Extracellular killing of Leishmania promastigotes and amastigotes by macrophage precursors derived from bone marrow cultures.

Flagellates of the genus Leishmania are obligate intracellular parasites of vertebrates including man. The microorganisms reside and multiply inside the phagolysosomes of cells of the mononuclear phagocyte lineage. We here report on the spontaneous leishmanicidal activity exerted extracellularly by immature cells of the mononuclear phagocyte lineage. Highly purified, bone marrow-derived macrophage precursor cells displayed a strong spontaneous leishmanicidal activity already at very low effector/target rations (3:1, 6:1). This leishmanicidal activity was effective against both promastigotes and amastigotes as targets. The cytotoxic effect was evident within 4 h and maximal after 12 h of effector-target organism cocultivation, as determined by a radiolabel-release assay. An intimate cell-cell contact seemed necessary for the parasites to be killed.

Cooperative effects of colony-stimulating factor 1 and recombinant interleukin 2 on proliferation and induction of cytotoxicity of macrophage precursors generated from mouse bone marrow cell cultures.

Precursor cells for NK activity, present in the light fraction of fresh mouse bone marrow, were cultivated in vitro in the presence of either CSF-1, IL-2, or a combination of both factors. In the presence of only CSF-1, strong proliferation was induced. Cells quickly passed the macrophage precursor stage and matured to typical macrophages. Neither granula formation nor NK activity were induced. Under culture conditions with only IL-2 NK activity had developed after 3 d, however, no significant proliferation occurred. In the presence of both factors strong proliferation was induced, and concomitantly, granula formation and NK activity developed. Apparently, proliferation depended on CSF-1 and granula formation, and NK cytotoxicity was induced by IL-2. When proliferating cells with strong anti-YAC-1 activity from a culture in CSF-1 plus IL-2 were further cultivated in only IL-2, the content of granula further increased, whereas proliferation gradually stopped. In contrast, when these cells from CSF-1 plus IL-2 culture were further cultivated in only CSF-1, granula disappeared and NK activity was lost, whereas sustained proliferation and differentiation to macrophages occurred. Only under culture conditions with both factors were proliferation and NK activity both maintained. More than 90% of cells from a 3-d culture in CSF-1 plus IL-2 expressed the NK 1.1. marker, whereas F4/80 was only marginally detected by FACS analysis. After two further days in culture, 70% of the cells expressed F4/80 and 60% coexpressed NK 1.1. and F4/80. By setting the size scatter in order to gate for large granular cells, a population was obtained with 100% coexpression of NK1.1. and F4/80. The data indicate that early cells of the macrophage lineage can develop into different functional and morphological directions depending on the varying influence of IL-2 and CSF-1.

Staphylococci surviving intracellularly in phagocytes from patients suffering from chronic granulomatous disease are killed in vitro by antibiotics encapsulated in liposomes.

Granulocytes and monocytes/macrophages from patients suffering from chronic granulomatous disease (CGD) are ineffective in killing specific kinds of phagocytized bacteria, e.g., Staphylococcus aureus, due to decreased or lacking ability to produce reactive oxygen intermediates. Commonly used antibiotics like flucloxacillin are of limited therapeutic value, because the staphylococci are protected against their action in the interior of phagocytes. However, encapsulation of flucloxacillin into liposomes could enable its entrance into the interior of neutrophils from two CGD patients to kill phagocytized bacteria there. The effect of rifampicin against intracellular staphylococci could be similarly enhanced by liposome encapsulation. Dose-response relations and kinetics of killing of intracellular bacteria by antibiotics in the free and encapsulated form were studied under different conditions using J 774 mouse macrophages, because phagocytes from CGD patients are not available in great amounts. Preincubation of phagocytes with either antibiotic in liposomes subsequently endowed the cells with a strongly enhanced ability to kill phagocytized bacteria. Our data show that a drug which normally will not reach a phagosome can be delivered to this intracellular compartment by a liposome. A possible clinical use is discussed.

Gamma interferon priming of mouse and human macrophages for induction of tumor necrosis factor production by bacterial lipopolysaccharide.

Priming of macrophages from both murine and human sources by recombinant immune interferons from Escherichia coli (r-IFN-gamma s) and activation by lipopolysaccharide (LPS) resulted in the production of tumor necrosis factor (TNF). r-IFN-gamma alone did not induce TNF production by macrophages; for this to occur, the second signal provided by small amounts (nanograms) of LPS was required. The small amounts of LPS alone were insufficient to activate the macrophages for TNF production. Priming by r-IFN-gamma was not necessary when larger amounts of LPS were employed, although an enhancement of yield resulted. Priming could also be demonstrated in vivo. Inoculation of r-IFN-gamma into mice resulted in increased yields of TNF following LPS challenge 12 hours later.

