Association of tyrosine hydroxylase expression in brain and tumor with increased tumor growth in sympathectomized mice.

OBJECTIVE: Lymphocytes express tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine, norepinephrine and epinephrine. This suggests a broader role for cathecholamines in lymphocyte function, as well as the potential secretion of catecholamines by tumors of lymphoid origin. Our aim was to evaluate the expression of Th by murine lymphoma cells in an in vivo mouse model. For this, L5178Y-R lymphoma cells were implanted in nerve-intact and sympathectomized male BALB/c mice. Relative Th gene expression in tumor and brain was determined by quantitative PCR. Body composition, tumor volume, and plasma TH1/TH2/TH17 cytokines were also evaluated as markers of tumor-host condition and anti-tumor immune response in absence of adrenergic innervation. RESULTS: We found a significant (p = 0.045) 3.3-fold decrease of Th gene expression in tumor and a non-significant (p = 0.60) 6.9-fold increase in brain after sympathectomy. Sympathectomized mice also showed a significant increase in tumor mass at days 18 (p = 0.032) and 28 (p = 0.022) and increased interscapular fat (p = 0.04). TH1/TH2 and TH17 cytokines levels in plasma from sympathectomized tumor-bearing mice were not different from control mice. CONCLUSION: The L5178Y-R lymphoma does not express Th during in vivo progression.

Alterations of antitumor and metabolic responses in L5178Y-R lymphoma-bearing mice after only 30-minute daily chronic stress exposure.

AIM: In stress research, reducing times of stress induction may contribute to improving the well-being of experimental animals, especially in cancer models, already under physiological distress. To support this idea, we evaluated the effects of a short-timed stress protocol on endocrine, metabolic and immune indicators in mice bearing the L5178Y-R lymphoma. MATERIALS AND METHODS: A 30-minute daily stress protocol was applied for 28 days to healthy and lymphoma-bearing BALB/c mice; body weight, plasma levels of corticosterone, norepinephrine, Th1/Th2 cytokines, insulin, and leptin, were measured. RESULTS: We found a 12% significant decrease in body weight in non-tumor bearing mice under stress (p < 0.007). The disruption of weight evolution was accompanied by a stress induced 85% decrease in plasmatic leptin (p < 0.01) and total reduction of insulin. Tumor burden alone was associated to an increase in more than two-fold of plasmatic levels of norepinephrine (p < 0.008). Neither stress nor tumor or their combination, resulted in an elevation of systemic IL-6. IFN-γ levels were 20 times higher in lymphoma-bearing animals when compared with non-tumor bearing mice (p < 0.01); however, under stress, this response was reduced by half, indicating a suppressing effect of chronic stress on the antitumor immune response. CONCLUSION: A short-timed stress induction is enough to cause significant alterations in the metabolism and immunity of healthy and tumor-bearing mice, supporting the use of short-timed protocols as an efficient way to induce chronic stress that also considers concerns regarding the well-being of experimental animals in biomedical research.

Expression of Foxp3, CD25 and IL-2 in the B16F10 cancer cell line and melanoma is correlated with tumor growth in mice.

The forkhead box P3 (Foxp3) transcription factor is one of the most studied markers used to identify CD4+CD25+ regulatory T cells (Tregs), and has been identified as a key regulator in the development and function of Tregs. Foxp3 expression has been reported in a variety of solid human tumors, including melanoma. The aims of the present study were to analyze Foxp3 expression in B16F10 melanoma cells in vitro, to determine whether this expression was affected during tumor growth in a murine melanoma model and to correlate Foxp3 expression with CD25 expression, interleukin (IL)-2 production and tumor weight. Foxp3 expression was analyzed with quantitative (q)PCR, flow cytometry and confocal microscopy. CD25 expression was analyzed by flow cytometry, and cytokine production was measured by ELISA [IL-2, interferon (IFN)-γ, transforming growth factor (TGF)-ß and IL-10] and flow cytometry (IL-2, IFN-γ, IL-4 and IL-5). Foxp3 and CD25 expression was detected in the B16F10 cells in culture and in the intratumoral B16F10 cells. An increase in Foxp3 and CD25 expression was observed in a time-dependent manner during tumor growth at 7, 14 and 21 days. The production of the IL-2, IL-10, IFN-γ and TGF-ß cytokines was observed in the B16F10 cells and also detected in the tumoral microenvironment during tumor growth (7, 14 and 21 days). An increase in IL-2 and IL-10 production was observed, whereas IFN-γ production decreased in a time-dependent manner. The production of tumor necrosis factor (TNF)-α was not observed in culture, but was detected during tumor growth, whereas the production of IL-4 and IL-5 was not detected. These data showed a positive correlation between the expression of Foxp3, CD25 and IL-2 and tumor weight in murine melanoma. From these data, it may be suggested that Foxp3 participates in melanoma growth, the modulation of the IL-2, IFN-γ and TNF-α cytokines and CD25 expression, and that it also plays a possible role in immunosuppression.

