Objective The prevalence of drug-resistant Mycobacterium tuberculosis (Mtb) strains is exacerbating the global burden of tuberculosis (TB), highlighting the urgent need for new treatment strategies for TB. Methods The recombinant adenovirus vaccine expressing cyclic di-adenosine monophosphate (c-di-AMP) phosphodiesterase B (CnpB) (rAd-CnpB), was administered to normal mice via mucosal immunization, either alone or in combination with drug therapy, to treat Mtb respiratory infections in mice.Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of antibodies in serum and bronchoalveolar lavage fluid (BALF). Real-time quantitative PCR was performed to assess the transcription levels of cytokines interferon γ(IFN-γ) and interleukin 10(IL-10) in mouse lungs. Flow cytometry was used to determine the proportions of CD4⁺ and CD8⁺ T cell subsets in the lungs and spleens. ELISA was employed to measure the levels of cytokines IFN-γ, IL-2, IL-10, inflammatory factors IL-6, and tumor necrosis factor α (TNF-α) secreted by spleen cells following antigen stimulation. The bacteria loads in the lungs and spleens of Mtb-infected mice were enumerated by plate counting methods. Resluts Intranasal immunization with rAd-CnpB induced high titers of IgG in mouse serum and the production of IgG and IgA in BALF, along with alterations in T lymphocyte subsets in the lungs and spleens. Administration of rAd-CnpB, either alone or in combination with drugs, to Mtb-infected mice significantly increased serum IgG levels as well as IgA and IgG levels in BALF. rAd-CnpB immunization promoted the secretion of CnpB-specific cytokines and inflammatory factors by splenocytes in Mtb-infected mice. However, rAd-CnpB immunotherapy, either alone or combined with drugs, did not significantly affect the bacterial loads in the lungs and spleens of mice with Mtb respiratory infections. Conclusion Mucosal immunization with rAd-CnpB induced significant mucosal, humoral and cellular immune responses in mice, and significantly enhanced CnpB-specific cellular immune responses in Mtb-infected mice.
Animals ; Adenoviridae/immunology* ; Mycobacterium tuberculosis/genetics* ; Mice ; Female ; Phosphoric Diester Hydrolases/genetics* ; Tuberculosis Vaccines/administration & dosage* ; Tuberculosis/prevention & control* ; Mice, Inbred BALB C ; Cytokines ; Lung/microbiology* ; Immunization ; Bronchoalveolar Lavage Fluid/immunology* ; Immunity, Mucosal

Objectives: This study aims to describe the clinical profile of patients with tuberculosis of the spine admitted at PCMC from the year 2012-2022. Moreover, this study aims to describe the clinical profile (age, gender, BMI, area of residence) of the patients with tuberculosis of the spine admitted at PCMC from the years 2012-2022. It also aims to present the known BCG vaccination status, exposure and risk factors (nutritional factors, comorbidities), of these patients. This study presents the symptomatology (including the spinal level of involvement, and severity, sensory or motor dysfunction) and the medical and/or surgical treatment and the outcome of these identified patients.

Materials and Methods: A retrospective chart review at PCMC analyzed children under 19 diagnosed with Pott’s Disease from January 2013 to December 2022. The study, approved by the Institutional Review Board, included demographic data, clinical manifestations, BCG vaccination status, treatment details, and outcomes, while excluding non-Filipino patients and readmissions.

Results: This study examined 41 pediatric patients with Pott’s disease at the Philippine Children’s Medical Center from 2012 to 2022, primarily affecting males aged 10-15. Most patients were from low-income backgrounds. Symptoms included chronic back pain, fever, and neurological issues, with advanced imaging required for diagnosis. While 93% had received BCG vaccinations, the correlation with disease severity was inconclusive. Treatment involved anti-tuberculous agents, with surgery for severe cases. Despite improvements, none were disease-free, highlighting chronic disabilities. The findings emphasize the need for better management of spinal tuberculosis and increased BCG vaccination among children in high TB-burden areas.

Conclusion: The study evaluated the clinical profile and clinical features of children with Pott’s Disease who were treated at the Philippine Children’s Medical Center (PCMC) between the years 2012-2022. The data from the study identifies the BCG vaccine may not prevent the onset of Pott's disease.