Tumor metastases and cell-mediated immunity in a model system in DBA/2 mice. VII. Interaction of metastasizing and nonmetastasizing tumors with normal tissue in vitro.

Three mouse tumors known to metastasize in vivo and 3 nonmetastasizing mouse tumors that grow only locally in vivo were examined for their ability to adhere to and invade normal syngeneic lung organ cultures in vitro. All 3 metastasizing tumors adhered to and invaded the normal lung cultures. In contrast, tumors that grow only locally in vivo neither adhered to nor invaded the normal lung tissue. The described system is ideally suited to correlate the in vivo invasiveness of a given tumor with its potential to metastasize in vivo and to study in vitro how to influence the interaction of metastasizing tumor cells with normal tissue.

Macrophage activation by the polysaccharide arabinogalactan isolated from plant cell cultures of Echinacea purpurea.

In this study, acidic arabinogalactan, a highly purified polysaccharide from plant cell cultures of Echinacea purpurea, with a molecular weight of 75,000, was effective in activating macrophages to cytotoxicity against tumor cells and micro-organisms (Leishmania enriettii). Furthermore, this polysaccharide induced macrophages to produce tumor necrosis factor (TNF-alpha), interleukin-1 (IL-1), and interferon-beta 2. Arabinogalactan did not activate B cells and did not induce T cells to produce interleukin-2, interferon-beta 2, or interferon-gamma, but it did induce a slight increase in T-cell proliferation. When injected ip, this agent stimulated macrophages, a finding that may have therapeutic implications in the defense against tumors and infectious diseases.

In vitro and in vivo effects of pentoxifylline on macrophages and lymphocytes derived from autoimmune MRL-lpr/lpr mice.

MRL-lpr/lpr mice develop an autoimmune disease similar to human systemic lupus erythematosus (SLE). The main characteristics of this disease are increasing autoantibody formation, elevated plasma levels of immune complexes, a massive lymphoproliferation, a rising proteinuria, and arthritic symptoms. Finally, the mice die at an age of about 6 months due to a fatal immune complex glomerulonephritis. Macrophages are involved in the development of SLE due to their functions as antigen-presenting as well as cytokine-producing cells. T and B cells are involved in the disease by secreting cytokines and producing antibodies. Pentoxifylline (PTX), a xanthine derivative, is known to exert different effects on functions of leukocytes and erythrocytes and has been used in clinical studies, e.g., in septic shock syndrome. In our studies we first investigated the in vitro effect of PTX on macrophages and lymphocytes derived from MRL-lpr mice. Our investigations concerning production of superoxide anion and TNF-alpha by LPS and/or IFN-gamma activated bone marrow and peritoneal macrophages, MHC class II expression on these cells, and the proliferative capacity and Il-2 production of mitogen activated lymphocytes, revealed that PTX reduces the activation and the inflammatory response of these cells. Based on these results, we further investigated the effect of in vivo treatment with PTX. MRL-lpr mice treated with PTX showed diminished proteinuria, reduced titer of dsDNA-autoantibodies in the plasma and an increased survival rate. Our data clearly demonstrate that PTX is able to diminish the severity of the disease and to prolong the life of MRL-lpr/lpr mice.

Macrophage function in murine allogeneic bone marrow radiation chimeras in the early phase after transplantation.

We tested several of the functions of macrophages (M phi) in the early phase after allogeneic bone marrow transfer to get information about this important aspect of the nonspecific immune system in the T-cell-deficient recipient. On days 3-5 after transfer, the number of M phi was reduced in the spleen, liver, lungs, and peritoneal cavity (Pe). The phagocytosis of sheep red blood cells (SRBC) by these M phi was normal or even enhanced, as in the case of Pe-M phi. Already on days 8-12 after transfer, the number of M phi in spleen and liver exceeded that of controls, whereas the number was still reduced in lungs and Pe. We examined their ability to kill P815 tumor cells, to produce tumor necrosis factor-alpha (TNF alpha), to phagocytose SRBC, to produce reactive oxygen intermediates (ROI) in vitro and to kill Listeria monocytogenes in vivo. Most functions were normal and often even enhanced, depending on the organ origin, but the ability of Pe-M phi to produce ROI was reduced. Proliferative response to macrophage colony-stimulating factor (M-CSF) and killing of YAC-1 tumor cells revealed a high frequency of macrophage precursor cells in the spleen and liver and a high natural killer (NK) activity in the liver. Altogether, enhanced nonspecific immune function, especially preactivated M phi, may enable chimeras to survive attacks by opportunistic pathogens.

