Peripheral Blood Leukocyte Cultures Research Articles

Synthesis of Selenolo[2,3‐b]quinoline‐2‐carboxylic Ethyl Esters (III): Cytogenetic Studies on Human Peripheral Blood Leucocyte Cultures, and Antibacterial Studies, and Antifungal Studies of Their Effects.

Synthesis of Selenolo[2,3‐b]quinoline‐2‐carboxylic Ethyl Esters (III): Cytogenetic Studies on Human Peripheral Blood Leucocyte Cultures, and Antibacterial Studies, and Antifungal Studies of Their Effects.

Loss of 10p material in a child with human papillomavirus–positive disseminated bilateral retinoblastoma

Retinoblastoma (RB) is a malignant childhood tumor that results from loss or inactivation of both alleles of the RB1 gene. Human papillomavirus (HPV) DNA sequences have been found in RB tissue, suggesting a role of the viral infection with RB. We here describe a child with disseminated bilateral RB without familial history, who displayed a loss of material from 10p. Fluorescence in situ hybridization studies showed a somatic loss of both alleles of the RB1 gene. Moreover, sequences for HPV-6a were detected on DNA extracted from eye tumor tissue and from nonstimulated peripheral blood leukocyte cultures. The eye tumor tissue was also positive for HPV L1 viral proteins. Repeated loss of the short arm of chromosome 10 in HPV-transfected keratinocytes has been reported. Loss of heterozygosity in 10p14∼p15 is also frequent in cervical cancers. Therefore, it seems probable that the abnormalities on 10p detected in the present case are related to the HPV infection. Thus, HPV could be a cofactor in the progression of RB by promoting nonrandom additional mutations.

Synthesis of Selenolo(2,3-b)quinoline-2-carboxylic Ethyl Esters: Cytogenetic Studies on Human Peripheral Blood Leucocyte Cultures, and Anti-Bacterial Studies, and Anti-Fungal Studies of Their Effects

The compounds selenolo(2,3-b)quinoline-2-carboxylic ethyl esters were synthesized in good yields by the reaction of 3-(2-chloro-3-quinolyl)acrylic ethyl esters, with the nucleophilic reagent sodium diselenide in ethanol medium under a nitrogen atmosphere. Cytogenetic studies on human blood leucocytes in vitro were evaluated for some of the synthesized compounds. Most of the synthesized compounds were tested for their antibacterial and antifungal activities.

Lindane increases macrophage-activating factor production and intracellular calcium in rainbow trout ( Oncorhynchus mykiss) leukocytes

The authors studied the in vitro effects of lindane on macrophage-activating factor (MAF) production by peripheral blood leukocytes (PBLs) in rainbow trout. MAF production by PBLs induced normally by mitogens concanavalin A (ConA) and phorbol myristate acetate (PMA) was not modified by a pretreatment with lindane (from 2.5 to 50 μM ). Only a concentration of 100 μM lindane decreased MAF production, associated with cellular death. Moreover, MAF activities were detected in supernatants of PBL cultures treated with lindane from 2.5 to 10 μM in absence of ConA/PMA stimulation. Factors present in these supernatants remain to be identified. Lindane, at concentrations which did not induce MAF production (50 and 100 μM ) led to an increase in PBL calcium levels by acting on the endoplasmic reticulum calcium stores. Although the intracytosolic calcium concentration ([Ca 2+] i) increase seems to be associated with cell death, lindane-induced MAF production may be linked with other intracellular mechanisms.

Erythrocyte-rosetting properties of feline blood lymphocytes and their relationship to monoclonal antibodies to T lymphocytes.