Keywords: Pott’s Disease, Clinical Profiles, Treatment Outcomes

Human ; Male ; Female ; Infant Newborn: First 28 Days After Birth ; Infant: 1-23 Months ; Child Preschool: 2-5 Yrs Old ; Child: 6-12 Yrs Old ; General Surgery ; Child ; Bcg Vaccine ; Mycobacterium Bovis ; Patients ; Risk Factors ; Tuberculosis, Spinal ; Vaccination

Early secreted antigenic target of 6 kDa protein (ESAT-6) is the major virulence factor of Mycobacterium tuberculosis (MTB), which can resist the clearance of MTB in bodies by inhibiting macrophage phagocytosis and autophagy reaction, thus impeding the immune defense function of the body against MTB infection. In addition, ESAT-6-induced apoptosis of macrophage and massive necrosis of innate immune cells can foster MTB proliferation and colonization, leading to systemic MTB infection. Moreover, ESAT-6 hampers the protective immune response of Th1 cells, reducing the secretion of pro-inflammatory cytokines and contributing to immune dysfunction, thus accelerating the course of MTB infection. During the process, the high immunogenicity of ESAT-6 can be leveraged as a dominant antigen in the development of new TB vaccines, making it a promising candidate with broad prospects for further development.
Humans ; Mycobacterium tuberculosis ; Vaccines ; Cytokines ; Apoptosis ; Autophagy ; Sepsis

To prepare a lipid nanoparticle (LNP)-based subunit vaccine of Mycobacterium tuberculosis (Mtb) antigen EsxV and study its immunological characteristics, the LNP containing EsxV and c-di-AMP (EsxV: C: L) was prepared by thin film dispersion method, and its encapsulation rate, LNP morphology, particle size, surface charge and polyphase dispersion index were measured. BALB/c mice were immunized with EsxV: C: L by nasal drops. The levels of serum and mucosal antibodies, transcription and secretion of cytokines in lung and spleen, and the proportion of T cell subsets were detected after immunization. EsxV: C: L LNPs were obtained with uniform size and they were spherical and negatively charged. Compared with EsxV: C immunization, EsxV: C: L mucosal inoculation induced increased sIgA level in respiratory tract mucosa. Levels of IL-2 secreted from spleen and ratios of memory T cells and tissue-resident T cells in mice were also elevated. In conclusion, EsxV: C: L could induce stronger mucosal immunity and memory T cell immune responses, which may provide better protection against Mtb infection.
Animals ; Mice ; Mycobacterium tuberculosis ; Antigens, Bacterial ; Immunization ; Nanoparticles ; Vaccines, Subunit ; Mice, Inbred BALB C

This study aims to develop a method to detect bovine multi-cytokines based on flow cytometry. Previously we have prepared and screened monoclonal antibodies against bovine cytokines IFN-γ, IL-2, TNF-α, IP-10 and MCP-1. These bovine cytokine monoclonal antibodies were fluorescently labeled, and the combination of antibody and cell surface molecules were used to develop the method for detecting bovine multi-cytokines. Subsequently, the developed method was used to determine the cytokine expression profile of Mycobacterium bovis BCG infected bovine peripheral blood mononuclear cells in vitro, and evaluate the cytokine expression level of peripheral blood CD4⁺ T cells of tuberculosis-positive cattle. The bovine multi-cytokine flow cytometry detection method can effectively determine the cytokine expression of BCG-infected bovine peripheral blood T lymphocytes. Among them, the expression levels of IFN-γ, IL-2, and TNF-α continue to increase after 40 hours of infection, while the expression levels of IP-10 and MCP-1 decreased. The combined detection of IFN-γ, IL-2, and TNF-α on CD4⁺ T lymphocytes in peripheral blood of cattle can effectively distinguish tuberculosis-positive and tuberculosis-negative samples. This method may facilitate evaluating the level of cellular immune response after bovine pathogen infection and vaccine injection.
Cattle ; Animals ; Cytokines ; BCG Vaccine/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Interleukin-2 ; Flow Cytometry/methods* ; Chemokine CXCL10/metabolism* ; Leukocytes, Mononuclear ; CD4-Positive T-Lymphocytes/metabolism* ; Tuberculosis ; Antibodies, Monoclonal/metabolism*

OBJECTIVE: To investigate the inhibitory effects of levofloxacin (LEV) combined with cellulase against bacille CalmetteGuerin (BCG) biofilms in vitro. METHODS: The mature growth cycle of BCG biofilms was determined using the XTT method and crystal violet staining. BCG planktonic bacteria and BCG biofilms were treated with different concentrations of LEV and cellulose alone or jointly, and the changes in biofilm biomass were quantified with crystal violet staining. The mature BCG biofilm was then treated with cellulase alone for 24 h, and after staining with SYTO 9 and Calcofluor White Stain, the number of viable bacteria and the change in cellulose content in the biofilm were observed with confocal laser scanning microscopy. The structural changes of the treated biofilm were observed under scanning electron microscopy. RESULTS: The MIC, MBC and MBEC values of LEV determined by broth microdilution method were 4 μg/mL, 8 μg/mL and 1024 μg/mL, respectively. The combined treatment with 1/4×MIC LEV and 2.56, 5.12 or 10.24 U/mL cellulase resulted in a significant reduction in biofilm biomass (P < 0.001). Cellulase treatments at the concentrations of 10.24, 5.12 and 2.56 U/mL all produced significant dispersion effects on mature BCG biofilms (P < 0.001). CONCLUSION LEV combined with cellulose can effectively eradicate BCG biofilm infections, suggesting the potential of glycoside hydrolase therapy for improving the efficacy of antibiotics against biofilmassociated infections caused by Mycobacterium tuberculosis.
Levofloxacin/pharmacology* ; Gentian Violet/pharmacology* ; BCG Vaccine/pharmacology* ; Anti-Bacterial Agents/pharmacology* ; Biofilms ; Cellulases/pharmacology* ; Microbial Sensitivity Tests