Expansion of the liver-associated macrophage system in systemic lupus erythematosus-prone NZB/W mice.

Systemic lupus erythematosus is characterized by profound changes of the immune system. We report on alterations of the macrophage system in the murine NZB/W model of this disease. A greatly increased number of mature macrophages was isolated from the liver of NZB/W mice as compared to BALB/c mice and several other inbred strains used as healthy controls. In addition, the macrophage precursor compartment in the liver of NZB/W mice was expanded severalfold as measured by proliferation of light-fraction nonadherent nonparenchymal cells (NPCs) in response to colony-stimulating factors. Functional properties of the macrophages isolated from various anatomic sites of the lupus-prone mice were tested. Production of monokines by macrophages from liver, spleen, and peritoneal cavity, calculated on a per cell basis, was in the same range as in several healthy control strains tested. Yet the overall production of these immunoregulatory molecules by the increased liver macrophage system, the body's largest compartment of macrophages, is likely to result in increased levels of circulating monokines in the plasma of lupus-prone NZB/W mice. Indeed, significantly elevated levels of interleukin-6, interleukin-1, and colony-stimulating activity could be demonstrated in the plasma of these mice both spontaneously and after stimulation with lipopolysaccharide. A possible contribution of the expansion of the macrophage system to the development of the disease is discussed.

Heterogeneity in the mobilization of cytoplasmic calcium by human polymorphonuclear leukocytes in response to fMLP, C5a and IL-8/NAP-1.

The visible excitation and emission wave-lengths of the recently developed fluorescent Ca2+ indicator fluo-3 permit analysis of the intracellular Ca2+ concentration, [Ca2+]i, in flow cytometry with a 488-nm argon laser. The role of [Ca2+]i in human polymorphonuclear leukocyte heterogeneity was investigated in response to formyl-methionyl-leucyl-phenylalanine (fMLP), C5a, and interleukin 8/neutrophil attractant/activation protein 1 (IL-8/NAP-1) by flow cytometry. The [Ca2+]i changes in different subpopulations within a heterogeneous cell suspension were resolved upon stimulation with fMLP. Using an anti-CD16 phycoerythrin-conjugated antibody and fluo-3 simultaneously, neutrophils affected and nonaffected in Ca2+ mobilization were distinguished in two patients suffering from glycogen storage disease type 1b. In normal neutrophils, a different time course of Ca2+ mobilization of neutrophil subpopulations immediately after stimulation with fMLP was detected. In addition, after stimulation with a low concentration of IL-8/NAP-1 (10(-10) M) two subsets of neutrophils appeared; one of them showed an increase in [Ca2+]i, while the other did not. These results indicate heterogeneity in the neutrophil signal transduction process involved in Ca2+ mobilization. Therefore, flow cytometric analyses can resolve changes in single-cell [Ca2+]i distribution patterns, which is important for the understanding of [Ca2+]i in neutrophil heterogeneous activation processes.

Comparative study of cytotoxicity, tumor necrosis factor, and prostaglandin release after stimulation of rat Kupffer cells, murine Kupffer cells, and murine inflammatory liver macrophages.

Macrophages (Mphi) and Mphi-depleted (nonadherent) nonparenchymal cells (NPC) of the liver were examined for their cytotoxic potential against tumor cells, production of tumor necrosis factor (TNF), and release of prostaglandins (PG) following stimulation by lipopolysaccharide (LPS), interferon-gamma (IFN gamma), and zymosan. Resident murine liver macrophages had no natural cytotoxicity for the TNF-resistant target cell line P815. Activation of these cells was only obtained by a combination of IFN gamma and LPS. Inflammatory murine macrophages were in a primed stage and could be activated by LPS alone in the absence of IFN gamma. Rat resident macrophages resembled functionally the inflammatory macrophages of the mouse liver rather than the resident macrophages. They displayed natural cytotoxicity against all targets tested and were further activated by LPS in the absence of IFN gamma. Similar results were obtained with respect to macrophage-depleted nonadherent NPC: Mouse NPC had a low level of NK activity against Yac-1 cells. Treatment with pyran copolymer resulted in a strong increase of cytotoxicity against Yac-1; furthermore, a TNF-dependent killing of Wehi 164 and TNF-independent cytotoxicity against P815 cells were now acquired. In the rat NPC prepared from unstimulated animals expressed high levels of natural cytotoxicity against all targets. No major differences could be observed between inflammatory Mphi and Kupffer cells of rat and mouse liver with regard to TNF production and TNF-dependent killing of Wehi 164 tumor cells. The same was true for the spectrum of secreted prostanoids. Upon activation of all cell populations a marked shift toward the production of PGE2 occurred. Experiments involving the cyclooxygenase inhibitor indomethacin showed enhanced TNF-dependent tumor cell killing by nonactivated Mphi in the absence of prostanoid production.