Rosette formation of feline peripheral blood leukocytes with guinea pig (GP) and gerbil (G) erythrocytes (E) has been shown in an earlier study to identify T lymphocytes expressing helper and suppressor cell activity, respectively. This T lymphocyte distinction was based on the removal of the E-rosetting populations from peripheral blood leukocytes (PBL) and the subsequent functional evaluation of the remaining cells in a pokeweed mitogen (PWM)-induced synthesis of immunoglobulin (Ig). In the present study, we demonstrate a direct helper and suppressor function of GPE- and GE-rosetted cells, respectively, wherein the induction of Ig synthesis is altered in a positive or negative way by the addition of the cells to a control target population. A pan-T monoclonal antibody (mAb), CT843, and mAbs to the CD4 (CT248) and CD8 (CT87) subsets are also described; their specificities are established in functional assays, the PWM-induced Ig synthesis and the production of interleukin-2 following Concanavalin A stimulation of PBL, and a biochemical analysis of the surface membrane antigens detected by the mAbs. Immunoprecipitation and SDS-PAGE analyses showed CT248 to react with a approximately 60-kDa protein under both reducing and nonreducing conditions. Under reducing conditions, CT87 reacted with one subunit at approximately 35 kDa; a second faint band at approximately 39 kDa was poorly resolved. mAb CT843 detected a heterodimer of approximately 70 and approximately 60 kDa under both reducing and nonreducing conditions. The relationship of the mAbs to E-rosetting was examined in FACScan analyses and rosette inhibition studies. The percentage of GE-rosetting cells agreed with the percentage of cells stained with the CD8 mAb, whereas a comparison of GPE-rosetting and staining with the CD4 mAb showed variability. The binding of GE to PBL was blocked by pretreatment of PBL with the CD8 mAb, whereas no inhibition of GPE rosettes was observed with any of the mAbs. In a previous study, we had shown that an overnight culture of feline PBL at 37 degrees C leads to the development of a second population of GPE-rosetting cells, also having a helper function. The relationship of the two GPE-rosetting populations to the CD4 mAb, CT248, was examined in rosette depletion studies and FACScan analyses. It was found that depletion of the GPE-rosetting cells from fresh, i.e., Day 0 cells, removed only a small percentage of cells reactive with the CD4 mAb, whereas GPE-rosette depletions performed on Day 1 PBL, which contained both populations of GPE-rosetting cells, removed almost all cells reactive with this antibody. The latter study suggests that the GPE-rosetting phenomenon is detecting two subsets of CD4 cells with T helper function, those present in fresh blood and those acquiring the GPE receptor after an overnight culture.

Non-Redundant Roles for Interleukin-1α and Interleukin-1β in Regulating Human IgG2.

Serum concentrations of immunoglobulin G2 (IgG2) are elevated in localized aggressive periodontitis (LAgP) patients, and secretory products of monocytes from LAgP patients enhance IgG2 responses of lymphocytes from healthy subjects. Furthermore, genes regulating production of interleukin (IL)-1 influence the risk for both aggressive periodontitis (AgP) and chronic periodontitis. These observations, and the fact that IgG2 dominates responses to carbohydrates from Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis, prompted the hypothesis that IL-1α, IL-1β, and IL-RA may help regulate human IgG2 responses. Human peripheral blood leukocytes (PBL) were stimulated in culture with pokeweed mitogen (PWM); the levels of available IL-1 gene products were manipulated; and the effect on IgG2 production was monitored. Manipulations of IL-1 were accomplished by adding specific neutralizing monoclonal antibodies or recombinant IL-1RA, IL-1α, or IL-1β. Blocking the IL-1 receptor with IL-1RA or neutralizing IL-1α or IL-1β with specific antibody dramatically suppressed IgG2 production (50% to 70%). Additional IL-1α did not compensate for neutralized IL-1β, and additional IL-1β did not compensate for neutralized IL-1α, suggesting the 2 monokines have separate roles in promoting IgG2. Furthermore, combinations of anti-IL-1α and anti-IL-1β were more inhibitory than either antibody alone, and IL-1α and IL-1β in combination appeared to work additively in promoting IgG2. Moreover, PBL cultures from a group of LAgP patients with high IgG2 levels had elevated levels of IL-1β. IL-1α and IL-1β appear to have critical and nonredundant roles in the generation and regulation of potent IgG2 responses, which appear to be important in human responses to carbohydrate-bearing bacteria. J Periodontol 2001;72:1332-1339.

