Anti-inflammatory properties of ethanolic extract from Vatairea macrocarpa leaves.

ETHNOPHARMACOLOGICAL RELEVANCE: In Brazilian traditional folk medicine, the leaves and heartwood from Vatairea macrocarpa (Benth) Ducke (Angelim-of-Cerrado) (Fabaceae family) are used as remedy after cold maceration for the treatment of the condition popularly known as rheumatism, as well as for the general inflammatory aspects such as pains, injury and swelling. The rheumatological and rheumatic diseases affect 0.3-1.0% of the world population and all long-term treatment with conventional medications lead to adverse effects. AIM OF THE STUDY: To investigate the chemical composition and the anti-inflammatory properties and of the ethanolic extract from V. macrocarpa leaves (EEVM) in experimental models. MATERIAL AND METHODS: EEVM was chemically analyzed by spectrophotometry and the compounds characterization was performed by nuclear magnetic resonance and mass spectrometry. EEVM was evaluated in methylthiazolyldiphenyl-tetrazolium bromide (MTT) (3, 10, 30, and 90 µg/ml) and neutrophils phagocytic activity (1, 3, and 10 µg/ml) tests. For in vivo models, the EEVM (10, 30, 100, and 300 mg/kg) was orally administered (p.o.) for inflammatory evaluation in carrageenan-induced pleurisy in Swiss mice. The EEVM (30 and 100 mg/kg, p.o.) was tested against the Complete Freund Adjuvant (CFA)-induced paw persistent inflammation and Mycobacterium bovis (bacillus Calmette-Guerin - BCG)-induced pleurisy in C57bL6 mice. RESULTS: The chemical composition of EEVM showed 157.06 mg (GAE)/g in relation to phenolic compounds, 82.13 mg (RUE)/g in relation to flavonoids and 48.99 mg (TAE)/g in relation to tannins. The flavonoid compounds identified were catechin, epicatechin and kaempferol-3-O-a-l-rhamnopyranoside. EEVM did not present cytotoxicity in MTT assay, however EEVM reduced phagocytic neutrophils activity at all tested concentration. EEVM significantly inhibited leukocytes migration/proteins exudation in carrageenan-induced pleurisy model. The daily administration of EEVM inhibited the inflammatory parameters in BCG and CFA models. CONCLUSIONS: The present study showed anti-inflammatory features of EEVM (V. macrocarpa) as a natural agent against inflammatory diseases.

Toxicity evaluation of ConvitVax breast cancer immunotherapy.

ConvitVax is a personalized vaccine for the treatment of breast cancer, composed of autologous tumor cells, bacillus Calmette-Guérin (BCG) and low concentrations of formalin. Previous pre-clinical studies show that this therapy induces a potent activation of the immune system and achieves an effective response against tumor cells, reducing the size of the tumor and decreasing the percentage of immunosuppressive cells. In the present study, we evaluate the toxicity of ConvitVax in healthy BALB/c mice to determine potential adverse effects related to the vaccine and each of its components. We used standard guidelines for pain, discomfort and distress recognition, continuously evaluated the site of the injection, and completed blood and urine clinical tests. Endpoint necropsy was performed, measuring the weight of organs and processing liver, kidney, thymus and lung for histological examination. Results show that the vaccine in its therapeutic dose, at 3 times its therapeutic concentration, and its individual components did not cause death or behavioral or biological changes, including any abnormalities in whole-body or organ weights, and tissue damage. These results support the safety of ConvitVax with minimal to no side-effects.

Poly-dispersed Acid-Functionalized Single-Walled Carbon Nanotubes (AF-SWCNTs) Are Potent Inhibitor of BCG Induced Inflammatory Response in Macrophages.

The present study is focused on the modulation of Mycobacterium bovis BCG-induced inflammatory response by poly-dispersed acid-functionalized single-walled carbon nanotubes (AF-SWCNTs) in macrophages. Flow cytometric and confocal microscopy studies indicated that both BCG and AF-SWCNTs were efficiently internalized by RAW 264.7 and MH-S macrophage cell lines and were essentially localized in the cytoplasmic area. BCG-induced production of reactive oxygen species (ROS) and nitric oxide by the two cell lines was significantly inhibited by AF-SWCNTs. Using RT-PCR technique, a marked decline was observed in the expression of BCG-induced pro-inflammatory genes COX-2, iNOS, TNF-α, IL-6, and IL-1ß upon treatment with AF-SWCNTs. Results of gelatin zymography indicated that the AF-SWCNTs treatment also induced a marked decline in BCG-induced release of matrix metalloproteinases MMP-2 and MMP-9 by the two macrophage cell lines. The anti-inflammatory effect of AF-SWCNTs in downregulating BCG-induced inflammatory response was further validated in murine peritoneal macrophages. Treatment with AF-SWCNTs led to a steep decline in BCG-induced NO production in murine peritoneal macrophages in vitro as well as in vivo. Peritoneal macrophages isolated from mice treated with BCG and AF-SWCNTs had a significantly lower intracellular expression of COX-2 as compared to the peritoneal macrophages derived from mice treated with BCG alone. Taken together, our results demonstrate a potent anti-inflammatory effect of AF-SWCNTs in alleviating BCG-induced inflammatory responses in macrophages in vitro and in vivo.