Macrophage precursor cells produce perforin and perform Yac-1 lytic activity in response to stimulation with interleukin-2.

Macrophage precursor cells, derived from mouse bone marrow culture with granulocyte-macrophage colony-stimulating factor or colony-stimulating factor 1 (CSF-1) as growth factor and interleukin-2 (IL-2) as stimulating factor, were activated by IL-2 to exert strong cytolytic activity against Yac-1 cells. In response to IL-2 stimulation these bone marrow macrophage precursor cells produced perforin as lytic molecules. The purity of the precursor cells for the study was proved as homogeneous positivity for Mac-1, NK-1.1 and negativity for Lyt 1 and 2. The cells express CSF-1 receptors on their surface, are able to proliferate and differentiate into typical macrophages when stimulated with CSF-1, and are therefore members of the macrophage lineage. Perforin transcripts were identified by Northern blot analysis of IL-2-treated macrophage precursor cells, and the presence of perforin protein in the cytoplasmic granules was demonstrated by immunohistochemical staining using a monoclonal antiperforin antibody. In addition, the biological activity of the perforin contained in the macrophage precursor's granules could be documented as calcium-dependent lytic activity using Yac-1 and sheep red blood cells as targets. The results presented in this paper imply the existence of a bipotent precursor cell, which can mature into a typical macrophage if CSF-1 or phorbol 12-myristate 13-acetate is supplied as differentiation stimulating factor but develops into an NK/LAK cell when early activation with IL-2 is provided.

Analysis of leukocyte activation during acute rejection of pulmonary allografts in noninfected and cytomegalovirus-infected rats.

After human lung transplantation acute rejection and cytomegalovirus (CMV) infections may occur, probably contributing to the development of chronic rejection. We established a model of subacute allograft rejection in rats to analyze leukocyte activation and effects of a CMV infection. Histoincompatible lung transplants (BN/LEW) without immunosuppression (group A) and lungs of initially immunosuppressed animals (group B) were analyzed. The production of inflammatory mediators (interleukin-6, tumor necrosis factor alpha, nitric oxides) and the expression of MHC class II antigens by alveolar and lung tissue macrophages were significantly enhanced during the alloresponse. In recipients without immunosuppression (group A) allograft necrosis was detected by day 6, whereas group B allografts were fully rejected by day 25. In allografts of immunosuppressed, CMV-infected animals (group C) the CMV infection was clearly aggravated and the number of activated lung tissue macrophages was increased when compared with noninfected allografts or isografts. The subacute model provides the advantage of allowing us to study mechanisms of acute rejection without the effects of reperfusion injury. Furthermore these findings underline the role of inflammatory mediators produced by macrophages during rejection.

Abnormal regulation in the signal transduction in neutrophils from patients with severe congenital neutropenia: relation of impaired mobilization of cytosolic free calcium to altered chemotaxis, superoxide anion generation and F-actin content.