Prophylaktische Wirkungen von Propolis zur Immunstimulation: Eine klinische Pilotstudie

Objective: The aim of this pilot investigation was to show the evidence of the prophylactic immunostimulating effectiveness caused by propolis. The immune response was determined by the measurement of the cytokine level in vivo and ex vivo (TNF-α, IL-6, IL-8). Study Design:In an open prospective monocentric study, test persons received Propolis XNP. Patients: 10 healthy test persons aged between 18 and 45 years, with normal weight and of either sex. Interventions:Probands received over 13 days 500 mg Propolis XNP (2 capsules) for peroral application in the morning. Main Outcome Measures: Significant changes of the investigated cytokine secretion capacity during and after the treatment of propolis compared to the situation without medication. Results:Although the cytokine plasma levels did not significantly change during the study, propolis led to a significant increase of both the spontaneous (TNF-α, p < 0.05; IL-6, p < 0.01; IL-8, p < 0.02; IL-1β, not detectable) and LPS(lipopolysaccaride)-induced (TNF-α, p < 0.001; IL-6, p < 0.02; IL-8, p < 0.0005; IL-1β, p < 0.05) cytokine secretion capacity following short-term ex vivo culture of peripheral blood leukocytes. Whereas the IL-6 secretion capacity further increased during the 13-day application, the IL-8 and TNF-α secretion reached a plateau after day 4 and the TNF-α secretion even decreased, but the level at day 13 was still significantly higher than at day 0. Conclusion: As the cytokine secretion capacity but not the cytokine plasma levels increased significantly during therapy, the prophylactic application of propolis led to a time dependent enhanced immune reactivity without undesired side effects.

Sequential production of cytokines by dengue virus-infected human peripheral blood leukocyte cultures.

The study was undertaken to elucidate the sequence of appearance of T helper (Th)1- and Th2-type cytokines in human peripheral blood leucocyte cultures infected in vitro with dengue type 2 virus. Commercial sandwich enzyme-linked immunosorbent assay kits were used to assay the levels of tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin (IL)-2, IL-4, IL-5, IL-6, and IL-10 in culture supernatants. Culture supernatants were also screened for the cytotoxic factor and the dengue virus titres determined. The cytokines that appeared in the culture supernatants on the first day post-infection (p.i.) were cytotoxic factor, TNF-alpha, IL-2, and IL-6; their levels were highest on the second day p.i. IFN-gamma appeared on the second day with a peak on the third day p.i. The levels of these cytokines declined quickly, except for human cytotoxic factor (hCF) and IL-2. The cytokines that appeared later were IL-10 and IL-5 on the fourth day and IL-4 on the sixth day p.i. Dengue virus replicated in the peripheral blood leucocyte (PBL) cultures and was present throughout the course of the study. The findings of the present study show that dengue virus induced a predominant Th1-type cytokine response during the first 3 days of infection of PBL cultures that was replaced by a Th2-type response later.

Enhancing the immunotherapeutic potential of mycobacteria by transfection with tumour necrosis factor-alpha.

