Effects of dietary supplementation of different levels of vitamin B12 on the liver metabolism of laying hens.

BACKGROUND: Vitamin B12 plays an important role in lipid, protein, carbohydrate and nucleic acid metabolism. We investigated the effect of supplementing layers' diets with different vitamin B12 levels on liver metabolism using a liquid chromatography-mass spectrometry-based metabolomic approach to observe and analyse wide-target metabolomics in the liver. RESULTS: We assigned hens to three groups, namely blank control group without vitamin B12 diet (BCG), normal control group with 25 µg kg-1 vitamin B12 (NCG) and vitamin B12 supplement group I with 100 µg kg-1 vitamin (VBSG I). The VBSG I group layers had higher (P < 0.05) vitamin B12 concentration than those from other groups. The egg yolk vitamin B12 concentration increased (P < 0.01) with the increasing vitamin B12 dietary supplemental level. Between the NCG versus BCG, VBSG I versus BCG, and VBSG I versus NCG groups, 11, 20 and 11 metabolites were significantly changed, respectively. The KEGG pathway of vitamin B6 metabolism was significantly impacted in the NCG layers than those from BCG; seven and five pathways were significantly impacted in the VBSG I layers compared with those from BCG and NCG, including pyrimidine metabolism, vitamin B6 metabolism, glycerophospholipid metabolism, etc. CONCLUSION: We concluded that 25 µg kg-1 vitamin B12 supplementation in corn-soybean meal-based layer diet increased the egg yolk vitamin B12 concentration and impacted the vitamin B6 metabolic pathway, and 100 µg kg-1 of it increased the egg yolk and liver vitamin B12 concentrations and impacted vitamin B6 , lipid, nucleic acid and amino acid metabolic pathways. © 2022 Society of Chemical Industry.

Overexpression of the celA1 gene in BCG modifies surface pellicle, glucosamine content in biofilms, and affects in vivo replication.

Biofilm formed in vitro by mycobacteria has been associated with increased antibiotic tolerance as compared with planktonic cells. Cellulose has been identified as a component of DTT-exposed biofilms formed by M. tuberculosis. The celA1 gene of M. tuberculosis encodes a cellulase, which could affect the formation of biofilm by slow-growing mycobacteria. In this work, the celA1 gene of M. tuberculosis was cloned into the integrative pMV361 plasmid and then transformed into M. bovis BCG Pasteur to produce BCG:celA1, to have celA1 expressed from the strong promoter hsp60. We compared planktonic and biofilm growth, possible presence of CelA1 in whole protein extracts, quantitated biofilm, presence of monosaccharides, and bacillary burden in lungs after aerosol infection in BALB/c mice. Differences in the appearance of the surface pellicle and of the biofilm attached to the substrate were observed. In biofilms, we observed a significant decrease of glucosamine in BCG:celA1 compared with BCG:pMV361. Finally, BCG:celA1 had lower viable bacteria than the BCG:pMV361 strain after 24 h and 3 weeks post-infection, but no difference was found at 9 weeks post-infection.

Effect of nitric oxide inhibition in Bacillus Calmette-Guerin bladder cancer treatment.