Attenuation of oxidative stress and neurotoxicity involved in the antidepressant-like effect of the MK-801(dizocilpine) in Bacillus Calmette-Guerin-induced depression in mice.

Background The emerging line of research suggests that neuro-inflammation and oxidative stress are linked to the development of depression-like behavior. The tryptophan metabolizing enzyme, indolamine 2,3-dioxygenase (IDO), serves as an important interface between chronic inflammation and depression. IDO is induced by pro-inflammatory cytokines and diverts tryptophan towards the kynurenine pathway, decreasing serotonin synthesis. Further, the metabolites of kynurenine pathway increase brain oxidative stress and also cause N-methyl-D-aspartate (NMDA) receptor-mediated exitotoxicity. The resulting oxidative damage and dysfunction in glutamatergic neurotransmission alters the network connectivity of the brain, which may be the further mechanism for emergence of depression-like symptoms. Methods A depression-like illness was induced in mice by injecting Bacillus Calmette-Guerin (BCG) suspended in isotonic saline at a dose of 107 CFU I.P. The mice were then divided into different groups and were administered MK-801 or normal saline for the next 21 days, after which a battery of behavior and biochemical tests were conducted to assess them. Results The BCG group had significantly reduced sucrose preference index and an increase in immobility time in forced swim test (FST) and Tail Suspension Test (TST) as compared to the saline group. There was also a significant increase in the brain MDA levels and a decline in the brain GSH levels. The hippocampal tissue from the BCG group had significantly more comet cells than the saline group. The NMDA receptor antagonist, MK-801, was able to reverse the BCG-induced depression-like behaviour. MK-801 also showed significant decrease in brain oxidative stress but failed to show significant protection against BCG-induced neurotoxicity observed in comet assay. Conclusions The NMDA receptor antagonist, MK-801, mitigated BCG-induced, depressive-like behavior in mice by improving the sucrose preference and decreasing the duration of immobility time in TST and FST. The overall improvement in depression-like behavior was accompanied by a reduction in brain oxidative stress and comet cells, thus suggesting the antioxidant and neuroprotective action of MK-801.

Effect of Liposomal Delivery of Oxidized Dextran with Isonicotinic Acid Hydrazide on Component Profile of Pulmonary Extracellular Matrix in Mice with BCG-Induced Granulomatosis.

Intraperitoneal injections of isonicotinic acid hydrazide (INH), dextrazide (oxidized dextran+INH), or liposomes loaded with dextrazide (INH dose of 14 mg/kg) over 2 months to mice with BCG-induced granulomatosis started from postinfection day 90 induced qualitative and quantitative changes in composition of pulmonary extracellular matrix. Both dextrazide and its liposomal form decreased the levels of sulfated glycosaminoglycans and uronic acids. In contrast to INH, both preparations did not decrease the levels of total glycosaminoglycans, proteins, and galactose. This difference is explained by the fact both free and liposomal dextrazide activated MMP, but did not increase the content of TIMP-1 and TIMP-2, whereas injection of INH was followed by an increase in TIMP-2 content and a decrease in the level of free hydroxyproline, which attested to down-regulation of collagen degradation and maintenance of the conditions for pulmonary fibrosis in mice of this group.

Are There Differences in Toxicity and Efficacy between Various Bacillus Calmette-Guerin Strains in Bladder Cancer Patients? Analysis of 844 Patients.