In an attempt to enhance the anti-tumour properties of mycobacteria we have developed recombinant forms of Mycobacterium smegmatis which express and secrete biologically active human tumour necrosis factor-alpha (TNF-alpha). This was achieved by transfecting M. smegmatis using shuttle plasmids incorporating the cDNA sequence for the human TNF-alpha mature peptide. In vitro experiments on a panel of human bladder tumour cell lines (EJ18, MGH-U1, RT4, RT112) indicate that our genetically modified mycobacteria are more effective than wild-type at inducing or up-regulating the expression of intracellular adhesion molecule-1 and the secretion of an array of proinflammatory cytokines [interleukin-1 (IL-1), IL-6, IL-8, granulocyte-macrophage colony-stimulating factor]. We have also demonstrated increased adhesion molecule and cytokine expression in response to mycobacteria transfected with vector containing no gene insert. However, this was not as pronounced as that observed following tumour cell stimulation by the TNF-alpha-transfected strain. In contrast, in three out of four tumour cell lines all M. smegmatis strains were found to down-regulate the secretion of the anti-inflammatory cytokine transforming growth factor-beta1. Our studies have also confirmed that M. smegmatis is a powerful inhibitor of bladder tumour cell growth and revealed that its antiproliferative potency is enhanced by transfecting with human TNF-alpha and, to a lesser extent, with vector alone. All M. smegmatis strains were effective in the activation of peripheral blood leucocyte cultures. However, no differences were observed in the ability of the TNF-alpha-transfected, mock-transfected and wild-type mycobacteria to induce tumour cell killing activity. These results suggest that the immunomodulatory effects of M. smegmatis can be enhanced by transfection with vectors which allow the secretion of human TNF-alpha, thus increasing mycobacterial immunotherapeutic potential.

Histamine and Eicosanoid Levels in Plasma and Peripheral Blood Leucocyte Cultures During Experimental Infection of Cattle with Bluetongue Virus Serotype 11

Three calves were sensitized with three doses of inactivated BTV-11 UC8 strain and then experimentally infected with the homologous virus. In addition, four BTV-seronegative heifers were also experimentally infected with BTV-11. Granulocyte rich fractions of peripheral blood leucocyte (PBL-GRF) cultures from BTV-sensitized/infected calves and from control unexposed cattle were exposed in vitro with BTV-11. Histamine, leukotriene (LT) C4 and prostaglandin (PG) D2 were assayed in supernatant fluids. Plasma histamine levels increased in BTV-infected heifers from 10.1 +/- 2 ng/ml at Day 0 to 23.1 +/- 6.6 ng/ml at Day 12 following virus exposure. In addition, in this experimental group the concentration of PGF2 alpha (mean 551.97 +/- 243.54 pg/ml) increased significantly (P < or = 0.05) compared with control cattle (mean 467.3 +/- 73.9 pg/ml). Bluetongue virus induced histamine and LTC4 release after in vitro infection of PBL-GRF. Release of LTC4 was significantly (P < or = 0.05) higher in PBL-GRF cultures from sensitized and control animals than in unexposed PBL-GRF cultures. In contrast to these results, PGD2 was not released after BTV infection of PBL-GRF in vitro. The histamine release caused by BTV was virus-specific and mainly mediated by an immunological reaction, since the release was significantly reduced by removal of cell surface immunoglobulins.

Synthesis and Properties of 2‐Carboxyalkyl‐1,2‐benzisoselenazol‐3(2H)‐ones and Related Organoselenium Compounds as Nitric Oxide Synthase Inhibitors and Cytokine Inducers

AbstractA convenient synthesis of the 2‐carboxyalkyl‐1,2‐benzisoselenazol‐3(2H)‐ones 4a–k and their esters 41–p from 2‐(chloroseleno)benzoyl chloride (2) and amino acids or their carboxy esters is reported. In similar way other 2‐substituted 1,2‐benzisoselenazol‐3(2H)‐ones 4q–u were synthesized. The related bis[2‐(carbamoyl)phenyl] diselenides 5 were obtained by reductive conversion of 1,2‐benzisoselenazol‐3(2H)‐ones 4 or directly by the reaction of bis[2‐(chlorocarbonyl)phenyl] diselenide (3) with compounds having a primary amino group. It was found that some of compounds 4 and 5 are modest cytokine (TNF, IFN) inducers in human peripheral blood leucocyte cultures and block the constitutive endothelial nitric oxide synthase (ce NOS).

