Altered phenotype and function of dendritic cells in individuals with chronic periodontitis.

OBJECTIVE: To investigate the effects of periodontal bacterial lysates on maturation and function of mature monocyte-derived dendritic cells (m-MDDCs) derived from individuals with chronic periodontitis (CP) or healthy periodontal tissue (HP). DESIGN: m-MDDCs derived from peripheral blood monocytes, cultured for 7 days in presence of interleukin (IL)-4 and granulocyte-macrophage colony stimulating factor (GM-CSF), were stimulated with lysates of Streptococcus sanguinis, Prevotella intermedia, Porphyromonas gingivalis, or Treponema denticola on day 4, and were then phenotyped. IL-10, IL-12 and IFN-gamma concentration in the supernatant of cultures were measured. RESULTS: Expression of HLA-DR was lower in bacterial-unstimulated mature m-MDDC from CP compared to HP (p=0.04), while expression of CD1a and CD123 were higher in CP. The expression pattern of HLA-DR, CD11c, CD123, and CD1a did not change on bacterial stimulation, regardless of the bacteria. Stimulation with P. intermedia upregulated CD80 and CD86 in CP cells (p≤0.05). Production of IL-12p70 by bacterial-unstimulated m-MDDCs was 5.8-fold greater in CP compared to HP. Bacterial stimulation further increased IL-12p70 production while decreasing IL-10. Significantly more IFN-gamma was produced in co-cultures of CP m-MDDCs than with HP m-MDDCs when cells were stimulated with P. intermedia (p=0.009). CONCLUSIONS: Bacterial-unstimulated m-MDDC from CP exhibited a more immature phenotype but a cytokine profile biased towards proinflammatory response; this pattern was maintained/exacerbated after bacterial stimulation. P. intermedia upregulated co-stimulatory molecules and IFN-gamma expression in CP m-MDDC. These events might contribute to periodontitis pathogenesis.

Continuous and high-level in vivo delivery of endostatin from recombinant cells encapsulated in TheraCyte immunoisolation devices.

Endostatin (ES) is a potent inhibitor of angiogenesis and tumor growth. Continuous ES delivery of ES improves the efficacy and potency of the antitumoral therapy. The TheraCyte system is a polytetrafluoroethylene (PTFE) semipermeable membrane macroencapsulation system for implantation of genetically engineered cells specially designed for the in vivo delivery of therapeutic proteins, such as ES, which circumvents the problem of limited half-life and variation in circulating levels. In order to enable neovascularization at the tissues adjacent to the devices prior to ES secretion by the cells inside them, we designed a scheme in which empty TheraCyte devices were preimplanted SC into immunodeficient mice. Only after healing (17 days later) were Chinese hamster ovary cells expressing ES injected into the preimplanted devices. In another model for device implantation, the cells expressing ES where loaded into the immunoisolation devices prior to implantation into the animals, and the TheraCyte were then immediately implanted SC into the mice. Throughout the 2-month study, constant high ES levels of up to 3.7 microg/ml were detected in the plasma of the mice preimplanted with the devices, while lower but also constant levels of ES (up to 2.1 microg/ml plasma) were detected in the mice that had received devices preloaded with the ES-expressing cells. Immunohistochemistry using anti-ES antibody showed reaction within the device and outside it, demonstrating that ES, secreted by the confined recombinant cells, permeated through the membrane and reached the surrounding tissues.

Cohabitation with a B16F10 melanoma-bearer cage mate influences behavior and dendritic cell phenotype in mice.

This study evaluated the effects of cohabitation with a B16F10 melanoma-bearer cage mate on behavior and immune functions in mice. Five different experiments were conducted. In each of them, the female mice were divided into two groups: control and experimental. One mouse of each control pair was kept undisturbed and called "companion of health partner" (CHP). One mouse of each experimental pair was inoculated with B16F10 cells and the other, the subject of this study, was called "companion sick partner" (CSP). On Day 20 of cohabitation, behavior and immune parameters from CHP and CSP mice were analyzed. In comparison to the CHP, the CSP mice: (1) presented an increased general locomotion in the open field and a decreased exploration time and number of entries in the plus-maze open arms; (2) had an enhanced expression of the CD80 costimulatory molecule on Iab(+)CD11c(+) spleen cells, but no differences were found on lymph nodes cells; (3) presented an altered differentiation of bone marrow cells in the presence of GM-CSF, IL-4, and LPS in vitro, resulting in a lower percentage of Iab(+)CD80(+) cells; (4) had a deficit in the establishment of a Delayed Type of Hypersensitivity to ovalbumin, which was associated to an in vitro proliferation of an IL-10-producing lymphocyte subpopulation after ovalbumin stimulation. Corticosterone levels detected on Day 20 of cohabitation were similar in CHP and CSP mice. It is shown here that DCs phenotype in mice is affected by conditions associated with behavioral alterations indicative of an anxiety-like state induced by the cohabitation with a tumor-bearer conspecific. This phenomenon occurred probably through a nondependent corticosterone mechanism.

