Acute bilineage leukemia after chronic myelogenous leukemia.

Blastic transformation of chronic myelogenous leukemia may involve any hematopoietic lineage, including that of lymphocytes. In a minority of cases, the blasts express characteristics of both myeloid and lymphoid cells (biphenotypic) or comprise two separate populations of myeloid and lymphoid cells (bilineage leukemia). In a coordinated effort, we used a sequence of morphologic, cytochemical, immunocytochemical, and molecular biological studies to identify a case of bilineage blastic transformation of CML with a predominance of lymphoid blasts. The patient was treated for ALL and responded. The case presented illustrates the need for greater flexibility by the physician and laboratory to determine the specific diagnostic requirements for patients with hematologic malignancy with unusual phenotypic characteristics.

IFN-gamma-induced MHC class II gene expression is suppressed in endothelial cells by dextran sulfate.

IFN-gamma-activated endothelial cells actively participate in initiating immune responses by interacting with immunocompetent cells via class II MHC proteins. In this study, dextran sulfate, a synthetic heparin analogue, was shown to selectively inhibit IFN-gamma-induced surface expression of HLA-DR molecules by human umbilical cord vascular endothelial cells, but not other cytokine-induced molecules such as ELAM-1 or ICAM-1. Inhibition occurred regardless of whether dextran sulfate was added 24 h before, at the same time as, or 24 h after IFN-gamma stimulation of cells. In addition, both high (500 kDa) and low (5 kDa) molecular mass dextran sulfate molecules were able to block class II expression, whereas treating cells with naturally occurring polysulfated glycosaminoglycans such as heparin, heparan, and chondroitin sulfate did not produce any suppressive effects. Radiolabeling of cells with [35S]methionine followed by radioimmunoprecipitation using anti-HLA-DR alpha mAb demonstrated that biosynthesis of class II proteins was specifically blocked. RT-PCR and Southern blotting were utilized to examine transcription of the HLA-DR alpha gene and demonstrated an absence of HLA-DR alpha mRNA from dextran sulfate-treated and IFN-gamma-induced cells. Dextran sulfate also prevented transcription of the gene encoding CIITA, a transactivator protein required for IFN-gamma-inducible expression of class II genes. Thus, dextran sulfate apparently inhibited this step or an earlier one in the intracellular signaling pathway for IFN-gamma in human endothelial cells, subsequent to IFN-gamma binding to its cell surface receptor.

Interleukin-10 inhibits interleukin-2-induced tumor necrosis factor production but does not reduce toxicity in C3H/HeN mice.

Immunotherapy with interleukin-2 (IL-2) is limited by severe side effects thought to be mediated by the activation of immune effector cells and the induction of secondary cytokines including tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). In C3H/HeN mice the primary IL-2 toxicity is the production of pleural effusion with subsequent respiratory compromise. IL-10 is a cytokine that has been shown to inhibit the generation of secondary cytokines in vitro and in vivo. In this study, in C3H/HeN mice, we tested the ability of IL-10 to inhibit IL-2-induced mononuclear cell and alveolar macrophage activation and IL-2-induced increases in serum TNF-alpha and IFN-gamma, all of which may contribute to the generation of toxicity. IL-10 was ineffective at reducing IL-2-induced pleural effusion. However, IL-10 did inhibit IL-2-induced increases in serum TNF-alpha, which was accompanied by a decrease in Golgi apparatus and rough endoplasmic reticulum in alveolar macrophages. In addition, IL-10 combined with IL-2 increased mononuclear cell activation, which may limit the ability of IL-10 to inhibit IL-2-induced IFN-gamma production and pulmonary injury.

Topoisomerase II activity involved in cleaving DNA into topological domains is altered in a multiple drug-resistant Chinese hamster ovary cell line.