Effects of products of activated immune cells and recombinant cytokines on spontaneous and ionophore-induced acrosome reaction.

The purpose of this study was to examine whether the products of activated immune cells influence spontaneous and ionophore-induced sperm acrosome reaction. The spontaneous and ionophore-induced acrosome reaction were evaluated by staining with fluorescein isothiocyanate (FITC) Pisum sativam agglutinin after incubation in capacitating media supplemented with either supernatants from Con-A activated leukocyte cultures or human recombinant (r) IL-1 beta, TNF-alpha, and INF-gamma. The supernatants from Con A-activated peripheral blood leukocyte cultures at 1:1 and 1:10 dilution significantly increased the rate of spontaneous acrosome reaction (P < 0.001 and P < 0.01). Along with displayed abnormally elevated levels of spontaneous acrosome loss, sperm cells showed an insufficient ability to undergo acrosome reaction in response to the ionophore treatment. Recombinant IL-1 beta at increasing concentrations from 30 to 3 x 10(4) U/ml did not have an effect on spontaneous and ionophore-induced acrosome reaction. In contrast, spermatozoa that underwent capacitation in media with 7 x 10(3), 7 x 10(4), and 7 x 10(5) U/ml of rINF-gamma showed a significant increase in spontaneous and induced acrosome reaction compared to the control (P < 0.001). Recombinant TNF-alpha at concentrations of 3.5 x 10(3) U/ml and 3.5 x 10(4) U/ml significantly inhibited ionophore-induced acrosome reaction (P < 0.001). Both rINF-gamma and rTNF-alpha together revealed an effect on the acrosome reaction similar to Con-A generated supernatants (1:1 and 1:10 dilution) only at the highest concentrations. Some cases of infertility may result from a defective acrosome reaction (premature acrosome loss or insufficient acrosome response to the stimulants) caused by products of activated lymphocytes and macrophages that are released into the male and female reproductive tracts.

Virus-induced interferon production in leukocyte cultures from children with recurrent respiratory infections. A follow-up study

Background: Lowered yields of virus-induced interferon (IFN) by leukocyte cultures were previously suggested to be associated with recurrent respiratory infections in children (Pitkäranta et al. (1993) Clin. Diagn. Virol. 1, 101–108) Objectives: To investigate if the observed lowered IFN producing capacity was secondary to the underlying disease and, consequently, would be normalized after recovery of the child from the chain of infections. Study design: Forty-eight 3–12-year-old children suffering from recurrent upper respiratory tract infections (acute otitis media included) were followed-up for 2 years. Their clinical condition and virus-induced interferon production in cultures of peripheral blood leukocytes were examined at the beginning and end of this period. Results: In 24 children the health improved strikingly during the follow-up, in 12 children a mild improvement took place, while 12 children remained constantly ill. IFN yields in cultures stimulated with corona- and respiratory syncytical viruses improved along with the clinical situation of the children. Parallel cultures induced with adeno-, influenza A or rhinoviruses did not show a similar correlation. Conclusion: These results suggest that the relationship between interferon production by leukocyte cultures and recurrent infections is complex and may be virus-specific.

Anti-HIV michellamines from Ancistrocladus korupensis.

Here we report details of the isolation and determination of the absolute configurations and comparative anti-HIV activities of novel, atropisomeric naphthylisoquinoline alkaloid dimers, michellamines A, B, and C, from a newly described species of Ancistrocladus from the Korup rainforest of Cameroon. We further provide a more extensive analysis of the range of anti-HIV activity of michellamine B, the most potent and abundant member of the series. Michellamine B inhibited HIV-induced cell killing and viral replication in a variety of human cell lines, as well as in cultures of human peripheral blood leukocytes and monocytes. Michellamine B was active against a panel of biologically diverse laboratory and clinical strains of HIV-1, including the AZT-resistant strain G910-6 and the pyridinone-resistant strain A17; the compound also inhibited several strains of HIV-2.