Human papillomavirus 16/18 infections in lung cancer patients in Mexico.

BACKGROUND/AIMS: Human papillomavirus (HPV) is an epitheliotropic, double-stranded DNA virus, and its high-risk genotypes are associated with human cancer. HPV genome has been detected in lung carcinomas in certain places around the world, including Mexico; however, the prevalence of this is unclear. In this study, we examine the frequency of high-risk HPV 16/18 in lung cancer tissues from a Mexican population. METHODS: 39 lung cancer specimens were analyzed by polymerase chain reaction (PCR) using HPV GP5+/GP6+ primers and then were genotyped using specific primers to HPV 16/18. Additionally, in situ hybridization (ISH) was performed using BIO-labeled oligonucleotide probes. RESULTS: Our results identified 15 positive cases (38.46%) for HPV 16 and 1 positive case (2.56%) for HPV 18 by PCR. ISH showed the presence of HPV DNA in 13 of 16 (81%) samples, in agreement with the PCR results. CONCLUSIONS: In this study, we detected HPV 16/18 gene sequences in lung cancer samples obtained from Mexican patients by PCR and ISH. We found the highest prevalence of HPV 16 infection in lung adenocarcinomas, suggesting that HPV infection may be associated with lung cancer. However, further studies are needed to elucidate the role of HPV in lung carcinogenesis.

Differential responses of macrophages from bovines naturally resistant or susceptible to Mycobacterium bovis after classical and alternative activation.

It is known that macrophages from naturally resistant animals possess a strong immune response against bovine tuberculosis to control mycobacterial infections. In the present study, the macrophage phagocytic activity, intracellular bacterial survival, and cytokine gene expression induced by classical and alternative activators against Mycobacterium bovis in naturally resistant or susceptible bovines, were evaluated. Animals were classified as naturally resistant or susceptible based on the capacity of their macrophages to allow M. bovis (BCG) growth. Peripheral blood macrophages from naturally resistant and susceptible animals were activated by classical and alternative stimuli and challenged with either non-pathogenic M. bovis BCG strain or pathogenic 9926 strain. Naturally resistant animals showed the highest phagocytosis index and microbial control after classical and alternative stimuli, being this response higher against the strain 9926 than the non-virulent strain. In addition, the response of macrophages activated by the classical pathway was higher than that under the alternative activation against both types of strains. Furthermore, classical pathway-activated macrophages derived from naturally resistant animals expressed higher levels of the pro-inflammatory markers iNOS, IL-1ß, TNF-α, MIP-1 and MIP-3, and the anti-inflammatory markers ARGII and TGF-b, particularly to BCG. The results of this study showed that macrophages from naturally resistant animals produced stronger pro-inflammatory responses than those from susceptible ones to signals provided by classical pathway activators. Its role in innate immunity against M. bovis is yet to be determined.

Long-chain alkanes and ent-labdane-type diterpenes from Gymnosperma glutinosum with cytotoxic activity against the murine lymphoma L5178Y-R.

The antitumor potential of Gymnosperma glutinosum was previously reported using the in vitro and in vivo L5178Y-R lymphoma murine model. The present study was carried out to isolate and identify the cytotoxic compounds present in the Gymnosperma glutinosum leaf hexane extract. Gymnosperma glutinosum was collected in the semi-arid region of Escobedo, State of Nuevo León, México, but it is commonly found in northeastern Mexico; it is traditionally used as a treatment for diarrhea, ulcers and rheumatism. G. glutinosum leaves were extracted with hexane and further fractioned and subfractioned over silica gel by gradient elution with hexane, chloroform, ethyl acetate and methanol. The cytotoxicity of fractions and subfractions was assessed in vitro against L5178Y-R lymphoma cells. Structure elucidation of the active compounds was determined by spectroscopic methods. Fractions and subfractions showed significant (p < 0.05) and concentration-dependent 20% to 56% cytotoxicity against L5178Y-R cells at concentrations ranging from 7.8 µg/mL to 500 µg/mL. The bioassay-guided fractionation of the hexane extract resulted in the isolation and identification of the alkane hentriacontane and the diterpene ent-labd-7-en-13S,14R,15-triol as the metabolites responsible for the activity.