PURPOSE: To evaluate if there are differences in toxicity and efficacy between different Bacillus Calmette-Guerin (BCG) stains used for intravesical immunotherapy in patients with non-muscle invasive bladder cancer. METHODS: We retrospectively analysed a group of 844 patients who received TICE, RIVM and Moreau BCG strains. The allocation of the strain to each patient was random, stemming from differences in supply and distribution. The patients were analysed in terms of toxicity, recurrence-free (RFS), progression-free (PFS), cancer-specific (CSS) and overall survival (OS). RESULTS: In the survival analysis, statistical significance was not reached in any tumour group for any clinical event. TICE caused more local and mild adverse effects and severe complications were mainly associated with RIVM strain. In a group in which the strain was changed during the course of the therapy, significantly more severe complications were observed and, in most of these cases, complications appeared right after the strain change. CONCLUSIONS: There were no differences in terms of RFS, PFS, CSS and OS after use of TICE, RIVM and Moreau strains. The complication profile differed statistically between used strains with TICE causing mostly mild complications. Also, strain change during the therapy course was associated with the increased risk of moderate to severe toxicity occurrence.

Variable BCG efficacy in rhesus populations: Pulmonary BCG provides protection where standard intra-dermal vaccination fails.

M.bovis BCG vaccination against tuberculosis (TB) notoriously displays variable protective efficacy in different human populations. In non-human primate studies using rhesus macaques, despite efforts to standardise the model, we have also observed variable efficacy of BCG upon subsequent experimental M. tuberculosis challenge. In the present head-to-head study, we establish that the protective efficacy of standard parenteral BCG immunisation varies among different rhesus cohorts. This provides different dynamic ranges for evaluation of investigational vaccines, opportunities for identifying possible correlates of protective immunity and for determining why parenteral BCG immunisation sometimes fails. We also show that pulmonary mucosal BCG vaccination confers reduced local pathology and improves haematological and immunological parameters post-infection in animals that are not responsive to induction of protection by standard intra-dermal BCG. These results have important implications for pulmonary TB vaccination strategies in the future.

Comparison of fluoxetine and 1-methyl-L-tryptophan in treatment of depression-like illness in Bacillus Calmette-Guerin-induced inflammatory model of depression in mice.

BACKGROUND: The inflammatory response system has been implicated in the pathophysiology of major depression. The pro-inflammatory cytokines like interferon-γ induce the enzyme indoleamine-2,3-dioxygenase (IDO) of the kynurenine pathway of tryptophan metabolism. The induction of IDO reduces the availability of tryptophan for serotonin synthesis. Furthermore, the metabolites of kynurenine pathway have neurotoxic property, which along with decreased serotonin may account for depression-like illness. METHODS: The aim of this study was to compare the effects of treatment with fluoxetine and 1-methyl-L-tryptophan (1-MT) on Bacillus Calmette-Guerin (BCG)-induced inflammatory model of depression in mice. Behavioral tests included locomotor activity, forced swim test (FST) and tail suspension test (TST). Oxidative stress was assessed by examining the levels of thiobarbituric acid reactive species (TBARS) and non-protein thiols (NP-SH) in homogenized whole brain samples. Comet assays were performed to assess neurotoxicity. RESULTS: The results of this study demonstrate that BCG treatment resulted in an increase in duration of immobility in FST and TST as compared to the saline group. Further, it produced a significant increase in the brain TBARS levels and decrease in the brain NP-SH levels. The hippocampal tissue from BCG group had significantly more comet cells than the saline group. 1-MT and fluoxetine were able to reverse the BCG-induced depression-like behavior and the derangement in oxidative stress parameters. Fluoxetine and 1-MT also reversed the BCG-induced neurotoxicity in such mice. CONCLUSIONS: 1-Methyl-L-tryptophan exhibits antidepressant-like effect comparable to that of fluoxetine in treating BCG-induced depression-like behavior in mice.

Vaccination of cattle with a high dose of BCG vaccine 3 weeks after experimental infection with Mycobacterium bovis increased the inflammatory response, but not tuberculous pathology.

A study was undertaken to determine whether BCG vaccination of cattle post-challenge could have an effect on a very early Mycobacterium bovis infection. Three groups of calves (n = 12/group) were challenged endobronchially with M. bovis and slaughtered 13 weeks later to examine for tuberculous lesions. One group had been vaccinated prophylactically with BCG Danish vaccine 21 weeks prior to challenge; a second group was vaccinated with a 4-fold higher dose of BCG Danish 3 weeks post-challenge and the third group, remained non-vaccinated. Vaccination prior to challenge induced only minimal protection with just a significant reduction in the lymph node lesion scores. Compared to the non-vaccinated group, BCG vaccination post-challenge produced no reduction in gross pathology and histopathology, but did result in significant increases in mRNA expression of pro-inflammatory mediators (IFN-γ, IL-12p40, IL-17A, IRF-5, CXCL9, CXCL10, iNOs, and TNF-α) in the pulmonary lymph nodes. Although there was no significant differences in the gross pathology and histopathology between the post-challenge BCG and non-vaccinated groups, the enhanced pro-inflammatory immune responses observed in the post-challenge BCG group suggest caution in the use of high doses of BCG where there is a possibility that cattle may be infected with M. bovis prior to vaccination.