Selective induction of PDGF gene expression in peritoneal macrophages by interleukin-2

We have previously reported that culture of human peripheral blood leukocytes with interleukin-2 (IL-2) triggers the secretion of mediators which induce fibroblast proliferation and collagen synthesis. In addition, fibrogenic cytokines (transforming growth factor-β 1 (TGFβ 1) and platelet-derived growth factor (PDGF) A and B chain) are present in the peritoneal fluid of patients undergoing intraperitoneal immunotherapy (IL-2-activated killer cells and IL-2) who go on to develop peritoneal adhesions. To determine the role of IL-2 in the formation of these adhesions, we chose to investigate whether IL-2 can induce the expression of fibrogenic cytokine genes in resident rat peritoneal macrophages. Cells were cultured with or without IL-2 or lipopolysaccharide (LPS) and expression of PDGF A chain, PDGF B chain, and TGFβ 1 mRNAs was determined. PDGF A and B chain mRNAs are minimally expressed in macrophages prior to stimulation and are induced within 2 hours of treatment with IL-2. In contrast, TGFβ 1 mRNA is constitutively expressed and can not be upregulated. The studies suggest that peritoneal macrophage-derived PDGF plays a critical role in the production of adhesions in patients receiving intra-abdominal immunotherapy.

Aromatic and Azaaromatic Diselenides, Benzisoselenazolones and Related Compounds as Immunomodulators Active in Humans: Synthesis and Properties

AbstractConvenient syntheses of aryl diselenides 2, 1,2‐benzisoselenazol‐3(2H)‐ones 4 and their 1‐oxides 7 are reported. Reductive conversions of these compounds to bis(2‐carbamoyl)‐phenyl diselenides 5 and oxidative cyclization of 5 to 1,2‐benzisoselenazol‐3(2H)‐one 1‐oxides 7 as the methods for the synthesis of these compounds are reported. Their ability to induce cytokines, such as TNF and IFN‐γ, in human peripheral blood leucocyte cultures is described.

IL-4 Inhibits IL-2 Induction of LAK Cytotoxicity in Lymphocytes from a Variety of Lymphoid Tissues

The purpose of this study was to determine the influence of recombinant human interleukin-4 (IL-4) on the regulation of lymphokine-activated killer (LAK) and mixed lymphocyte culture cytotoxic activity. Lymphocytes from several lymphoid tissues were studied including human peripheral blood leukocytes (PBL), spleen cells, thymocytes, and thoracic duct lymphocytes. Cells were cultured with IL-4 in the presence or absence of recombinant human interleukin-2 (IL-2) in 4-day cultures. LAK and natural killer (NK) activities were measured in a standard chromium release cytotoxicity assay against LAK-sensitive, NK-resistant M14 melanoma targets and NK-sensitive K562 erythroleukemic cells. IL-4 alone does not increase NK or LAK activity under the conditions studied. However, IL-4 does inhibit the induction of cytotoxic activity by IL-2. IL-4 inhibits IL-2-induced thymidine incorporation in 3-day PBL cultures, suggesting that the inhibition of cytotoxicity is not a dilution effect due to the proliferation of noncytotoxic cell populations. IL-4 also inhibits the development of LAK and NK-like activities generated in mixed lymphocyte culture (MLC) while augmenting MLC-generated allospecific cytotoxic T lymphocyte activity. Thus, IL-4 appears to inhibit the induction of nonspecific cytotoxic effectors while augmenting the generation of MHC-specific responses. This confirms an important regulatory function for this lymphokine in the generation of cytotoxic effectors.