Severe congenital neutropenia (SCN) can be corrected in vivo by treatment with pharmacological dosages of recombinant human granulocyte colony-stimulating factor (rhG-CSF). In order to analyze the decreased chemotaxis of neutrophils from SCN patients receiving rhG-CSF, neutrophil functions essential for chemotaxis were investigated. The mobilization of cytosolic calcium ([Ca2+]i) and the functional state of cytoskeletal proteins in neutrophils from SCN patients were compared with either neutrophils from healthy donors (or, in selected experiments, from patients with cyclic neutropenia) and neutrophils from patients with chemotherapy-induced neutropenia also receiving rhG-CSF. Using flow cytometric analysis, two neutrophil subpopulations were detected in SCN patients in response to N-formylmethionine leucyl-phenylalanine (FMLP) (10(-9) M to 10(-7) M), one of which was unable to respond to this stimulus with an increase in [Ca2+]i. Whereas a homogeneous increase in [Ca2+]i in normal neutrophils occurred at 10(-9) M FMLP, neutrophils from SCN patients required 10(-6) M FMLP to respond homogeneously with an increase in [Ca2+]i. In contrast, G-CSF induced neutrophils from patients with cyclic neutropenia and from patients with chemotherapy-induced neutropenia showed a normal increase in [Ca2+]i after stimulation. The [Ca2+]i-dependent superoxide anion (O2-) generation in response to FMLP was also significantly diminished in neutrophils from SCN patients compared to normal neutrophils. However, O2- generation elicited by phorbolester (PMA), which directly activates protein kinase C (PKC), was not affected in SCN neutrophils. The total immunoreactive actin content and basal F-actin content in neutrophils from SCN patients were elevated as compared to normal neutrophils and neutrophils from patients with chemotherapy-induced neutropenia. The increase in F-actin content following FMLP activation was much lower in neutrophils from SCN patients as compared with normal neutrophils. These data suggest a defect in the signal transduction pathway in neutrophils from SCN patients between FMLP ligand-receptor interaction and Ca2+ mobilization, whereas upstream of PKC, triggered events seem to be unaffected. Therefore, [Ca2+]i-dependent neutrophil function in response to FMLP, such as actin disassembly, chemotaxis and O2- generation are diminished in SCN neutrophils. The pathomechanism responsible for the defective [Ca2+]i increase might be an initial step in understanding the underlying pathophysiology of SCN.

Identification of the antigen recognized by the monoclonal antibody 31D8.

The monoclonal antibody (mAb) 31D8 has previously been described to bind more avidly to functionally active neutrophils and proved to be useful as a differentiation marker of neutrophils. However, attempts to further characterize the antigen recognized by 31D8 have not been successful. Studying the altered Fcgamma-receptor expression of human neutrophils induced by granulocyte colony-stimulating factor (G-CSF) in vivo, we could demonstrate a parallel decrease in the expression of 31D8 and the CD16 antigen. Furthermore, 31D8 showed a binding pattern on leukocyte subsets similar to that of clustered CD16 antibodies, exhibiting identical cells in double-staining experiments. Preincubation of neutrophils with 31D8 resulted in a dose-dependent inhibition of the binding of immune complexes. A decreased expression of the 31D8 antigen was found on the same cell clones to the same extent as found for the 3G8 antigen on neutrophils from patient with paroxysmal nocturnal hemoglobinuria (PNH). Treatment of polymorphonuclear leukocytes (PMN) with PIPLC resulted in a dose-dependent decrease of mAb 31D8 binding, showing that the 31D8 antigen is phosphatidylinositolglycan (PIG)-anchored. Moreover, 31D8 competed with the binding of antibodies (such as mAb 3G8) directed against the binding site of FcgammaRIII for the Fc-part of IgG. However, this mAb did not influence the binding of a CD16 antibody (mAb B73.1) which recognizes an epitope elsewhere on the CD16 antigen. We conclude from our experiments that the mAb 31D8 binds with high avidity to fcgammaRIII (CD16 antigen). Furthermore, our data indicate that its binding site is most probably located at the binding site for the Fc-part of IgG.

The interferon-inducible GBP1 gene: structure and mapping to human chromosome 1.

We have isolated DNA clones containing the interferon (IFN)-inducible guanylate-binding protein-1-encoding gene (GBP1) from a human genomic library. Two overlapping phage clones contained the entire GBP1 gene. The 2880 bp corresponding to the GBP1 cDNA were subdivided into eleven exons which were interrupted by a total of 9500 bp of intron DNA. All exon/intron junctions contained consensus splice donor and acceptor sequences. Using hybrid rodent cell lines containing human chromosomes, GBP1 was mapped to human chromosome 1. In addition to GBP1, we detected and partially characterized a novel gene, GBP3, with a structure related to GBP1 and a high degree of sequence homology to both GBP1 and GBP2. Our data suggest that the GBP family of genes contains members other than the previously characterized GBP1 and GBP2.

The capacity to produce IFN-gamma rather than the presence of interleukin-4 determines the resistance and the degree of susceptibility to Leishmania donovani infection in mice.