Local secretion/shedding of tumor-derived CD83 molecules as a novel tumor escape mechanism.

CD83, a characteristic marker of activated dendritic cells, is also expressed by tumor cell lines from various origins and by primary lung cancers. Here, we show that CD83+ tumor cells (from a primary lung cancer and from an established breast cancer cell line) release in their culture supernatants a soluble factor that is able to block, in a dose-dependent manner, CD4+ and CD8+ T cell proliferative responses to allogeneic dendritic cells. This factor was removed from the medium by incubation in anti-CD83 covered plates, indicating that it could be one of the known soluble forms of the CD83 molecule, released in the medium by the cultured tumor cells. This phenomenon, happening in vivo, in the tumor microenvironment, could affect profoundly anti-tumor immune responses and should, therefore, be considered in immunotherapeutic approaches to cancer.

Paullinia cupana Mart var. sorbilis, guaraná, reduces cell proliferation and increases apoptosis of B16/F10 melanoma lung metastases in mice.

We showed that guaraná (Paullinia cupana Mart var. sorbilis) had a chemopreventive effect on mouse hepatocarcinogenesis and reduced diethylnitrosamine-induced DNA damage. In the present experiment, we evaluated the effects of guaraná in an experimental metastasis model. Cultured B16/F10 melanoma cells (5 x 10(5) cells/animal) were injected into the tail vein of mice on the 7th day of guaraná treatment (2.0 mg P. cupana/g body weight, per gavage) and the animals were treated with guaraná daily up to 14 days until euthanasia (total treatment time: 21 days). Lung sections were obtained for morphometric analysis, apoptotic bodies were counted to calculate the apoptotic index and proliferating cell nuclear antigen-positive cells were counted to determine the proliferation index. Guaraná-treated (GUA) animals presented a 68.6% reduction in tumor burden area compared to control (CO) animals which were not treated with guaraná (CO: 0.84 +/- 0.26, N = 6; GUA: 0.27 +/- 0.24, N = 6; P = 0.0043), a 57.9% reduction in tumor proliferation index (CO: 23.75 +/- 20.54, N = 6; GUA: 9.99 +/- 3.93, N = 6; P = 0.026) and a 4.85-fold increase in apoptotic index (CO: 66.95 +/- 22.95, N = 6; GUA: 324.37 +/- 266.74 AB/mm(2), N = 6; P = 0.0152). In this mouse model, guaraná treatment decreased proliferation and increased apoptosis of tumor cells, consequently reducing the tumor burden area. We are currently investigating the molecular pathways of the effects of guaraná in cultured melanoma cells, regarding principally the cell cycle inhibitors and cyclins.

Paullinia cupana Mart var. sorbilis, guaraná, reduces cell proliferation and increases apoptosis of B16/F10 melanoma lung metastases in mice

We showed that guaraná (Paullinia cupana Mart var. sorbilis) had a chemopreventive effect on mouse hepatocarcinogenesis and reduced diethylnitrosamine-induced DNA damage. In the present experiment, we evaluated the effects of guaraná in an experimental metastasis model. Cultured B16/F10 melanoma cells (5 x 10(5) cells/animal) were injected into the tail vein of mice on the 7th day of guaraná treatment (2.0 mg P. cupana/g body weight, per gavage) and the animals were treated with guaraná daily up to 14 days until euthanasia (total treatment time: 21 days). Lung sections were obtained for morphometric analysis, apoptotic bodies were counted to calculate the apoptotic index and proliferating cell nuclear antigen-positive cells were counted to determine the proliferation index. Guaraná-treated (GUA) animals presented a 68.6 percent reduction in tumor burden area compared to control (CO) animals which were not treated with guaraná (CO: 0.84 ± 0.26, N = 6; GUA: 0.27 ± 0.24, N = 6; P = 0.0043), a 57.9 percent reduction in tumor proliferation index (CO: 23.75 ± 20.54, N = 6; GUA: 9.99 ± 3.93, N = 6; P = 0.026) and a 4.85-fold increase in apoptotic index (CO: 66.95 ± 22.95, N = 6; GUA: 324.37 ± 266.74 AB/mm², N = 6; P = 0.0152). In this mouse model, guaraná treatment decreased proliferation and increased apoptosis of tumor cells, consequently reducing the tumor burden area. We are currently investigating the molecular pathways of the effects of guaraná in cultured melanoma cells, regarding principally the cell cycle inhibitors and cyclins.