Diarylsulfones, a new chemical class of nonnucleoside antiviral inhibitors of human immunodeficiency virus type 1 reverse transcriptase

A series of variously substituted diarylsulfones and related derivatives were found to prevent human immunodeficiency virus type 1 (HIV-1) replication and HIV-1-induced cell killing in vitro. One of the more potent derivatives, 2-nitrophenyl phenyl sulfone (NPPS), completely protected human CEM-SS lymphoblastoid cells from the cytopathic effects of HIV-1 in cell culture at 1 to 5 microM concentrations. HIV-1 replication, as assessed by the production of infectious virions, viral p24 antigen, and virion reverse transcriptase (RT), was inhibited by NPPS at similar concentrations. There was no evidence of direct cytotoxicity of the drug at concentrations below 100 microM. A variety of other CD4+ T-cell lines as well as cultures of peripheral blood leukocytes and monocytes were protected from HIV-1-induced cytopathicity and/or viral replication. NPPS also inhibited several distinctly different strains of HIV-1 but was ineffective against three strains of HIV-2. Biochemical studies revealed that NPPS inhibited HIV-1 RT but not HIV-2 RT. NPPS had no direct effect on HIV-1 virions, nor did it block the initial binding of HIV-1 to target cells. Time-limited treatments of cells with NPPS found that NPPS had to be present continuously in culture to provide maximum antiviral protection. In addition, HIV-1 replication in cells in which infection was already fully established or in chronically infected cells was also unaffected by NPPS. We conclude that NPPS acts in a reversible manner as a nonnucleoside HIV-1-specific RT inhibitor. Although markedly different in structure from a larger, structurally diverse group of known HIV-1-specific nonnucleoside RT inhibitors, NPPS shares several of the biological properties that characterize this emerging new pharmacologic class.

Measles virus-induced changes in leukocyte function antigen 1 expression and leukocyte aggregation: possible role in measles virus pathogenesis.

Measles virus (MV) infection of U937 cell or peripheral blood leukocyte cultures was shown to induce changes in the expression of leukocyte function antigen 1 (LFA-1) and cause marked aggregation of these cells. Addition of selected monoclonal antibodies specific for LFA-1 epitopes that did not neutralize MV in standard neutralization assays were found to block both virus-induced leukocyte aggregation and virus dissemination. These data suggest that MV modulation of LFA-1 expression on leukocytes may be an important step in MV pathogenesis.

Evidence of embryo- and trophoblast-toxic cellular immune response(s) in women with recurrent spontaneous abortion

The purpose of this study was to determine whether reproductive antigens stimulate lymphocytes and macrophages from women with recurrent abortion to secrete factors that are toxic to preimplantation embryos or trophoblast cells in vitro. Mononuclear cells were isolated from 30 fertile controls and 300 nonpregnant women being evaluated for recurrent abortion. Supernatants generated from these cells after separate culture with sperm and trophoblast antigen extracts were added to two-cell mouse embryo cultures and trophoblast proliferation assays. Toxicity was assumed when the median percentage of embryos developing to blastocysts or trophoblast proliferation was less than or equal to 50% of control values. Both parametric and nonparametric statistical methods were used where appropriate. Mouse embryo development and/or trophoblast proliferation were significantly inhibited by supernatants from trophoblast and/or sperm antigen-activated peripheral blood leukocyte cultures from a majority of 300 women with recurrent abortion but not from 30 women with normal reproductive histories. The mouse blastocyst development assay was more sensitive than the trophoblast proliferation assay in determining toxic factor production. Embryo-toxic factors were produced by activated leukocyte cultures from 90% of 180 women with a history of recurrent abortion of unexplained etiology, whereas trophoblast-inhibitory factors were detected in 50% of women from the same group. The embryo-toxic factor(s) was heat labile, had a molecular weight(s) between 10 and 30 kd, and was absorbed out by passage through affinity columns containing anti-interferon gamma beads. We conclude that recurrent abortion in some women is associated with embryo- and/or trophoblast-toxic factor production in response to stimulation by sperm or trophoblast antigens and that the principal factor may involve the 18 kd, heat-labile, T-lymphocyte cytokine interferon gamma. This study suggests a new cause of recurrent abortion.