BACKGROUND: Bacillus Calmette-Guerin (BCG) is the standard treatment for patients with high-risk non-muscle invasive bladder cancer (BC). Despite its success, about 30-50% of patients are refractory. It was reported that inducible nitric oxide synthase (iNOS) tumor expression is presented in 50% of human BC, associated with bad prognosis and BCG failure. OBJECTIVE: to evaluate in human bladder tumors the association between iNOS expression and the tumor microenvironment focusing on the immunosuppressive protein S100A9. Also, investigate in a preclinical murine MB49-BC model the tumor immunoresponse induced by BCG in combination with the nitric oxide production inhibitor l-NAME. RESULTS: In human bladder tumors, we detected a positive association between iNOS and S100A9 tumor expression, suggesting a relationship between both immunomodulatory proteins. We also found a positive correlation between iNOS tumor expression and the presence of S100A9+ tumor-infiltrating cells, suggesting an immunosuppressive tumor microenvironment induced by the nitric oxide production. Using the subcutaneous murine BC model, we show that similarly to the human pathology, MB49 tumors constitutively expressed iNOS and S100A9 protein. MB49 tumor-bearing mice presented an immunosuppressive systemic profile characterized by fewer cytotoxic cells (CD8+ and NK) and higher suppressor cells (Treg and myeloid-derived suppressor cells -MDSC-) compared to normal mice. BCG treatment reduced tumor growth, increasing local CD8+-infiltrating cells and induced a systemic increase in CD8+ and a reduction in Treg. BCG combined with l-NAME, significantly reduced tumor growth compared to BCG alone, diminishing iNOS and S100A9 tumor expression and increasing CD8+-infiltrating cells in tumor microenvironment. This local response was accompanied by the systemic increase in CD8+ and NK cells, and the reduction in Treg and MDSC, even more than BCG alone. Similar results were obtained using the orthotopic BC model, where an increase in specific cytotoxicity against MB49 tumor cells was detected. CONCLUSION: The present study provides preclinical information where NO inhibition in iNOS-expressing bladder tumors could contribute to improve BCG antitumor immune response. The association between iNOS and S100A9 in human BC supports the hypothesis that iNOS expression is a negative prognostic factor and a promising therapeutic target.

Alternative Therapies to Bacillus Calmette-Guérin Shortage for Nonmuscle Invasive Bladder Cancer in Brazil and Other Underdeveloped Countries: Management Considerations.

Bacillus Calmette-Guérin (BCG) plays a cornerstone role in the management of nonmuscle invasive urothelial carcinoma of the bladder. However, there has been a worldwide intermittent BCG shortage in recent years that may affect the care of patients with bladder cancer and pose difficult clinical decisions to urologists and clinical oncologists. This literature review aims to clarify alternatives to BCG during a shortage and propose measures to replace BCG, mainly in Brazil and probably in other low- and middle-income countries, where not all studied and commonly suggested treatments are available.

Effects of oral butyrate supplementation on inflammatory potential of circulating peripheral blood mononuclear cells in healthy and obese males.

Sodium butyrate is well-known for its immune-modulatory properties. Studies until now only focused on the in vitro effects of butyrate or assessed local effects in the gut upon butyrate administration. In this trial, we studied the systemic anti-inflammatory effects induced by sodium butyrate supplementation in humans. Nine healthy (Lean) and ten obese (metabolic syndrome group, MetSyn) males were given 4 grams sodium butyrate daily for 4 weeks. PBMCs were isolated before and after supplementation for direct stimulation experiments and induction of trained immunity by oxidized low-density lipoprotein (oxLDL), ß-glucan, or Bacillus Calmette-Guérin vaccine (BCG). Butyrate supplementation moderately affected some of the cytokine responses in the MetSyn group. In the direct stimulation setup, effects of butyrate supplementation were limited. Interestingly, butyrate supplementation decreased oxLDL-induced trained immunity in the MetSyn group for LPS-induced IL-6 responses and Pam3CSK4-induced TNF-α responses. Induction of trained immunity by ß-glucan was decreased by butyrate in the MetSyn group for Pam3CSK4-induced IL-10 production. In this study, while having only limited effects on the direct stimulation of cytokine production, butyrate supplementation significantly affected trained immunity in monocytes of obese individuals with metabolic complications. Therefore, oral butyrate supplementation may be beneficial in reducing the overall inflammatory status of circulating monocytes in patients with metabolic syndrome.

A new tool for tuberculosis vaccine screening: Ex vivo Mycobacterial Growth Inhibition Assay indicates BCG-mediated protection in a murine model of tuberculosis.