Drug resistance to inhibitors of DNA topoisomerase II can result from qualitative or quantitative alterations in the target enzyme, topoisomerase II, or from perturbations in drug transport that may or may not involve P-glycoprotein. In the present study, a drug-resistant Chinese hamster ovary cell line, SMR16, was selected in the presence of an epipodophyllotoxin (VP-16) and was found to be cross-resistant to all classes of topoisomerase II inhibitors (3-35-fold). The 3-fold level of resistance of these cells to vincristine is likely due to diminished uptake of this drug, and this is not mediated by overexpression of P-glycoprotein. No alteration in transport of VP-16 was observed. Immunoblotting with several polyclonal anti-topoisomerase II antibodies demonstrated that the resistant cells contain approximately two-thirds of the parental enzyme amount. The topoisomerase II catalytic activity present in 0.35 M NaCl nuclear extracts paralleled this decrease. VP-16- and 4'-(9-acridinylamino)methanesulfon-m-anisidide-induced DNA damage, mediated by topoisomerase II, was found to be decreased 10-12-fold in both intact SMR16 cells and nuclei isolated from these cells, when measured by alkaline filter elution. However, the VP-16-induced DNA cleavage activity present in 0.35 M NaCl nuclear extracts of the resistant cells was attenuated only 2-fold, relative to wild-type cells. Homogeneous preparations of the enzyme obtained from resistant cells demonstrated the same cleavage and catalytic activity as purified wild-type topoisomerase II. Analysis by pulse-field gel electrophoresis of the DNA isolated from VM-26- and 4'-(9-acridinylamino)methanesulfon-m-anisidide-treated sensitive and resistant cells demonstrated significantly less conversion of SMR16 chromosomal DNA into 50-150-kilobase DNA fragments. Chinese hamster ovary SMR16 cells are apparently resistant to topoisomerase II poisons because the topoisomerase II that defines the DNA topological domains is either decreased in amount or insensitive to drug action.

Flow cytometric analysis of altered mononuclear cell transmembrane potential induced by cyclosporin.

Immunosuppression by the fungal metabolite cyclosporin A (CsA) is characterized by functional inhibition, rather than destruction of cells. Because activation of immune cells involves intracellular signalling events associated with modulations of cell transmembrane potential (TMP), we tested the ability of cyclosporin A (CsA) to modulate immune mononuclear cell TMP in vitro using a TMP sensitive cationic dye, dihexyloxacarbocyanine (DIOC6(3)). All analyses were performed by flow cytometry. CsA increased TMP in monocytes and lymphocytes isolated from the blood of healthy human volunteers. CsA-induced hyperpolarization was time and concentration dependent in monocytes while the lymphocyte hyperpolarization, although time dependent, was evident over the entire range of CsA concentrations tested. CsA-induced hyperpolarization of lymphocytes was dependent on potassium ion (K+) efflux as indicated by the absence of hyperpolarization in 154 mM KCl or with pretreatment with 100 microM quinine (an inhibitor of K+ channels). Monocyte hyperpolarization by CsA was not inhibited in either system. Dihydrocyclosporin C (DH-CsC), an immunosuppressive analog of CsA, also hyperpolarized mononuclear cells. The anionic TMP sensitive dye bis oxonal (diBA-C4) indicated that CsA treatment depolarized mononuclear cell plasma membranes. The mitochondrial poison carbonyl cyanide p-trifluoromethoxy-phenyl-hydrazine (FCCP) eliminated CsA induced hyperpolarization and also indicated that CsA caused plasma membrane depolarization. We conclude that brief in vitro exposure to cyclosporin alters the transmembrane electrical potential of human lymphocytes and monocytes.

Flow microfluorometric analysis of murine spermatozoa fails to detect H-2 antigens.