High frequency of immature dendritic cells and altered in situ production of interleukin-4 and tumor necrosis factor-alpha in lung cancer.

INTRODUCTION: Antigen-presenting cells, like dendritic cells (DCs) and macrophages, play a significant role in the induction of an immune response and an imbalance in the proportion of macrophages, immature and mature DCs within the tumor could affect significantly the immune response to cancer. DCs and macrophages can differentiate from monocytes, depending on the milieu, where cytokines, like interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) induce DC differentiation and tumor necrosis factor (TNF)-alpha induce DC maturation. Thus, the aim of this work was to analyze by immunohistochemistry the presence of DCs (S100+ or CD1a+), macrophages (CD68+), IL-4 and TNF-alpha within the microenvironment of primary lung carcinomas. RESULTS: Higher frequencies of both immature DCs and macrophages were detected in the tumor-affected lung, when compared to the non-affected lung. Also, TNF-alpha-positive cells were more frequent, while IL-4-positive cells were less frequent in neoplastic tissues. This decreased frequency of mature DCs within the tumor was further confirmed by the lower frequency of CD14-CD80+ cells in cell suspensions obtained from the same lung tissues analyzed by flow cytometry. CONCLUSION: These data are discussed and interpreted as the result of an environment that does not oppose monocyte differentiation into DCs, but that could impair DC maturation, thus affecting the induction of effective immune responses against the tumor.

Expression of a dendritic cell maturation marker CD83 on tumor cells from lung cancer patients and several human tumor cell lines: is there a biological meaning behind it?

The present paper shows, for the first time, the membrane expression of the dendritic cell maturation marker CD83 on tumor cells from lung cancer patients. CD83 was also detected on freshly cultured fibroblast-like cells from these tissues and on several adherent human tumor cell lines (lung adenocarcinomas P9, A459 and A549, melanomas A375 and C81-61, breast adenocarcinomas SKBR-3 and MCF-7 and colon carcinoma AR42-J), but not in the non-adherent MOT leukemia cell line. CD83 may have immunosuppressive properties and its expression by cancer cells could have a role in facilitating tumor growth.

Impaired dendritic cell differentiation and maturation in the absence of C3.

Human monocytes can be differentiated into immature dendritic cells (DCs) in the presence of serum and cytokines. One of the main functions of immature DCs is to capture and process antigens. Following maturation, they differentiate into antigen presenting cells. The role of complement in the differentiation process from monocytes towards immature DCs remains elusive. Here we demonstrate that complement 3 (C3) has a regulatory impact on the expression of specific DC surface molecules and DC-derived cytokine production during DC differentiation. We isolated human adherent peripheral blood mononuclear cells, which were cultured in the presence of GM-CSF plus IL-4 in medium supplemented with normal human serum or C3 deficient serum. The lack of C3 during DC differentiation negatively impacted the expression of C-type lectin receptor DC-SIGN, the antigen presenting molecules HLA-DR and CD1a, and the costimulatory molecules CD80 and CD86. Further, the spontaneous production of IL-6 and IL-12 was reduced in the absence of C3. Moreover, the maturation of immature DCs in response to LPS challenge was impaired in the absence of C3 as evidenced by reduced MHC-II, co-stimulatory molecule expression as well as modulated IL-12 and TNF-alpha production. Collectively, our results provide evidence for a novel role of C3 as a critical cofactor in human DC differentiation and maturation.

Human monocyte-derived dendritic cells are a source of several complement proteins.