BACKGROUND: In the absence of a validated animal model and/or an immune correlate which predict vaccine-mediated protection, large-scale clinical trials are currently the only option to prove efficacy of new tuberculosis candidate vaccines. Tools to facilitate testing of new tuberculosis (TB) vaccines are therefore urgently needed. METHODS: We present here an optimized ex vivo mycobacterial growth inhibition assay (MGIA) using a murine Mycobacterium tuberculosis infection model. This assay assesses the combined ability of host immune cells to inhibit mycobacterial growth in response to vaccination. C57BL/6 mice were immunized with Bacillus Calmette-Guérin (BCG) and growth inhibition of mycobacteria by splenocytes was assessed. Mice were also challenged with Mycobacterium tuberculosis Erdman, and bacterial burden was assessed in lungs and spleen. RESULTS: Using the growth inhibition assay, we find a reduction in BCG CFU of 0.3-0.8 log10 after co-culture with murine splenocytes from BCG vaccinated versus naïve C57BL/6 mice. BCG vaccination in our hands led to a reduction in bacterial burden after challenge with Mycobacterium tuberculosis of approx. 0.7 log10 CFU in lung and approx. 1 log10 CFU in spleen. This effect was also seen when using Mycobacterium smegmatis as the target of growth inhibition. An increase in mycobacterial numbers was found when splenocytes from interferon gamma-deficient mice were used, compared to wild type controls, indicating that immune mechanisms may also be investigated using this assay. CONCLUSIONS: We believe that the ex vivo mycobacterial growth inhibition assay could be a useful tool to help assess vaccine efficacy in future, alongside other established methods. It could also be a valuable tool for determination of underlying immune mechanisms.

Novel approaches to preclinical research and TB vaccine development.

The 4th Global Forum on TB Vaccines, convened in Shanghai, China, from 21 - 24 April 2015, brought together a wide and diverse community involved in tuberculosis vaccine research and development to discuss the current status of, and future directions for this critical effort. This paper summarizes the sessions on Low-Dose NHP Challenge Models, Novel Approaches to Animal Models for TB Vaccine R&D, Novel Antigen Delivery Strategies, and Next Generation TB Vaccines and Vaccine Concepts. Summaries of all sessions from the 4th Global Forum are compiled in a special supplement of Tuberculosis. [August 2016, Vol 99, Supp S1, S1-S30].

Antiandrogen Therapy with Hydroxyflutamide or Androgen Receptor Degradation Enhancer ASC-J9 Enhances BCG Efficacy to Better Suppress Bladder Cancer Progression.

Recent studies suggest that the androgen receptor (AR) might play important roles in influencing bladder cancer progression, yet its clinical application remains unclear. Here, we developed a new combined therapy with Bacillus Calmette-Guérin (BCG) and the AR degradation enhancer ASC-J9 or antiandrogen hydroxyflutamide (HF) to better suppress bladder cancer progression. Mechanism dissection revealed that ASC-J9 treatment enhanced BCG efficacy to suppress bladder cancer cell proliferation via increasing the recruitment of monocytes/macrophages that involved the promotion of BCG attachment/internalization to the bladder cancer cells through increased integrin-α5ß1 expression and IL6 release. Such consequences might then enhance BCG-induced bladder cancer cell death via increased TNFα release. Interestingly, we also found that ASC-J9 treatment could directly promote BCG-induced HMGB1 release to enhance the BCG cytotoxic effects for suppression of bladder cancer cell growth. In vivo approaches also concluded that ASC-J9 could enhance the efficacy of BCG to better suppress bladder cancer progression in BBN-induced bladder cancer mouse models. Together, these results suggest that the newly developed therapy combining BCG plus ASC-J9 may become a novel therapy to better suppress bladder cancer progress.

A review of preclinical animal models utilised for TB vaccine evaluation in the context of recent human efficacy data.

There is an urgent need for an improved TB vaccine. Vaccine development is hindered by the lack of immune correlates and uncertain predictive value of preclinical animal models. As data become available from human efficacy trials, there is an opportunity to evaluate the predictive value of the criteria used to select candidate vaccines. Here we review the efficacy in animal models of the MVA85A candidate vaccine in light of recent human efficacy data and propose refinements to the preclinical models with the aim of increasing their predictive value for human efficacy.

1 alpha, 25-dihydroxylvitamin D3 promotes Bacillus Calmette-Guérin immunotherapy of bladder cancer.