Neuropeptide Y and peptide YY protect from weight loss caused by Bacille Calmette-Guérin in mice.

BACKGROUND AND PURPOSE: Immune challenge of mice with Bacille Calmette-Guérin (BCG) has been reported to cause transient weight loss and a behavioural sickness response. Although BCG-induced depression involves the kynurenine pathway, weight loss occurs independently of this factor. Because neuropeptide Y (NPY) and peptide YY (PYY) are involved in the regulation of food intake, we hypothesized that they play a role in the BCG-induced weight loss. EXPERIMENTAL APPROACH: Male wild-type, PYY knockout (PYY-/-), NPY knockout (NPY-/-) and NPY-/-;PYY-/- double knockout mice were injected with vehicle or BCG (approximately 10(8) colony-forming units per mouse), and their weight, locomotion, exploration and ingestion were recorded for 2 weeks post-treatment. KEY RESULTS: Deletion of PYY and NPY aggravated the BCG-induced loss of body weight, which was most pronounced in NPY-/-;PYY-/- mice (maximum loss: 15%). The weight loss in NPY-/-;PYY-/- mice did not normalize during the 2 week observation period. BCG suppressed the circadian pattern of locomotion, exploration and food intake. However, these changes took a different time course than the prolonged weight loss caused by BCG in NPY-/-;PYY-/- mice. The effect of BCG to increase circulating IL-6 (measured 16 days post-treatment) remained unaltered by knockout of PYY, NPY or NPY plus PYY. CONCLUSIONS AND IMPLICATIONS: These data show that NPY and PYY are both required to protect from the action of BCG-evoked immune challenge to cause prolonged weight loss and disturb energy balance. The findings attest to an important role of NPY and PYY in orchestrating homeostatic reactions to infection and immune stimulation.

[Comparison of the effectiveness between long-term instillation of mitomycin C and short-term prophylaxis with MMC or bacille Calmette-Guérin. Study of patients with non-muscle-invasive urothelial cancer of the urinary bladder]. / Vergleich der Effektivität der Langzeitinstillation mit Mitomycin C gegen Kurzzeitprophylaxen mit MMC oder Bacillus Calmette-Guerin. Untersuchung bei Patienten mit nicht muskelinvasivem Urothelkarzinom der Harnblase.

BACKGROUND: Adjuvant instillation therapy with chemo- or immunotherapeutic agents is an integral component in the treatment of non-muscle-invasive bladder cancer. There is, however, no general consensus on the choice of medication and the optimal duration of therapy. This multicenter trial compared a long-term treatment regimen with mitomycin C (MMC) with two short-term treatment approaches with MMC or bacille Calmette-Guérin (BCG) for intermediate-/high-risk bladder tumor after transurethral resection. In patients with low-risk bladder tumors, the effectiveness of six weekly MMC instillations was determined and compared with the results of patients not receiving adjuvant treatment. MATERIAL AND METHODS: A total of 495 patients with intermediate-/high-risk bladder tumor (recurrent and/or multifocal pTaG1, pTaG2-3, or pT1G1-3) were randomly administered either BCG-RIVM 2x108 CFU in six weekly instillations, MMC 20 mg in six weekly instillations, or MMC 20 mg in six weekly instillations with subsequent monthly instillations for 3 years. A total of 132 low-risk patients (first diagnosis of a unifocal pTaG1 bladder tumor) were randomly allocated to two treatment arms. In the first arm, 20 mg MMC were instilled weekly six times. In the control arm, the patients received no adjuvant therapy. RESULTS: The 3-year recurrence-free rate in the patients of the intermediate-/high-risk group was 65.5% (95% CI: 55.9-73.5%) in the BCG arm and 68.6% (95% CI: 59.9-75.7%) in the MMC short-term arm. In the MMC long-term arm, the 3-year recurrence-free rate was significantly higher at 86.1% (95% CI: 77.9-91.4%, log-rank test: p=0.001). There was no increased toxicity observed with long-term administration of MMC. In the low-risk group, the 3-year recurrence-free rate after adjuvant therapy was 74% (95% CI: 60.0-83.8%) and in the patients receiving no adjuvant treatment 63% (95% CI: 46.6-75.5%, corresponding to a hazard ratio of 0.58 (95% CI: 0.28-1.18%). The difference between the treatment arms was not significant. CONCLUSION: Long-term prophylaxis with MMC results in a significantly reduced recurrence rate in intermediate-/high-risk bladder cancer with a comparable toxicity profile in comparison to short-term MMC or short-term BCG. Our study showed no significant decrease of the recurrence rate in low-risk tumors with six adjuvant MMC instillations. This treatment approach thus does not represent an alternative to early instillation.