Antitumor properties of Gymnosperma glutinosum leaf extracts.

The present study was undertaken to validate the antitumor potential of Gymnosperma glutinosum from regional people's account, using the in vitro and in vivo L5178Y-R lymphoma murine model. Non-polar G. glutinosum crude extracts were tested on L5178Y-R cells. We found significant (p < 0.05) cytotoxic activity (up to 40%) of the hexane extract, which was further fractioned; fraction 1 (F1) was then observed to produce up to 51% apoptosis-mediated L5178Y-R cytotoxicity in vitro at concentrations lower than 0.98 microg/ml, and possess significant in vivo antitumor activity. This study may support further evaluation of active F1 in clinical trials.

IMMUNEPOTENT CRP (bovine dialyzable leukocyte extract) adjuvant immunotherapy: a phase I study in non-small cell lung cancer patients.

BACKGROUND: IMMUNEPOTENT CRP is a mixture of low molecular weight substances, some of which have been shown to be capable of modifying the immune response. We evaluated the response and adjuvant effect of IMMUNEPOTENT CRP on non-small cell lung cancer (NSCLC) patients in a phase I clinical trial. METHODS: Twenty-four NSCLC patients were included in the study and divided into two groups. Group 1 received a conventional treatment of 5400 cGy external radiotherapy in 28 fractions and chemotherapy consisting of intravenous cisplatin (40 mg/m(2)) delivered weekly for 6 weeks. Group 2 received the conventional treatment plus IMMUNEPOTENT CRP (5 U) administered daily. We performed clinical evaluation by CT scan and radiography analysis, and determined the quality of life of the patients with the Karnofsky performance scale. A complete blood count (red and white blood cell tests), including flow cytometry analysis, blood work (alkaline phosphatase test) and a delayed-type hypersensitivity (DTH) skin test for PPD, Varidase and Candida were performed. RESULTS: The administration of IMMUNEPOTENT CRP induced immunomodulatory activity (increasing the total leukocytes and T-lymphocyte subpopulations CD4(+), CD8(+), CD16(+) and CD56(+), and maintaining DHT) and increased the quality of the patients' lives, suggesting immunologic protection against chemotherapeutic side-effects in NSCLC patients. DISCUSSION: Our results suggest the possibility of using IMMUNEPOTENT CRP alongside radiation and chemotherapy for maintaining the immune system and increasing the quality of life of the patients.

Detection of genes encoding antimicrobial peptides in Mexican strains of Trichoplusia ni (Hübner) exposed to Bacillus thuringiensis.

The systemic immune response of Trichoplusia ni after Bacillus thuringiensis (Bt) exposure was evaluated by comparing the expression of genes encoding antimicrobial peptides (AMPs) in Bt-susceptible and -resistant T. ni strains that were either exposed or not to XenTari (Bt-XT). AMP genes were detected by RT-PCR using primers for attacin, gloverin, lebocin, lysozyme, and peptidoglycan recognition peptide (PGRP). In general, AMP genes were detected more frequently in Mexican field strains previously exposed to Bt (SALX and GTOX) than in a Mexican laboratory strain (NL), but expression was similar to the AMP expression in USA laboratory strains (US and USX). Among the AMPs, transcripts for lebocin were the least detected (11.7%) and those for lysozyme were the most detected (84.8%) in all samples. Lebocin was detected only in 2nd instar and pupa. All untreated controls expressed attacin. Attacin and gloverin were not detected in any midgut sample, and their highest detection was in pupa. Lysozyme was rarely detected in 2nd instar larvae from any strain or treatment but was detected in almost all midgut and hemolymph samples. Overall, AMPs were found more in T. ni strains previously exposed to Bt-XT, especially lebocin and globerin (1.8-fold increase) and PGRP (3.8-fold increase). The data suggest that the expression of AMPs in T. ni correlates to previous Bt exposure.

Bovine dialyzable leukocyte extract modulates AP-1 DNA-binding activity and nuclear transcription factor expression in MCF-7 breast cancer cells.