OBJECTIVE: Little is known about the role of local production of complement components by dendritic cells (DCs) during the generation of specific immune responses. In this study, we demonstrate that human DCs are an extrahepatic source of several soluble complement proteins. METHODS: Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot were used to evaluate the expression and production of several complement proteins. RESULTS: We show that DCs produce C3, C5, C9, Factor (F)I, FH, FB, FD and properdin at levels similar to macrophages. Treatment of DCs with lipopolysaccharide (LPS) promoted an increase in the expression of C3 and FI mRNAs and a decrease in C5 mRNA, while C9, FH, FB, FD and properdin mRNA levels were not affected. Treatment with interleukin (IL) -1 or dexamethasone induced a modest increase in C3 mRNA levels and did not affect the expression of other complement components. CONCLUSION: DCs are a source of complement proteins whose synthesis may be regulated in response to different inflammatory stimuli.

Absence of peripheral blood mononuclear cells priming in hemodialysis patients.

As a consequence of the proinflammatory environment occurring in dialytic patients, cytokine overproduction has been implicated in hemodialysis co-morbidity. However, there are discrepancies among the various studies that have analyzed TNF-alpha synthesis and the presence of peripheral blood mononuclear cell (PBMC) priming in this clinical setting. We measured bioactive cytokine by the L929 cell bioassay, and evaluated PBMC TNF-alpha production by 32 hemodialysis patients (HP) and 51 controls. No difference in TNF-alpha secretion was observed between controls and HP (859 +/- 141 vs 697 +/- 130 U/10(6) cells). Lipopolysaccharide (5 microg/ml) did not induce any further TNF-alpha release, showing no PBMC priming. Paraformaldehyde-fixed HP PBMC were not cytotoxic to L929 cells, suggesting the absence of membrane-anchored TNF-alpha. Cycloheximide inhibited PBMC cytotoxicity in HP and controls, indicating lack of a PBMC TNF-alpha pool, and dependence on de novo cytokine synthesis. Actinomycin D reduced TNF-alpha production in HP, but had no effect on controls. Therefore, our data imply that TNF-a production is an intrinsic activity of normal PBMC and is not altered in HP. Moreover, TNF-alpha is a product of de novo synthesis by PBMC and is not constitutively expressed on HP cell membranes. The effect of actinomycin D suggests a putative tighter control of TNF-alpha mRNA turnover in HP. This increased dependence on TNF-alpha RNA transcription in HP may reflect an adaptive response to hemodialysis stimuli.

Absence of peripheral blood mononuclear cells priming in hemodialysis patients

As a consequence of the proinflammatory environment occurring in dialytic patients, cytokine overproduction has been implicated in hemodialysis co-morbidity. However, there are discrepancies among the various studies that have analyzed TNF-alpha synthesis and the presence of peripheral blood mononuclear cell (PBMC) priming in this clinical setting. We measured bioactive cytokine by the L929 cell bioassay, and evaluated PBMC TNF-alpha production by 32 hemodialysis patients (HP) and 51 controls. No difference in TNF-alpha secretion was observed between controls and HP (859 ± 141 vs 697 ± 130 U/10(6) cells). Lipopolysaccharide (5 æg/ml) did not induce any further TNF-alpha release, showing no PBMC priming. Paraformaldehyde-fixed HP PBMC were not cytotoxic to L929 cells, suggesting the absence of membrane-anchored TNF-alpha. Cycloheximide inhibited PBMC cytotoxicity in HP and controls, indicating lack of a PBMC TNF-alpha pool, and dependence on de novo cytokine synthesis. Actinomycin D reduced TNF-alpha production in HP, but had no effect on controls. Therefore, our data imply that TNF-alpha production is an intrinsic activity of normal PBMC and is not altered in HP. Moreover, TNF-alpha is a product of de novo synthesis by PBMC and is not constitutively expressed on HP cell membranes. The effect of actinomycin D suggests a putative tighter control of TNF-alpha mRNA turnover in HP. This increased dependence on TNF-alpha RNA transcription in HP may reflect an adaptive response to hemodialysis stimuli

Effects of social isolation on ehrlich tumor growth and tumor leukocyte infiltration in mice: evidence of participation of the submaxillary salivary gland.