The immune response against Leishmania donovani infection has been investigated in one resistant mouse strain (C3H/HeJ) and three susceptible mouse strains (C57BL/6, BALB/c, and B10D2/n). In order to correlate the strain-specific course of infection with the individual T cell response phenotype, the ex vivo cytokine secretion patterns of splenic lymphocytes were assessed by ELISA (interferon-y [IFN-gamma], interleukin-4 [IL-4], IL-10) or by bioassay (IL-2). The strain-dependent differences in the course of infection correlated closely with the potency of T cells to produce IFN-gamma. C3H/HeJ mice produced high amounts of IFN-gamma before and during infection, whereas susceptible mice produced low amounts of IFN-gamma early during L. donovani infection. However, C57BL/6 mice, which recovered from the infection rapidly after the acute stage, developed marked IFN-gamma response within the first 30 days of infection. In contrast, in BALB/c and B10D2/n mice, the IFN-gamma production diminished during the acute stage, and this was associated with a delay in recovery and with subsequent switching into the chronic stage. Interestingly, CD8+ T cells contributed significantly to IFN-gamma production during this phase. In contrast to IFN-y, the levels of IL-4 in response to antigen or mitogen ex vivo were always very low. Moreover, neutralization of endogenous IL-4 in vivo by treatment with soluble murine IL-4 receptor did not result in significant decreases in the parasite burdens in spleen and liver but did cause a decrease in the serum IgE level of L. donovani-infected BALB/c mice. These results confirm that in visceral leishmaniasis a Thl-dominated immune response is protective against the L. donovani parasites and, furthermore, that the capacity to produce IFN-gamma rather than the presence of IL-4 determines the efficacy of the immune response in susceptible mice. The data show that CD8+ T cells represent an important source of IFN-gamma during L. donovani infection in susceptible mice, implying a role for this cell type in healing and development of protective immunity.

A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity.

A simple way of measuring and evaluating lactate dehydrogenase release from lysed tumor cells is described. LDH activity was determined as NADH oxidation or INT reduction over a defined time interval, which was limited by stopping the enzymatic reaction with the inhibitor oxamate. Reaction products were then assayed using a microplate reader. The principle of measuring LDH activity of cellular culture supernatants as a measure of cytotoxicity was successfully applied to a number of murine and human effector-target cell combinations (macrophages, monocytes, NK cells and cytotoxic T cells with P815, A375, K562 and Yac-1 tumor cells) as well as to the determination of TNF activity on L929 cells. Comparison with 51Cr release assays suggests that LDH release assays are an appropriate and possibly preferable means of measuring cellular cytotoxic reactions. This LDH release assay combines the advantages of reliability and simple evaluation characteristic of radioisotope release assays with the convenience of speed and avoidance of radioactivity.

A new and rapid method for immunoglobulin class and subclass determination of mouse monoclonal antibodies using a solid-phase immunoradiometric assay.

A solid-phase immunoradiometric assay is described for the detection of mouse immunoglobulin classes and subclasses in unpurified and unconcentrated supernatants of hybridomas. IgG fractions from rabbit antisera specific for mouse immunoglobulin classes and subclasses are used for coating the wells of flexible microtiter plates. Monoclonal antibody present in hybridoma supernatants is bound only to wells that contain the appropriate anti-subclass antibody. The binding of hybridoma antibodies to corresponding IgG subclasses or IgM is then detected by a labeled rabbit anti-mouse antibody binding to all mouse immunoglobulins (heavy and light chains). Thus, only 1 labeled antibody is needed for all assays. The advantages of the method described are the following: results are obtained within a few hours and antibody containing hybridoma supernatants may be used without a concentration step since minute amounts of antibody are detected by the immunoradiometric assay. Cultures producing several subclasses may be early recognized as oligo/polyclonal.

A fast and objective assay for cell mediated intra- and extracellular killing of Leishmania promastigotes.

An in vitro radiometric assay is described for the detection of cytolytic activity against the obligate intracellular parasite Leishmania enriettii. The assay system can be equally well applied to non-cellular (humoral), cellular non-phagocytic, and cellular phagocytic effector situations. Leishmania promastigote organisms are DNA-labeled with [methyl-3H]thymidine [( 3H]dThd) with high efficiency. Spontaneous label release remains very low even in non-ideal culture conditions. We have modeled a leishmanicidal situation by co-cultivating L. enriettii with starch-elicited murine peritoneal macrophages for different time periods. At an effector-to-target ratio of 1:6 a highly significant [3H]dThd release can be observed after 4 h of co-cultivation, reaching 60% after 18 h. The principle advantages of this assay system are speed, high sensitivity and objectivity. This makes it suitable for mass screening of, e.g., immunomodulatory or parasiticidal agents and equally useful in both phagocytic and non-phagocytic situations.