BACKGROUND: We have previously demonstrated that bovine dialyzable leukocyte extract (bDLE) induces death through an apoptosis mechanism in MCF-7 breast cancer cells. Depending on the cell type and stimulus, activating protein-1 (AP-1) has been shown to regulate cell proliferation and differentiation, the stress response, apoptosis and survival. It remains unknown whether AP-1 and other transcription factors are mechanisms by which bDLE induces cell death. METHODS: To determine whether bDLE modulates the AP-1 DNA binding and gene expression, MCF-7 breast cancer cells were treated with bDLE (0, 1, 5, 10 U) for 72 h and evaluated by electrophoretic mobility shift assay, reverse transcriptase-polymerase chain reaction and Western blot assays. RESULTS: bDLE induced inhibition of cell growth, suppressed the AP-1 DNA-binding activity, decreased c-Jun protein expression and modulated NFATx, NFATc, NFkappaB, c-Jun and c-Fos transcription factor gene expression in MCF-7 breast cancer cells. DISCUSSION: The present data indicate that bDLE can block the AP-1 DNA-binding activity and expression of several transcriptions factors in breast cancer cells, which will have great potential in improving cancer therapy.

Mouse mammary tumor virus-like gene sequences in breast cancer samples of Mexican women.

BACKGROUND: Previous reports related the presence of mouse mammary tumor virus (MMTV)-like gene sequences to human breast carcinoma. The aim of this study was to determine whether MMTV-like env gene sequences are present in breast cancer samples of Mexican women and in breast and lung cancer cell lines. METHODS: Using specific primers for MMTV, we tested 3 breast cancer cell lines, 4 non-small lung cancer cell lines and 119 breast cancer samples from Mexican women. RESULTS: MMTV-like gene sequences were amplified in the lung cancer cell INER-51, but not in the MCF-7 cell line that has been used as a positive control in other reports and in 5 of 119 (4.2%) breast cancer biopsy tissues. Furthermore, the identity of sequences of PCR products from INER-51 and a breast cancer-positive sample are 98 and 99% when compared with the env region of MMTV (GenBank accession No. AY161347). CONCLUSION: These results indicate that MMTV-like gene sequences are present in the Mexican population.

Bovine dialyzable leukocyte extract modulates cytokines and nitric oxide production in lipopolysaccharide-stimulated human blood cells.

BACKGROUND: In the current study, we determined whether bovine dialyzable leukocyte extract (bDLE) modulates lipopolysaccharide (LPS)-induced nitric oxide and cytokine overproduction. METHODS: Human whole blood cells were treated with LPS (50 ng) + bDLE (1 U). RESULTS: The bDLE treatment decreased nitric oxide as well as TNF-alpha, IL-6 and IL-10 (P <0.01) cytokine production. In addition, it decreased TNF-alpha, IL-1beta and IL-6 mRNA expression and suppressed IL-10 and IL-12p40 mRNA expression, but did not modulate IL-8 mRNA expression in LPS-stimulated human blood cells. DISCUSSION: Our results suggest that bDLE may effectively modulate the fatal symptoms of hypotensive shock associated with endotoxin (LPS)-induced nitric oxide and cytokine production, and this may offer therapeutic potential for the treatment of endotoxic shock.

In vitro effects of bovine dialyzable leukocyte extract (bDLE) in cancer cells.

BACKGROUND: Bovine dialyzable leukocyte extract (bDLE) is a dialyzate of a heterogeneous mixture of low molecular weight substances released from disintegrated blood leukocytes or lymphoid tissue obtained from homogenized bovine spleen. The purpose of this study was to determine if bDLE had cytotoxic effects and modulated apoptosis gene expression in breast cancer cells. METHODS: The MCF-7, BT-474, MDA-MB-453, A-427, Calu-1, U937 and L5178Y cancer cell lines and PBMC human cells were treated with bDLE (0-0.66 U/mL) for 72 h. The bDLE effect on cell growth proliferation was evaluated by MTT assay, and the MCF-7 was evaluated by ethidium bromide-acridine orange staining; total DNA was evaluated for DNA fragmentation, and total RNA was isolated for p53, bag-1, c-myc, bim, bax, bcl-2 and bad mRNA expression. RESULTS: The bDLE had dose-dependent cytotoxic effects and demonstrated an IC50 at a dosage of 0.06 U/mL (P<0.05). The bDLE did not affect the viability of normal human PBMC. The bDLE induced DNA fragmentation at doses of 0.06 and 0.13 U/mL in MCF-7 breast cancer cells. The bDLE induced cytotoxic effects and suppressed the p53, bag-1, c-myc, bax, bcl-2, and bad mRNA expression that influences apoptosis in MCF-7 breast cancer cells. Bim mRNA expression was not detected. DISCUSSION: This may open up interesting prospects for the treatment of human breast cancer.