OBJECTIVE: Considering previous studies on stress and Ehrlich tumor growth from our laboratory and also known cell growth and behavior-related properties of submaxillary salivary gland products, we studied the effects of stress by social isolation, in combination or not with sialectomy (submaxillary gland ablation), on growth and leukocyte infiltration into Ehrlich adenocarcinomas. METHODS: 5 x 10(7) tumor cells/ml were inoculated into the footpads of mice and tumor growth was evaluated by measuring paw thickness on alternate days. Ten days after inoculation, tumors were harvested and processed for immunocytochemical analysis of tumor-infiltrating leukocytes (TIL) and nerve-like growth factor (NGF)/epidermal growth factor (EGF)-positive tumor cells. T and B lymphocyte, NK cell and macrophage percentages were calculated. RESULTS: Sialectomy slightly reduced tumor growth and caused a significant increase in NK cell infiltration of tumors (p < 0.05). Social isolation caused a highly significant enhancement of both tumor growth (p < or = 0.05) and percentage of macrophages infiltrating tumors (p < 0.05). Sialectomized isolated animals showed few significant changes in tumor growth rate (p < or = 0.05), but presented increased percentages of NK cells, macrophages and B lymphocytes (p < 0.05). A reduction in the percentage of NGF+ tumor cells was also observed (p < 0.05). All of these effects were seen only in 8-month-old mice, but not in younger animals. CONCLUSION: A possible link between salivary gland factors, tumor growth and TIL patterns was considered, suggesting that growth factors may modulate tumor leukocyte infiltration as well as tumor growth rate.

A priori estimation of accuracy and of the number of wells to be employed in limiting dilution assays.

The use of limiting dilution assay (LDA) for assessing the frequency of responders in a cell population is a method extensively used by immunologists. A series of studies addressing the statistical method of choice in an LDA have been published. However, none of these studies has addressed the point of how many wells should be employed in a given assay. The objective of this study was to demonstrate how a researcher can predict the number of wells that should be employed in order to obtain results with a given accuracy, and, therefore, to help in choosing a better experimental design to fulfill one's expectations. We present the rationale underlying the expected relative error computation based on simple binomial distributions. A series of simulated in machina experiments were performed to test the validity of the a priori computation of expected errors, thus confirming the predictions. The step-by-step procedure of the relative error estimation is given. We also discuss the constraints under which an LDA must be performed.

Discrimination between NK and LAK cytotoxic activities of murine spleen cells by MTT assay: differential inhibition by PGE(2) and EDTA.

In the present study we propose a mathematical approach to improve the analysis of NK and LAK activities measured by MTT assay adapted for murine cells. We found that to calculate NK activity, high E:T ratios should be used (up to 50:1) and the phenomenon fits to a linear least-squares analysis. However, 5-fold less effector cells (10:1, E:T) should be used to detect LAK activity and the phenomenon has a nonlinear exponential behavior. Using this approach, we showed that EDTA inhibits LAK but not NK activity whereas PGE(2) inhibits NK but not LAK activity. In conclusion, this analytical approach allowed the discrimination between NK and LAK activities and exposed differences between these two cytotoxic activities.

A priori estimation of accuracy and of the number of wells to be employed in limiting dilution assays

The use of limiting dilution assay (LDA) for assessing the frequency of responders in a cell population is a method extensively used by immunologists. A series of studies addressing the statistical method of choice in an LDA have been published. However, none of these studies has addressed the point of how many wells should be employed in a given assay. The objective of this study was to demonstrate how a researcher can predict the number of wells that should be employed in order to obtain results with a given accuracy, and, therefore, to help in choosing a better experimental design to fulfill one's expectations. We present the rationale underlying the expected relative error computation based on simple binomial distributions. A series of simulated in machina experiments were performed to test the validity of the a priori computation of expected errors, thus confirming the predictions. The step-by-step procedure of the relative error estimation is given. We also discuss the constraints under which an LDA must be performed

Mycoplasma arginini enhances cytotoxicity of thioglycollate-elicited murine macrophages toward YAC-1 tumor cells through production of NO.

Bacterial products stimulate macrophage tumoricidal activity through release of tumor necrosis factor (TNF) and nitric oxide (NO). We show here that thioglycollate-elicited macrophages acquire cytotoxic activity when cocultured with Mycoplasma arginini-infected YAC-1 tumor cells and release TNF and NO. Fixed mycoplasma-infected cells, supernatants from infected-cell cultures, or purified heat-killed mycoplasma obtained from cell-free cultures were all able to induce TNF and NO production. Thus, the mycoplasma per se and not a product of infected cells induce the release of these molecules. Addition of prostaglandin E2 (PGE2) to the cocultures, which reduced TNF release, or antibodies to TNF, did not affect macrophage cytotoxicity nor NO release. Inhibition of NO production by L-NAME or aminoguanidine reduced the cytotoxicity, and treatment with a NO donor was toxic to YAC-1 cells. These results indicate that M. arginini activates thioglycollate-elicited murine macrophages for NO and TNF release increasing their cytotoxic activity toward YAC-1 cells and that this activity is dependent on NO but not TNF release.