The presence of class 1 and class 2 histocompatibility antigens on murine sperm was investigated by flow microfluorometry. Monoclonal anti-H-2Kk (class 1), anti-Iak (specificity 2, class 2) and allo-anti-Iak (class 2) antisera were used in direct or indirect fluorescence labelling experiments to probe the expression of class 1 and class 2 antigens on epididymal mouse spermatozoa. Sperm-specific antibodies were generated by intraperitoneal immunization of both male and female C3H/HeN mice with syngeneic spermatozoa. Sperm-specific antigens were detected in 68-85% of syngeneic mouse sperm and 65-90% of allogeneic mouse sperm examined. Conversely, these antibodies did not stain syngeneic or allogeneic lymphocytes above the background of the negative control. Mouse sperm samples failed to exhibit specific fluorescence above the background of negative control values with antibodies against either class 1 or class 2 MHC antigens. We have established the sensitive, objective and economical assay of sperm membrane antigens with fluorochrome-labelled antibodies by flow microfluorometry. By use of this sensitive and objective technique we have not detected MHC antigens on murine sperm. We conclude that these MHC antigens are not expressed on sperm at a level to be practically detectable.

HLA-DR antigen expression on peripheral blood monocytes correlates with surgical infection.

Monocyte human leukocyte antigen-DR (HLA-DR) expression has correlated closely with clinical outcome in severely injured patients at high risk for infection. Monocytes from 77 asymptomatic volunteers expressed HLA-DR antigen with minimal variability in respect to age, gender, race, time of day or year, or serum alcohol level. Patients who developed infection after elective laparotomy had a significantly lower mean percentage of monocytes expressing HLA-DR antigen and a lower mean fluorescent intensity than uninfected patients (p less than 0.05). Severely infected nonsurgical patients had significantly lower values than normal volunteers (p less than 0.01), and the mean fluorescent intensity of those who died from infection was significantly lower than that of those who survived (p less than 0.05). Patients on immunosuppressive regimens after renal transplantation had levels of HLA-DR expression similar to those of the volunteers. Monocyte HLA-DR expression was found to be a reliable marker of clinical infection and showed remarkable reproducibility within the normal uninfected study population.

Effect of extracorporeal membrane oxygenation on leukocyte function in neonates.

The impact of extracorporeal membrane oxygenation (ECMO) on neonatal leukocyte content and function was examined in six patients. Patients were treated with ECMO for a mean of 134 h (range 44-246 h). Absolute neutrophil counts decreased from 14679 +/- 2291/mm3 to 7791 +/- 1672/mm3 after 2 h of ECMO. However, neutrophil phagocytosis and oxidative burst remained unchanged during the first 48 h of bypass. Monocyte counts also decreased during bypass, and at times were undetectable in 50% of patients. Monocyte HLA-DR content was decreased compared to normal cord blood prior to initiation of ECMO, and remained low throughout ECMO. However, the content increased significantly after termination of bypass. Plasma C3a levels increased transiently, paralleled by an increase in neutrophil CR3 expression. While moribund infants had some impairment of host defenses prior to ECMO, there was no further impact of ECMO per se on the parameters measured, other than transient complement activation and decreased monocyte counts.

Effects of spaceflight on levels and activity of immune cells.

Experiments were carried out on cells from rats that had been flown on Soviet Biosputnik Cosmos 1887 to explore the effects of spaceflight on immune responses. Rat bone marrow cells were examined for their response to colony stimulating factor-M. Rat spleen and bone marrow cells were stained with antibodies directed against cell surface antigenic markers. The results of the studies indicate that bone marrow cells from flown rats showed a decreased response to colony stimulating factor. There was a higher percentage of spleen cells from flown rats staining positively for pan-T-cell, suppressor-T-cell and interleukin-2 receptor cell surface antigens. A small increase in the percentage of cells staining positively for helper-T-cell antigens was also noted. In addition, a higher percentage of cells that appeared to be part of the myelogenous population of bone marrow cells from flown rats stained positively for surface immunoglobulin.

Interactions between adherent mononuclear cells and lymphocytes from granulomas of mice with schistosomiasis mansoni.