In vitro immunopotentiating properties and tumour cell toxicity induced by Lophophora williamsii (peyote) cactus methanolic extract.

Lophophora williamsii, also known as peyote, is found primarily in dry regions from Central Mexico, including the Mexican States of Nayarit, San Luis Potosí, Zacatecas, Nuevo León, Chihuahua, Coahuila and Tamaulipas, to Texas particularly in regions along Rio Grande. Peyote extracts have been associated with stimulating the central nervous system and regulating blood pressure, sleep, hunger and thirst. However, there is no evidence of any effect of peyote on the immune system or against tumour cell growth. The present study was designed to evaluate the in vitro effects of peyote methanolic extracts on some parameters of mouse and human leukocyte immunocompetence and tumour cell growth. Peyote extract (0.18-18 micro g/mL) activated nitric oxide production by murine macrophages, and stimulated up to 2.4-fold proliferation of murine thymic lymphocytes. In addition, peyote extract induced up to 1.85-, 2.29- and 1.89-fold increases in mRNA signal of IL-1, IL-6 and IL-8 by human leukocytes. Also examined were the effects of peyote extracts on murine lymphoma L5178Y-R and fi broblastoma L929, and human myeloid U937 and mammary gland MCF7 tumour cell growth using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Peyote extracts were toxic for MCF7, L5178Y-R, U937 and L929 (18 mg/mL peyote extract caused 1.3%, 8%, 45% and 60% viability respectively) cell lines.

Chemotaxis of human and rat leukocytes by the delta-selective non-peptidic opioid SNC 80.

Opioids like morphine, represent a major source of relief for most chronic moderate to severe nonmalignant pain. However, opioid abuse may lead to infections such as hepatitis and AIDS because opioids have been associated with suppressing various parameters of immune function including antimicrobial resistance, antibody production, monocyte-mediated phagocytosis, and both neutrophil and monocyte chemotaxis. We have previously reported immunopotentiating properties of non-peptidic opioid receptor selective agonists and antagonists. In this study, we evaluated the effects of the nonpeptidic delta-opioid receptor agonist (+)-4-((alpha R)-alpha-((2S, 5R)-4-allyl-2, 5-dimethyl-1-piperazinyl)-3-methoxybenzyl)-N, N-diethyl-benzamide (SNC 80) on chemotaxis of rat thymic and human peripheral blood mononuclear cells by using a modified Wilkinson chamber. Cell recruitment is an essential process in acute and chronic inflammatory responses. We observed that SNC 80 at concentrations of 10(-10), 10(-9), 10(-8), 10(-7), and 10(-6) M, significantly (p < 0.01) stimulated rat thymic (1.3, 1.55, 1.58, 1.75, and 1.8-fold increases respectively) and human leukocyte (1.13, 1.37, 1.43, 1.7, 1.83 fold-increases respectively) chemotaxis (demonstrated by checkerboard assays), compared with untreated control. The effects of SNC 80 on chemotaxis of rat and human leukocytes were antagonized by naloxone, indicating that the modulation of chemotaxis by SNC 80 is via a classic opioid receptor. The development and use of non-peptidic opioids like SNC 80 could have an immediate impact not only as potent analgesics, but in immunoregulation.

The molecular epidemiology of HIV type 1 of men in Mexico.

Genotypic characteristics of human immunodeficiency virus type 1 (HIV-1) in Mexico were investigated in a multicenter study that involved centers in five geographic regions of the country. Study samples (n = 65) collected from male patients in 1998-1999 were sequenced within the C2-V5 region of the gp120 env gene. Phylogenetic analysis revealed that subtype B predominates in Mexico. The level of interpatient nucleotide diversity (mean value of 8.9%) was congruent with multiple introductions of the virus and the "aging" epidemic in Mexico. One-third of samples (30.8% of cases) showed polymorphism within the crown of the V3 loop demonstrating non-GPGR motifs. Two new motifs in the V3 loop crown - HPGG and GPEG - were observed. The evolution of the AIDS epidemic in Mexico should be closely monitored since non-B HIV-1 subtypes might be introduced. The nucleotide sequences were deposited in the GenBank under accession numbers AF200855-AF200869, AF200871-AF200892, and AF200894-AF200921.

Acute effects of 60-Hz electromagnetic fields on ex vivo murine lymphocyte and macrophage functions, and in vitro tumor cell growth.