Bacillus Calmette-Guérin (BCG), a vaccine against tuberculosis(TB), has been used and proven to be one of the most effective treatments for non-muscle invasive bladder cancer (BCa). However, the mechanisms of BCG action have not been completely understood, thereby limiting the improvement of BCG therapy. Vitamin D deficiency has been associated with a high risk of TB infection, and the beneficial effect of UV exposure in TB patients was proven to be mediated via activation of vitamin D signals of innate immune cells. Thus, vitamin D signals might be involved in mediating BCG immunotherapy. To test this hypothesis, we examined the impact of 1 alpha, 25-dihydroxyvitamin D3 (1,25-VD) on BCG-induced response in BCa cells and macrophage cells. Our data revealed that 1,25-VD promotes BCG-induced interleukin 8 (IL-8) secretion by BCa cells, consequently inducing the migration of macrophage, THP-1. This THP-1 cell migration promoted by 1,25-VD can be blocked by IL-8 neutralized antibody. Furthermore, 1,25-VD increased BCG-induced expression of macrophage markers in THP-1 cell, and enhanced the BCG-induced THP-1 cytotoxicity against low-grade BCa cells. Importantly, a pre-clinical trial using the N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced BCa mouse model revealed that intravesical co-treatment of 1,25-VD with BCG can prolong mice survival. These data demonstrate a novel mechanism by which 1,25-VD promotes BCG-mediated anti-BCa pathways and provides a platform for improving BCG efficacy with combination of 1,25-VD.

Adaptive immunity in the colostrum-deprived calf: response to early vaccination with Mycobacterium bovis strain bacille Calmette Guerin and ovalbumin.

Responses of the newborn calf to vaccination are frequently characterized by marginal antibody (Ab) responses. The present study evaluated effects of colostrum ingestion on the adaptive immune response of the preruminant calf to early vaccination. Colostrum-fed (CF) and colostrum-deprived (CD) calves were vaccinated at 2 d of age with Mycobacterium bovis, Pasteur strain of bacille Calmette Guerin (BCG), and ovalbumin (OVA) to track development of the adaptive immune response during the first 8 wk of life. Dams were also vaccinated with BCG prepartum. At wk 0, serum IgG(1), IgG(2), IgA, and IgM were elevated in CF calves, with IgG(1) predominating. In these calves, IgG(2), IgA, and IgM concentrations decreased with age. The CD calves, in contrast, had very low or undetectable serum immunoglobulin concentrations at wk 0 followed by an age-related increase in IgG(1), IgG(2), and IgM concentrations, suggesting endogenous production of these immunoglobulin classes. Immunoblot and ELISA analyses of Ab response to BCG vaccination indicated that colostrum ingestion was associated with measurable serum anti-mycobacterial Ab in CF calves during the first month postpartum, with substantially lower levels at 7 wk of age. Although mycobacteria-specific Ab was undetectable in CD calves at wk 0, it was present at 4 and 7 wk of age, suggesting that these calves, unlike CF calves, were capable of generating an Ab response to BCG vaccination. Antibody responses of CF and CD calves to vaccination with OVA, an antigen not present in the natural environment of dairy cattle, were of comparable magnitude and characterized by a progressive increase in Ab levels from birth (wk 0) to 7 wk of age. The disparate Ab responses of CF calves to BCG and OVA suggest that maternal antigenic experience or exposure influences Ab responses of the colostrum-fed preruminant calf to early vaccination. Ex vivo, antigen [OVA and M. bovis-derived purified protein derivative (PPDb)]-induced IFN-γ and nitric oxide responses of blood mononuclear cells (PBMC) from CF and CD calves were comparable at wk 0 and wk 7. As expected, responses were very low or nonexistent at wk 0. Responses for all calves were greater at wk 7 than at wk 0, suggesting a colostrum-independent maturation of the cell-mediated immune response capacity of the preruminant calf. The consistently greater proliferative responses of antigen-stimulated T-cell subsets at wk 7 versus wk 0 indicate the development of antigen-specific lymphocyte responses to early vaccination. Total numbers of blood leukocytes as well as numbers of lymphocytes and monocytes were unaffected by colostrum feeding; however, granulocyte numbers were higher in CD than in CF calves at wk 0. Granulocyte numbers decreased and monocyte numbers increased with age in all calves. Within the lymphocyte population, only natural killer (NK(+)) cell percentages were affected by colostrum ingestion, with higher percentages of NK(+) cells in CD calves at wk 0 and wk 7. Antigen-induced proliferation of lymphocyte subsets including IgM(+) cells was unaffected by colostrum ingestion. In conclusion, ingestion of colostrum within hours after birth inhibited the capacity of the calf to produce antigen-specific immunoglobulin (i.e., antibody) in response to vaccination, with little or no effect on cell-mediated immune responses. Although colostrum appeared to block endogenous antibody production, certain B-cell functions were retained. These findings will aid in development of new vaccination strategies for improving health of the preruminant calf.

