1 (also known as PM-81) showed the highest binding to each case. Neuraminidase was added to cells from seven AML patients that we had previously found to be low in CD15 expression, in order to determine if cryptic CD15 was present on these cells. Neuraminidase enhanced binding of each of the entire panel of mAb on five patients' cells, thus demonstrating the ubiquitous expression of CD15 on AML cells. In two cases, binding of only some of the mAb was increased, indicating exposure of unusual epitopes on those cells. Subpopulations of normal peripheral blood lymphocytes, cells not associated with CD15 expression, also substantially increased their level of binding to some of the mAb after the addition of neuraminidase. Two-color flow cytometry was used to determine the immunologic phenotype of the lymphocytic population that expressed CD15. This technique revealed that 9.5% normal lymphocytes coexpressed the CD15 and CD3 (T cell) antigens. In addition, by gating on large granular lymphocytes we found that 24.4% of these cells coexpressed CD15 (detected by PM-81) and CD2 (sheep erythrocyte receptor), while 50.3% expressed CD15 and CD16 (type III Fc receptor, natural killer cell-associated). This is consistent with the notion that sialylated CD15 is expressed on some natural killer cells and T cells.

1,25-Dihydroxyvitamin D3 induces a myelomonocytic phenotype with enhanced effector cell function in the HL-60 promyelocytic leukemia cell line.

Human promyelocytic leukemia (HL-60) cells were induced by 1,25-dihydroxyvitamin D3 (calcitriol) to differentiate and examined using a panel of monoclonal antibodies (MoAbs) and functional assays. Although morphologically and histochemically these cells appeared to be of the monocyte-macrophage phenotype, there was a decline in Fc receptors for IgGl and no induction of class II HLA antigens. There was, however, dramatic induction of the antigen detected by the myeloid-specific MoAb AML-2-23. These data suggest that the phenotypic changes induced by calcitriol in HL-60 cells are consistent with myelomonocytic differentiation in that the resultant cells possess characteristics of both monocytes (morphology, non-specific esterase staining, high levels of AML-2-23 reactivity) and granulocytes (PMN 29 binding, decreased Fc receptors for IgGl, absence of class II HLA antigens). Perhaps more important, the ability of calcitriol-treated cells to perform antibody-dependent cellular cytotoxicity and phagocytosis was markedly augmented. Lysis of antibody-coated erythrocytes by HL-60 cells increased from 5% in controls to 30-35% with calcitriol treatment for 4 days. This enhanced effector cell function was seen despite a decline in Fc receptors measured by cytofluorography. These data suggest that calcitriol may be involved in both differential and functional activation of myeloid cells.

Gamma interferon and 1,25 dihydroxyvitamin D3 cooperate in the induction of monocytoid differentiation but not in the functional activation of the HL-60 promyelocytic leukemia cell line.

The physiological agents gamma interferon and 1,25 dihydroxyvitamin D3 (calcitriol) are both potent inducers of monocytoid differentiation in the HL-60 promyelocytic leukemia cell line. However, the populations resulting from treatment with either inducer have differing properties, suggesting that these agents do not have identical modes of action. In this study we have compared the effects of gamma interferon (IFN gamma) and calcitriol and examined the effect of exposing HL-60 cells to both compounds simultaneously. In addition, we have examined the effects of inhibitors of protein and RNA synthesis on induction of cell surface antigens in order to gain insight into the mechanisms of action of these compounds. We found that combining IFN gamma and calcitriol resulted in greater monocytic differentiation of HL-60 cells than did either compound alone. In addition, a monocyte-associated cell surface antigen, My23, and class-I HLA antigen were induced by both agents in at least an additive manner. In contrast, combined treatment with both compounds did not augment antibody-dependent cellular cytotoxicity (ADCC) any better than did IFN gamma alone, but it did cause a decrease in the density of receptors for the Fc portion of immunoglobulin. In studies examining the effects of IFN gamma and calcitriol on the cloning potential of HL-60 cells, we found that incubation of cells with either compound alone significantly reduced the number of HL-60 colonies, and both compounds added together caused an almost total elimination of colony-forming cells. Inhibition of RNA synthesis with alpha amanitin had no effect on the action of IFN gamma on cell surface HLA, but did block the induction of My23 antigen, suggesting that the mechanism of HLA induction may involve posttranscriptional phenomena. RNA-synthesis blockade inhibited the ability of calcitriol to induce My23. These studies demonstrate that multiple and separable events accompany the monocytoid differentiation of HL-60 cells induced by different chemical mediators and that different intracellular pathways may be involved.