Thioglycollate-elicited murine macrophages are cytotoxic to Mycoplasma arginini-infected YAC-1 tumor cells

Macrophages are important components of natural immunity involved in inhibition of tumor growth and destruction of tumor cells. It is known that these cells can be activated for tumoricidal activity by lymphokines and bacterial products. We investigated whether YAC-1 tumor cells infected with Mycoplasma arginini stimulate nitric oxide (NO) release and macrophage cytotoxic activity. Thioglycollate-elicited macrophages from male BALB/c mice were co-cultured for 20 h with YAC-1 tumor cells infected or not with Mycoplasma arginini. The cytotoxic activity was evaluated by MTT assay and nitrite levels were determined with the Griess reagent. Thioglycollate-elicited macrophages co-cultured with noninfected YAC-1 cells showed low cytotoxic activity (34.7 ñ 8.6per cent) and low production of NO (4.7 ñ 3.1 µM NO2-). These macrophages co-cultured with mycoplasma-infected YAC-1 cells showed significantly higher cytotoxic activity (61.4 ñ 9.1 per cent; P=0.05) and higher NO production (48.5 ñ 13 µM NO2-; P=0.05). Addition of L-NAME (10 mM), an inhibitor of NO synthesis, to these co-cultures reduced the cytotoxic activity to 37.4 ñ 2per cent (P=0.05) and NO production to 3 ñ 4 µM NO2- (P=0.05). The present data show that Mycoplasma arginini is able to induce macrophage cytotoxic activity and that this activity is partially mediated by NO.

Central nervous system metastases from breast carcinoma. A clinical and laboratorial study in 47 patients.

In this retrospective study, 47 patients with clinical diagnosis of central nervous system metastases of breast cancer were evaluated by computerized tomography (CT), magnetic resonance imaging (MRI) and cerebrospinal fluid (CSF) examination. The patients were divided in 2 groups: 1, without leptomeningeal neoplasm and 2, with leptomeningeal neoplasm. In the group 2, the time interval between the primary disease and the central nervous system metastasis as well as the survival time were shorter than in group 1 (40 and 4.3 months in group 2 versus 57 and 10 months respectively, in group 1). In both groups the most common neurological symptoms and signs were intracranial hypertension and motor deficits. The most sensitive diagnostic methods were CT and MRI in group 1, and the CSF examination in group 2. The use of the tumor markers CEA and CA-15.3 in the routine examination of CSF showed promising results, mainly in leptomeningeal forms.

Thioglycollate-elicited murine macrophages are cytotoxic to Mycoplasma arginini-infected YAC-1 tumor cells.

Macrophages are important components of natural immunity involved in inhibition of tumor growth and destruction of tumor cells. It is known that these cells can be activated for tumoricidal activity by lymphokines and bacterial products. We investigated whether YAC-1 tumor cells infected with Mycoplasma arginini stimulate nitric oxide (NO) release and macrophage cytotoxic activity. Thioglycollate-elicited macrophages from male BALB/c mice were co-cultured for 20 h with YAC-1 tumor cells infected or not with Mycoplasma arginini. The cytotoxic activity was evaluated by MTT assay and nitrite levels were determined with the Griess reagent. Thioglycollate-elicited macrophages co-cultured with noninfected YAC-1 cells showed low cytotoxic activity (34.7 +/- 8.6%) and low production of NO (4.7 +/- 3.1 microM NO2-). These macrophages co-cultured with mycoplasma-infected YAC-1 cells showed significantly higher cytotoxic activity (61.4 +/- 9.1%; P < 0.05) and higher NO production (48.5 +/- 13 microM NO2-; P < 0.05). Addition of L-NAME (10 mM), an inhibitor of NO synthesis, to these co-cultures reduced the cytotoxic activity to 37.4 +/- 2% (P < 0.05) and NO production to 3 +/- 4 microM NO2- (P < 0.05). The present data show that Mycoplasma arginini is able to induce macrophage cytotoxic activity and that this activity is partially mediated by NO.