T-lymphocyte-adherent mononuclear cell interaction was analyzed in the vigorous and immunomodulated liver granulomas of Schistosoma mansoni-infected mice. Collagenase-dispersed granulomas contained 15% lymphocytes, 30% macrophages, 50% eosinophilis, and some neutrophils. Dispersed granuloma cells stimulated with concanavalin A or soluble worm egg antigens (SEA) did not proliferate unless plate-adherent, esterase-positive mononuclear cells were removed before culture. To analyze the granuloma adherent cell-mediated suppression, vigorous granuloma cell cultures partially depleted of adherent mononuclear cells were supplemented with indomethacin, catalase, superoxide dismutase, levamisole, and anti-murine alpha/beta interferon antiserum. In concanavalin A and SEA-stimulated cultures, only the addition of indomethacin or anti-alpha/beta interferon antiserum alleviated the adherent cell-mediated suppression of vigorous granuloma lymphocyte response. In contrast, these agents only minimally alleviated the suppressed response of SEA-stimulated, immunomodulated granuloma lymphocytes. Moreover, coculture of equal numbers of vigorous and immunomodulated granuloma cells partially depleted of adherent suppressor cells abrogated the alleviated response of vigorous granuloma lymphocytes. These findings indicate that, within the schistosome egg-induced vigorous granulomas, the adherent mononuclear cells exert regulation over lymphocyte responsiveness by alpha/beta-interferon and an indomethacin-sensitive, probably prostaglandin-mediated pathway. Within the immunomodulated granulomas, the adherent suppressor cell-mediated regulation of lymphocyte proliferation appears to play a lesser role.

Challenge of the mononuclear phagocytic system with colloidal carbon alters lymphocyte distribution and responsiveness.

The objective of this study was to determine whether or not a particulate challenge to the mononuclear phagocytic system (MPS) would modulate lymphocyte populations. For this purpose subset distributions were measured in mice subsequent to colloidal carbon challenge of the MPS. The thymus underwent acute cortical atrophy followed by cellular replenishment to levels which exceeded those of the control group. The spleen and lymph nodes underwent acute expansion of all lymphocyte subsets. In lymph nodes this nonspecific expansion was disproportionately greater for B lymphocytes and Ts lymphocytes and subsided by day 30. Splenic lymphocyte expansion was equivalent for all subsets and included an increase in nonspecific esterase positive cells. The systemic MPS clearance rate underwent enhancement. Administration of colloidal carbon during topical application of DNFB depressed the cellular response to this immunogen. The cellular response to DNFB applied 28 days after carbon administration was normal. These observations indicate that distribution and responsiveness of lymphocyte subsets may be modulated by administration of a particulate material that constitutes a challenge to the MPS.

Monocyte HLA-DR antigen expression characterizes clinical outcome in the trauma patient.

Immunological assessment is important to characterize the host defence response of trauma patients as infection is the most common cause of severe morbidity and late death. Sixty trauma patients were followed serially and divided into three groups: those with an uneventful recovery (n = 17), those with recovery after major sepsis (n = 27) and those who died (n = 16). The ability of peripheral blood monocytes to express the antigen HLA-DR was measured and compared to the results from 77 asymptomatic volunteers. After initial injury, there was a significant reduction from normal in the three trauma groups. It took one week for HLA-DR antigen expression to return to the normal range in the first group, three weeks in the second group, and in the third group it never returned to normal. Monocyte HLA-DR antigen expression, after incubation with lipopolysaccharide, distinguished those patients who survived from those who died. There was no difference in HLA-DR antigen expression between a high transfusion group of 31 patients who received 10 or more units of blood and a low transfusion group of 29 patients who received less than 10 units. The ability of monocytes to express HLA-DR antigen correlated directly with the clinical course in these patients and its measurement identified a group of patients at high risk of infection and death following trauma.

Role of intracellular calcium in priming of human peripheral blood monocytes by bacterial lipopolysaccharide.