Electromagnetic fields (EMFs) have been associated with impairing health. There are data that associate EMFs exposure to incidence of cancer, but there are conflicting results between epidemiological and laboratory studies. Similarly studies on the effect of EMF on the immune system have produced variable results. In the present study, we evaluated the acute effects of 60 Hz EMFs exposure at 1.0 mT, on proliferation of murine thymic lymphocytes, production of nitric oxide and phagocytosis of Candida albicans by peritoneal murine macrophages, as well as the effect of 8 h/day of EMF exposure during 6 days on proliferation of murine lymphoma L5178Y-R cell growth. We observed that exposure to EMF did not alter lymphocyte and macrophage functions, and did not affect in vitro cell growth of the murine lymphoma cell line L5178Y-R.

Immunoenhancing properties of Plantago major leaf extract.

Plantago major (PM), also known as plantain, is a weed found in temperate zones worldwide. PM leaves have been associated with various biological properties ranging from antiinflammatory, antimicrobial and antitumour to wound healing. However, its mechanism of action associated with boosting of the immune function remains to be elucidated. We found that endotoxin-free methanol extracts from PM leaves, at doses of 50, 100, 250, and 500 microg/mL, were associated with 4.4 +/- 1, 6 +/- 1, 12 +/- 0.4, and 18 +/- 0.4-fold increases of nitric oxide (NO) production, and increased TNF-alpha production (621 +/- 31, 721 +/- 36, 727 +/- 36, and 1056 +/- 52 U/mL, respectively) by rat peritoneal macrophages, in the absence of IFN-gamma or LPS. NO and TNF-alpha production by untreated macrophages was negligible. In addition, PM extracts potentiated Con A-induced lymphoproliferation (3- to 12-fold increases) in a dose-dependent fashion, compared with the effect of Con A alone. The regulation of immune parameters induced by plant extracts may be clinically relevant in numerous diseases including chronic viral infections, tuberculosis, AIDS and cancer.

Bidirectional effects of IFN-gamma on growth of Mycobacterium avium complex in murine peritoneal macrophages.

The effects of macrophage stimulation with interferon-gamma (IFN-gamma) before or after infection on the intracellular growth of Mycobacterium avium complex (MAC) were investigated. Treatment of murine peritoneal macrophages before infection with IFN-gamma (50 U/ml) for 24 h and 48 h, but not for 72 h, was associated with 41% and 52% significant MAC growth inhibition, respectively. NG-monomethyl-L-arginine (NMA) did not affect the preinfection antimycobacterial activity of IFN-gamma, thus indicating that nitric oxide was not involved in this phenomenon. In contrast, treatment of macrophages with IFN-gamma (50 U/ml) for 24 h and 48 h after infection was ineffective, whereas treatment for 72 h caused some MAC growth promotion. The use of NMA suppressed the IFN-gamma-mediated MAC growth, suggesting that nitric oxide may affect postinfection microbicidal function of macrophages. These results suggest that activation of macrophages with IFN-gamma before or after infection may direct the course of the infection and that nitric oxide may be detrimental more than beneficial for MAC-infected macrophages.

Nitric oxide and TNF-alpha production by murine peritoneal macrophages activated with a novel 20-kDa protein isolated from Bacillus thuringiensis var. thuringiensis parasporal bodies.

The effects of a novel 20-kDa protein isolated from Bacillus thuringiensis var. thuringiensis (BTp20) parasporal bodies on macrophage activation were investigated and compared with the activity of LPS. Murine resident or IFN-gamma-primed peritoneal macrophages (50 U/ml of IFN-gamma for 16 h) were treated with 50 microg/ml of BTp20, alone or in combination with LPS (1 to 100 ng/ml). BTp20 was not toxic for macrophages as determined by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reduction assay, however, 16% reduction of viability was observed when macrophages were treated with a combination of IFN-gamma, BTp20, and LPS at 100 ng/ml. BTp20 was able to stimulate significant cellular adherence and spreading, and significant production of nitrite and TNF-alpha by resident macrophages after 1.5 h of treatment; nitrite release, however, was induced with only 10 min of macrophage exposure to BTp20. BTp20 activities were significantly potentiated by IFN-gamma pretreatment. It was also observed that nitrite release by IFN-gamma-primed macrophages activated with LPS (1-100 ng/ml) was 20 times lower than that induced by IFN-gamma + BTp20. BTp20 and LPS independently stimulated significant TNF-alpha production by IFN-gamma-primed macrophages, the effect of BTp20 + LPS (1 and 10 ng/ml) combination was additive. In summary, this study demonstrated that BTp20 activates macrophage functions in the absence of IFN-gamma or LPS, and that IFN-gamma enhances BTp20 activities.