Commonly used prophylactic vaccines as an alternative for synthetically produced TLR ligands to mature monocyte-derived dendritic cells.

Currently dendritic cell (DC)-based vaccines are explored in clinical trials, predominantly in cancer patients. Murine studies showed that only maturation with Toll-like receptor (TLR) ligands generates mature DCs that produce interleukin-12 and promote optimal T-cell help. Unfortunately, the limited availability of clinical-grade TLR ligands significantly hampers the translation of these findings into DC-based vaccines. Therefore, we explored 15 commonly used preventive vaccines as a possible source of TLR ligands. We have identified a cocktail of the vaccines BCG-SSI, Influvac, and Typhim that contains TLR ligands and is capable of optimally maturing DCs. These DCs (vaccine DCs) showed high expression of CD80, CD86, and CD83 and secreted interleukin-12. Although vaccine DCs exhibited an impaired migratory capacity, this could be restored by addition of prostaglandin E(2) (PGE(2); vaccine PGE(2) DCs). Vaccine PGE(2) DCs are potent inducers of T-cell proliferation and induce Th1 polarization. In addition, vaccine PGE(2) DCs are potent inducers of tumor antigen-specific CD8(+) effector T cells. Finally, vaccine PGE(2)-induced DC maturation is compatible with different antigen-loading strategies, including RNA electroporation. These data thus identify a new clinical application for a mixture of commonly used preventive vaccines in the generation of Th1-inducing clinical-grade mature DCs.

Reducing the activity and secretion of microbial antioxidants enhances the immunogenicity of BCG.

BACKGROUND: In early clinical studies, the live tuberculosis vaccine Mycobacterium bovis BCG exhibited 80% protective efficacy against pulmonary tuberculosis (TB). Although BCG still exhibits reliable protection against TB meningitis and miliary TB in early childhood it has become less reliable in protecting against pulmonary TB. During decades of in vitro cultivation BCG not only lost some genes due to deletions of regions of the chromosome but also underwent gene duplication and other mutations resulting in increased antioxidant production. METHODOLOGY/PRINCIPAL FINDINGS: To determine whether microbial antioxidants influence vaccine immunogenicity, we eliminated duplicated alleles encoding the oxidative stress sigma factor SigH in BCG Tice and reduced the activity and secretion of iron co-factored superoxide dismutase. We then used assays of gene expression and flow cytometry with intracellular cytokine staining to compare BCG-specific immune responses in mice after vaccination with BCG Tice or the modified BCG vaccine. Compared to BCG, the modified vaccine induced greater IL-12p40, RANTES, and IL-21 mRNA in the spleens of mice at three days post-immunization, more cytokine-producing CD8+ lymphocytes at the peak of the primary immune response, and more IL-2-producing CD4+ lymphocytes during the memory phase. The modified vaccine also induced stronger secondary CD4+ lymphocyte responses and greater clearance of challenge bacilli. CONCLUSIONS/SIGNIFICANCE: We conclude that antioxidants produced by BCG suppress host immune responses. These findings challenge the hypothesis that the failure of extensively cultivated BCG vaccines to prevent pulmonary tuberculosis is due to over-attenuation and suggest instead a new model in which BCG evolved to produce more immunity-suppressing antioxidants. By targeting these antioxidants it may be possible to restore BCG's ability to protect against pulmonary TB.

Bacillus Calmette-Guérin-pulsed dendritic cells stimulate natural killer T cells and gammadeltaT cells.