To determine the role of intracellular calcium ([Ca2+]i) in the priming of monocytes (M phi) by bacterial lipopolysaccharide (LPS), the membrane expression of two functional proteins and phagocytosis and respiratory burst were examined by microfluorimetry. LPS induced a significant increase in HLA-DR and C3bi receptor (CR3) expression within 2 h of its addition to whole blood. The enhanced expression of both antigens by LPS was dose-dependent, with concentrations as low as 0.1 ng/ml producing a response. The involvement of [Ca2+]i was demonstrated by loading isolated M phi with the intracellular calcium chelator quin-2 or the inhibitor of intracellular calcium redistribution TMB-8 prior to addition of LPS. Both compounds inhibited the LPS-induced increase in HLA-DR and CR3 expression. No role for extracellular calcium, for calcium slow channel flux, or for the calcium-calmodulin complex in LPS priming was demonstrated when LPS was added in the presence of EGTA, trifluperazine (TFP), or verapamil. The addition of the calcium ionophores A23187 or ionomycin failed to increase expression of either antigen. Prior exposure to LPS primed M phi for enhanced phagocytosis and respiratory burst activity. These functions were inhibited by TMB-8, but not by TFP or verapamil. Addition of LPS to isolated M phi increased [Ca2+]i by 23% at 30 sec and 42% at 5 min, as measured by the calcium-sensitive, intracellular probe indo-1. These results suggest that intracellular Ca2+ mobilization is necessary, but not sufficient, for LPS-induced priming of human peripheral blood monocytes.

Interferon-gamma treatment increases HLA-DR expression on monocytes in severely injured patients.

Traumatic injury and sepsis reduce HLA-DR expression on monocytes. This study assessed the effect of interferon-gamma (IFN-gamma) on the expression of HLA-DR on monocytes taken from severely injured patients. The mononuclear layer of each blood sample was incubated with either 500 units IFN-gamma or control media. The mean baseline HLA-DR expression of the trauma group (n = 11) was 32%, significantly less than the mean HLA-DR expression of 91% for the control group (n = 10). In the trauma group, HLA-DR expression after culturing with IFN-gamma was 81%, significantly greater than both the mean baseline HLA-DR expression (32%). In the control group, HLA-DR expression after culturing with IFN-gamma was 94%, similar to the mean baseline HLA-DR expression of 91%. These data confirm that HLA-DR on monocytes of severely injured patients is markedly reduced and show that it can be increased significantly in vitro by IFN-gamma.

Experimental and clinical significance of endotoxin-dependent HLA-DR expression on monocytes.

For much of the last decade, an increasing number of surgeons have been interested in objective assessment of cellular contributors to host defense function. In order to study many of these processes, it is apparently desirable that the cells be isolated to the extent feasible for the purpose of analyzing a more or less pure population of cellular elements. The purpose of this paper is to describe the physiologic activation of mononuclear cells that occurs as a result of the isolation process. Therefore, it follows logically that such cells are therein intrinsically less responsive to further physiologic manipulation in vitro. Analyses of such data without an awareness of this intrinsic aberration will undoubtedly lead to misinterpretation of the capacity of such cells for further modulation by immunostimulants or by the intrinsic processes related to injury, anesthesia, and operation. Furthermore, it may indicate that certain agents, e.g., cytokines, are unable to stimulate cellular function when, in fact, the defense function of the cell has been initially stimulated by the isolation procedure. Fractionation of human peripheral blood over Hypaque-Ficoll and subsequent purification of monocytes by adherence to plastic lead to an increase in the relative density of HLA-DR on monocytes. This increase occurred when carried out in endotoxin lipopolysaccharide (LPS)-contaminated or LPS-depleted reagents. LPS, added experimentally to whole blood, enhanced HLA-DR expression on monocytes without further manipulation. Monocyte HLA-DR expression measured in whole blood was reduced in patients with major sepsis (n = 19) compared to normal subjects (n = 10).(ABSTRACT TRUNCATED AT 250 WORDS)

Polymorphonuclear leukocyte function during hemodialysis: relationship to complement activation.