Content of steroid hormones in the blood and adrenal glands of mice in the dynamics of BCG- and SiO2-induced granulomatous inflammation.

Individual or combined administration of BCG vaccine and silicon dioxide to male mice induced a nonspecific stress response of the adrenal glands and gonads judging from changes in the concentration of blood cortisol and testosterone. The dynamics of cortisol concentration in the adrenal glands was similar, while changes in progesterone concentration were in antiphase to those in the blood. After combined administration of both inductors of granulomatous inflammation, changes in the concentrations of the studied hormones to a greater extent corresponded to their dynamics after injection of BCG.

Effects of Ganoderma lucidum polysaccharide on CYP2E1, CYP1A2 and CYP3A activities in BCG-immune hepatic injury in rats.

The purpose of the present study was to investigate the effect of Ganoderma lucidum polysaccharide (GLPS), a major active component in Chinese medicinal fungus, on cytochrome P450 metabolic activity in Bacillus Calmette Guérin (BCG)-induced immune hepatic injury in rats. The enzyme kinetics of the probes including chlorzoxazone (CYP2E1), phenacetin (CYP1A2) and nifedipine (CYP3A) were evaluated by HPLC. The results showed that BCG-pretreatment (125 mg/kg) significantly increased serum levels of alanine transaminase (ALT), nitrite and malondialdehyde (MDA), inhibited activities of superoxide dismutase (SOD) and decreased P450 total content in microsomes (p<0.05). Administration of GLPS (50 and 200 mg/kg) reversed above hepatic injury stimulated by BCG in vivo. Moreover, GLPS dose-dependently inhibited activities of CYP2E1, CYP1A2 and CYP3A in hepatic microsomes in vitro, suggesting that inhibition of GLPS on P450 oxidative metabolism might participate in the hepatoprotective mechanism, and also suggested that pharmacokinetics might be changed by drug-herb interaction.

Evidence for synaptic stripping by cortical microglia.

Recent studies have described significant demyelination and microglial activation in the cerebral cortex of brains from multiple sclerosis patients. To date, however, experimental models of cortical demyelination or cortical inflammation have not been extensively studied. In this report we describe focal cortical inflammation induced by stereotaxic injection of killed bacteria (BCG), followed 1 month later by subcutaneous injection of the same antigen, a protocol that overcomes the immune privilege of the cortex. Intracerebral BCG injection produced focal microglial activation at the injection site (termed acute lesion). Ten days after peripheral challenge (termed immune-mediated lesion), larger areas and higher densities of activated microglia were found near the injection site. In both paradigms, activated microglia and/or their processes closely apposed neuronal perikarya and apical dendrites. In the immune-mediated lesions, approximately 45% of the axosomatic synapses was displaced by activated microglia. Upon activation, therefore, cortical microglial migrate to and strip synapses from neuronal perikarya. Since neuronal pathology was not a feature of either the acute or immune-mediated lesion, synaptic stripping by activated microglia may have neuroprotective consequences.

Mononuclear phagocyte system in mice with intrauterine Candida albicans infection and postnatal experimental tuberculosis.

Intrauterine Candida albicans infection in mouse fetuses affected the type of granulomatous inflammation induced by BCG vaccine during the postnatal period. It manifested in increased formation of granulomas and variations in their cellular composition.

Comparative study of the high molecular mass fraction and low molecular mass fraction of Sho-saiko-to in a murine immunologically induced liver injury model.

We compared the pharmacological actions of the high and low molecular mass fractions of Sho-saiko-to using a murine immunologically induced liver injury model to estimate the roles of these fractions in the expression of the pharmacological action. In a Bacillus Calmette-Guerin (BCG)/lipopolysaccharide (LPS)-induced liver injury model, Sho-saiko-to and both of its fractions significantly reduced the increases in the aminotranseferase levels in serum. They also reduced the increase in the nitric oxide (NOx) level in serum. On the other hand, Sho-saiko-to and its high molecular mass fraction suppressed the increase in plasma NOx level in an LPS-induced endotoxin shock model but its low molecular mass fraction did not. These results suggest the possibility that both fractions act hepatoprotectively in a different manner. We believe that these results can help to elucidate the mechanism of action of ingredients in Sho-saiko-to.