BACKGROUND: Immunotherapy with bacillus Calmette-Guérin (BCG) for bladder cancer is successful, although the precise mechanism is unclear. Natural killer (NK) cells are a candidate for BCG-activated killer cells, but the roles of other T lymphocytes, such as NKT cells and gammadeltaT cells, are not fully understood. Mycobacterium tuberculosis is a potent activator of both NKT cells and gammadeltaT cells. However, it is known that the patient's prognosis is good if there are increased numbers of dendritic cells (DCs) in the urine after BCG therapy. Therefore, we investigated whether DCs are matured by BCG and whether BCG-pulsed DCs stimulate NKT cells and gammadeltaT cells. METHODS: Naïve Pan T cells were isolated form peripheral blood mononuclear cells (PBMCs) and DCs were obtained by culturing CD14(+) monocytes with granulocyte-macrophage colony-stimulating factor and interleukin-4. The DCs were pulsed with BCG and their maturation was measured by fluorescence-activated cell sorter analysis using the CD86 antibody. Naïve T lymphocytes were stimulated by coculture with BCG-pulsed DCs in vitro, followed by FACS analysis to estimate the ratio and activation of NKT cells and the ratio of gammadeltaT cells. The (51)Cr (chromium) release assay was used to estimate the cytotoxic activity of the stimulated T cells. Cytolytic proteins in the patient's PBMCs were measured during BCG therapy using semiquantitative reverse transcriptase-polymerase chain reaction. RESULTS: The DCs were matured by BCG stimulation and the number of NKT cells and gammadeltaT cells increased after culturing with BCG-pulsed DCs. The BCG-pulsed DCs also activated the NKT cells and gammadeltaT cells. Also, the lymphocytes that were cocultured with the BCG-pulsed DCs showed unspecific cytotoxic activity against a bladder cancer cell line. CONCLUSION: Sensitization of NKT cells and gammadeltaT cells by BCG-pulsed DCs might be one of the mechanisms of BCG immunotherapy.

Lactoferrin augments BCG vaccine efficacy to generate T helper response and subsequent protection against challenge with virulent Mycobacterium tuberculosis.

The ability to control intracellular Mycobacterium tuberculosis (MTB) infection relies on cellular immunity and generation of a strong T-cell helper 1 (T(H)1) response. Lactoferrin, an iron-binding protein with immune regulatory functions, was investigated as an adjuvant to boost Mycobacterium bovis Bacillus Calmette-Guerin (BCG) efficacy. Lactoferrin was initially shown to augment IL-12(p40) production from macrophages stimulated with LPS. A single immunization of mice with Lactoferrin as an adjunct adjuvant resulted in amplified splenocyte proliferative response to heat-killed BCG, and elevated IL-12(p40) production with increased relative ratios of IL-12/IL-10. Furthermore, splenocyte recall response to HK-BCG was augmented for proinflammatory mediators, TNF-alpha, IL-1beta, and IL-6, approaching responses generated to complete Freund's adjuvant (CFA) immunized controls. Specific responses were identified, with significant elevation of IFN-gamma generated during antigenic recall. Subsequent aerosol challenge of Lactoferrin adjuvant immunized mice with virulent M. tuberculosis revealed decreased mycobacterial loads in the lung, and limitation of organism dissemination to a peripheral organ (spleen). These studies indicate that Lactoferrin can act as an adjunct adjuvant to augment cellular immunity and boost BCG efficacy for protection against subsequent challenge with virulent MTB.

N-acetylcysteine augments the cellular redox changes and cytotoxic activity of internalized mycobacterium bovis in human bladder cancer cells.

PURPOSE: We determined whether changes in cellular reactive oxygen species correlated with mycobacteria internalization and bladder cancer cell death. MATERIALS AND METHODS: Reactive oxygen species and thiols in RT112 and MGH bladder cancer cells were determined using the fluorescence probes 5-(and 6)-carboxy-2', 7' dichlorodihydrofluorescein diacetate and monobromobimane. Superoxide and nitrite production were measured using bis-N-methylarcridinium nitrate and Griess reagents. Cytotoxicity was determined by the release of 14C-thymidine from cells with 14C labeled DNA. RESULTS: MGH cells that internalize bacillus Calmette-Guerin (BCG) had decreased cellular reactive oxygen species and thiols, although superoxide and nitric oxide production increased. RT112 cells, which do not internalize BCG, did not show a decrease in reactive oxygen species after incubation with BCG. Blocking BCG uptake in MGH cells abrogated reactive oxygen species reduction, confirming that the changes in reactive oxygen species were internalization dependent events. Treating cells with BCG and the antioxidant N-acetylcysteine caused a greater reduction in reactive oxygen species, and induced earlier and greater cytotoxicity in MGH but not in RT112 cells. CONCLUSIONS: The induction of bladder cancer cell killing by BCG parallels the ability of cells to internalize BCG, which in turn indicates that the susceptibility of tumor cells to the cytotoxic effects of BCG may be related to changes in cellular levels of reactive oxygen species and thiols. Supplementation with an antioxidant could enhance the antitumor effect of BCG.