Phagocytosis, H2O2 production, and C3bi receptor (CR3) expression by polymorphonuclear leukocytes (PMN) obtained from patients before, during, and after a hemodialysis treatment were evaluated by flow microfluorometry. The results were compared to changes in plasma levels of C3ades Arg and C5ades Arg. Prior to hemodialysis C3ades Arg and C5ades Arg levels, CR3 expression and phagocytosis were not different from normal controls. However, both basal and phagocytosis-induced H2O2 production were increased. C3ades Arg and C5ades Arg were increased after 15 min of dialysis; this was accompanied by transient but significant reductions in PMN count and phagocytosis and increased CR3 expression. No changes in basal or stimulated H2O2 production were observed. We conclude that PMN of hemodialysis patients are primed for an enhanced respiratory burst before dialysis is initiated. Dialysis-induced complement activation after the initiation of dialysis does not further stimulate H2O2 production or enhance the response to phagocytosis. However, complement activation may cause leukopenia and CR3 expression.

Infant orthotopic cardiac transplantation.

Infant orthotopic cardiac transplantation has been recently applied to various forms of congenital heart disease with encouraging short-term results. Between June 1986 and September 1987 we evaluated 16 infants for orthotopic cardiac transplantation. Fourteen had hypoplastic left heart syndrome, one had endocardial fibroelastosis with aortic atresia, and one had anomalous pulmonary arterial origin of the left main coronary. Eight families accepted the treatment program and eight families refused (two because of associated anomalies and six on philosophical grounds). Of the eight patients who were candidates for orthotopic cardiac transplantation, one died 6 hours after diagnosis, one was allowed to die after 60 days because of acquired neurologic complications, and another had congenital cytomegalic virus infection. The remaining five patients (four with hypoplastic left heart syndrome, one with anomalous pulmonary arterial origin of the left main coronary) had orthotopic cardiac transplantation. The operation was performed with absorbable polydioxanone suture with deep hypothermia and circulatory arrest in four neonates for hypoplastic left heart syndrome (average time 47 minutes) and bicaval cannulation and continuous bypass in one 11-month-old infant for anomalous origin of the left main coronary. In-house retrieval was used in all. One neonate died of complications as a result of pretransplant donor heart dysfunction and size discrepancy, whereas the remaining three neonates and one infant survived and are home 23 months, 12 months, and 8 months (the patients with hypoplastic left heart syndrome) and 17 months (the patient with anomalous origin of the left main coronary) postoperatively. Triple-drug immunosuppression included cyclosporine, azathioprine, and prednisone. Rejection was diagnosed by clinical evaluation of child activity and monocyte cell cycle analysis from peripheral blood samples without myocardial biopsies. Routine echocardiograms, electrocardiograms, and chest x-ray films were not helpful. Six episodes of rejection were successfully treated in four patients. Twelve-month postoperative catherization in one patient (hypoplastic left heart syndrome) showed appropriate graft growth, no aortic or pulmonary anastomotic strictures, normal right and left ventricular function, and no coronary artery disease. We conclude that infant orthotopic cardiac transplantation is an acceptable procedure for severe forms of untreatable congenital heart disease. The excellent short-term results warrant continued application of orthotopic cardiac transplantation.

Depressed interferon gamma production and monocyte HLA-DR expression after severe injury.

Monocyte HLA-DR antigen expression and mitogen-stimulated interferon gamma production were measured sequentially on days 1, 3, 7, 14, and 21 after admission in 20 multiply injured patients (mean Injury Severity Score, 33). Ten patients recovered uneventfully and ten developed a major infection, three of whom died. Trauma resulted in immediate and profound depression of both interferon gamma production and monocyte HLA-DR antigen expression compared with controls. Interferon gamma remained below control levels for all days on which it was measured in all patients. In uninfected patients, interferon gamma production began to recover after day 7 and interferon gamma levels on day 21 were greater than on all other days. Monocyte HLA-DR antigen expression returned to normal between days 7 and 14 in uninfected patients, despite subnormal production of interferon gamma. Failure to increase interferon gamma production and monocyte HLA-DR antigen expression was associated with an episode of major infection. We postulate that stimulation of the immune system early after injury may reverse the defects reported and decrease the incidence of infection after severe trauma.