Distinct ability to accumulate eosinophils during the inflammatory cellular response to M. bovis BCG in the mouse pleural cavity.

OBJECTIVE AND DESIGN: The host response to Mycobacteria focuses on the development of cell-mediated immunity and granuloma formation. Here, we investigated the onset of cellular responses to mycobacteria in murine pleurisy. MATERIAL: Distinct mouse strains previously described as Bcg susceptible or resistant were inoculated intrathoracically with different doses of live M. bovis BCG. METHODS: At various time intervals, cells harvested from the inflammatory site were identified and ultra-structurally analysed. RESULTS: BCG-induced pleurisy had two peaks of cellular influx at 1 and 15 days after infection. At the first half hour, macrophages were found to be heavily infected. Neutrophil arrival started after 2 h of infection and peaked at 4 h. At this time, neutrophils were found ingesting mycobacteria exclusively with a high infecting dose. BCG was potently more eosinophilotactic in Bcg susceptible mice than in the resistant ones and to other well known eosinophilia inducers: IL-5, PAF-acether or LPS. CONCLUSIONS: Mycobacterial load and mouse susceptibility seem to determine the early granulocyte dynamics in the lesion.

Quantitative analysis of apoptotic cell death in granulomatous inflammation induced by intravenous challenge with Cryptococcus neoformans and bacillus Calmette-Guérin vaccine.

Apoptotic cell death of macrophage has become recognized as a significant mechanism responsible for the resolution of inflammation. The purpose of this study was to examine how the apoptotic cell death involves the formation and resolution of granulomas in rats intravenously inoculated with Cryptococcus neoformans (Cr. neoformans) and Mycobacterium bovis-derived bacillus Calmette-Guérin (BCG) vaccine. The number and size of granulomas in the livers obtained on days 5, 10, 15, 20 and 25 after inoculation were examined by morphometric image analysis, as well as the occurrence of apoptotic cell death quantitatively analyzed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) procedure on tissue sections. In both groups the number and size of granulomas were maximized on day 10, then the granulomas were almost resolved until day 25 when the inoculated Cr. neoformans and BCG almost disappeared. From the induction to the resolving stages of granulomatous inflammation, TUNEL-positive cells constantly appeared in granulomas, and the highest frequency of apoptotic cells in granulomas was observed in the earlier stage of granuloma formation. These results indicate that the maintenance and resolution of infectious granulomas are regulated by the balance between the influx of newly recruited macrophages and the apoptotic elimination of granuloma macrophages. The apoptosis of granuloma macrophages actively involves the cellular turnover in both granuloma formation and resolution.

Immunogenicity and safety of Mycobacterium tuberculosis culture filtrate proteins in non-human primates.

The development of improved vaccines is considered a high priority in the effort to control tuberculosis (TB) world wide. Results from several studies performed in relevant animal models have demonstrated that Mycobacterium tuberculosis secreted antigens may represent major components of improved TB vaccines. To characterize further the M. tuberculosis secreted antigens as they relate to specific features important for vaccine development, rhesus macaques were immunized with either one of two different preparations containing M. tuberculosis culture filtrate (CF) proteins. These preparations differed in relative protein content and in the presence or absence of lipoarabinomannan. Animals received a total of three monthly intramuscular injections consisting of CF proteins resuspended in RIBI adjuvant and were tested for development of specific antibody and cellular proliferative responses. In addition, all animals were constantly monitored for local and systemic reactions as well as for the development of DTH reactions to intradermal tuberculin injection. Results from this study show that the two CF preparations are relatively safe and immunogenic in non-human primates. These two CF preparations differed in their ability to induce specific antibody responses, but were comparable in their ability to induce specific cellular proliferative responses. Induction of both humoral and cellular responses occurred even in presence of pre-existing antibodies directed against M. tuberculosis antigens. However, these responses appeared to be short-lived. Only one of the four animals produced interferon-gamma (IFN-gamma) in response to immunization with CF proteins. No DTH reaction to intradermal tuberculin injection was observed in any immunized animal. Although it is clear that additional studies are required to design strategies for the improvement of the immunogenicity of CF proteins, our observations support the currently accepted view that secreted protein-based preparations may represent promising vaccine candidates for TB.