Bacillus Calmette-Guerin induces long-term local formation of nitric oxide in the bladder via the induction of nitric oxide synthase activity in urothelial cells.

PURPOSE: Bladder instillation of bacillus Calmette-Guerin (BCG) is effective therapy for recurrent superficial bladder cancer and carcinoma in situ. BCG induces nitric oxide synthase activity in the bladder. Nitric oxide is formed from L-arginine by nitric oxide synthase. We investigated nitric oxide formation and its localization in bladder cancer patients treated with intravesical BCG instillation. MATERIALS AND METHODS: The L-citrulline conversion assay was done to assess nitric oxide synthase activity in BCG treated T24 human bladder cancer cells and cultured normal human urothelial cells. Nitrite and nitrate in cell culture medium, urine and plasma were measured by capillary electrophoresis. Nitric oxide formation in the bladder was measured by chemiluminescence. RESULTS: A 24-hour treatment with BCG induced calcium independent nitric oxide synthase activity in T24 cells in a dose dependent manner. Nitrite and nitrate production by T24 cells also increased in a dose dependent manner after 24-hour BCG treatment. BCG treatment of cultured normal human urothelial cells resulted in the induction of calcium dependent and independent nitric oxide synthase activity. Nitrite in the urine of patients receiving BCG for the first time was increased 5-fold 24 hours after instillation. Furthermore, BCG increased luminal nitric oxide in the bladder. The increase was noted after a single treatment and sustained for 6 months. No changes in plasma nitrite or nitrate were observed after BCG treatment. CONCLUSIONS: BCG induces the local formation of nitric oxide in the bladder, whereas no evidence for systemic nitric oxide formation was noted. Increased nitric oxide production in the bladder is likely due to the induction of nitric oxide synthase activity in urothelial cells.

No effect of exposure to static and sinusoidal magnetic fields on nitric oxide production by macrophages.

The effects of exposure to static (1 - 100 mT) or sinusoidal (1 Hz, 1.6 mT) magnetic fields on the production of nitric oxide (NO) by murine BCG-activated macrophages were investigated. In these cells, the inducible isoform of NO synthase is present. No significant differences were observed in nitrite levels among exposed, sham-exposed, or control macrophages after exposure for 14 h to static fields of 1, 10, 50, and 100 mT and to sinusoidal 1.6 mT, 1 Hz magnetic fields.

[The effects of zhuling-duotang, hepatitis B vaccine and bacillus Calmette-Guerin on immunoactivities of peripheral blood mononuclear cells: an in vitro research].

"Zhuling-duotang (a polysaccharide preparation of the Chinese traditional herb medicine polyporusum bellatus) and hepatitis B vaccine (HB vaccine)" and "Persantin and bacillus calmeteguerin (BCG)" have been used to treat chronic hepatitis B. To elucidate their therapeutic mechanism, we studied the effects of zhuling-duotang, HB vaccine and BCG on immunoactivities of nomal human peripheral blood mononuclear cells (PBMCs). Cytotoxicity of PBMCs incubated with such drugs against target cells K562, HepG2 or 2.2.15 was detected by using 3H-TdR release assay. IL-2 and IFN-gamma activities of the supernatant were assayed. Immunofluorescent analysis of molecular CD4, CD8, CD16, IL-2R on the surface of PBMCs were done. The results showed that: (1) Zhuling-duotang and BCG could significantly increase the cytotoxicity of PBMCs. They could induce PBMCs to produce IL-2 but not IFN-gamma. They also could stimulate PBMCs to express IL-2R. Zhuling-duotang and BCG did not affect the percentage of CD4+, CD8+, CD16+ cells among PBMCs. (2) HB vaccine did not significantly increase the cytotoxicity of PBMCs. (3) The cytotoxicity of PBMCs induced by Zhuling-duotang combined with HB vaccine was not significantly higher than that of PBMCs induced by Zhuling-duotang alone.