Objectives To develop a multi-stage and multi-epitope vaccine, which consists of epitopes from the early secretory and latency-associated antigens of Mycobacterium tuberculosis (MTB). Methods The B-cell, cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL) epitopes of 12 proteins were predicted using an immunoinformatics. The epitopes with antigenicity, without cytotoxicity and sensitization, were further screened to construct the multi-epitope vaccine. Furthermore, the proposed vaccine underwent physicochemical properties analysis and secondary structure prediction as well as 3D structure modeling, refinement and validation. Then the refined model was docked with TLR4. Finally, an immune simulation of the vaccine was carried out. Results The proposed vaccine, which consists of 12 B-cell, 11 CTL and 12 HTL epitopes, had a flexible and stable globular conformation as well as a thermostable and hydrophilic structure. A stable interaction of the vaccine with TLR4 was confirmed by molecular docking. The efficiency of the candidate vaccine to trigger effective cellular and humoral immune responses was assessed by immune simulation. Conclusion A multi-stage multi-epitope MTB vaccine construction strategy based on immunoinformatics is proposed, which is expected to prevent both active and latent MTB infection.
Mycobacterium tuberculosis/metabolism* ; Molecular Docking Simulation ; Toll-Like Receptor 4 ; Epitopes, T-Lymphocyte/chemistry* ; Epitopes, B-Lymphocyte/chemistry* ; Vaccines, Subunit/chemistry* ; Computational Biology/methods*

INTRODUCTION: Usage of metformin is associated with improved survival in lung, breast and prostate cancer, and metformin has been shown to inhibit cancer cell growth and proliferation in in vitro studies. Given the lack of clinical data on metformin use in patients with bladder cancer, we aimed to evaluate the role of metformin in their oncological outcomes. METHODS: Medication use data from a prospectively maintained database of 122 patients with non-muscle-invasive bladder cancer treated with intravesical Bacille Calmette-Guerin (BCG), who were recruited under a randomised, double-blinded, controlled clinical trial, was collected and analysed. Kaplan-Meier curves were used to assess overall survival (OS) and disease-specific survival (DSS). RESULTS: At a median follow-up duration of 102 (range 3-357) months, 53 (43.4%) patients experienced disease recurrence and 21 (17.2%) experienced disease progression. There was no significant difference in mortality between patients with and without diabetes mellitus. There was significant difference in OS between patients without diabetes mellitus, patients with diabetes mellitus on metformin and patients with diabetes mellitus but not on metformin (p = 0.033); patients with diabetes mellitus on metformin had the best prognosis. Metformin use was associated with significantly lower DSS (p = 0.042). Other oral hypoglycaemic agents, insulin or statins were not associated with disease recurrence or progression. CONCLUSION Metformin use was associated with improved oncological outcomes in patients with non-muscle-invasive bladder cancer treated with intravesical BCG. Prospective studies with larger patient populations are needed to validate the role of metformin as potential therapy for bladder cancer.
Adjuvants, Immunologic/therapeutic use* ; Administration, Intravesical ; BCG Vaccine/therapeutic use* ; Diabetes Mellitus ; Disease Progression ; Humans ; Male ; Metformin/therapeutic use* ; Neoplasm Invasiveness ; Neoplasm Recurrence, Local ; Prospective Studies ; Retrospective Studies ; Urinary Bladder Neoplasms/drug therapy*

Objective: To investigate the economic burden of bacillus Calmette-Guérin (BCG) lymphadenitis in Shandong Province. Methods: From May 2011 to December 2019, 304 patients applying for the province-level compensation of BCG lymphadenitis was selected from Shandong Province in this study. The basic situation, vaccination, outpatient (inpatient) records, cost and relevant information of those patients were collected to calculate the direct economic burden (including direct medical costs and direct non-medical costs), indirect economic burden and total economic burden. Comparison of the difference of economic burden of cases with different characteristics was taken. Results: The M(Q₁,Q₃) of age of BCG lymphadenitis patients was 3 (2, 4) months, among which 239 cases (78.6%) were male, 71 cases (23.4%) had lymphadenopathy, and 227 cases (74.7%) underwent surgery.The number of outpatient only, inpatient only and outpatient then inpatient was 25.7% (78 cases), 7.2% (22 cases) and 67.1% (204 cases), respectively. The M(Q₁,Q₃) of direct, indirect and total economic burden of single case after discount was 9 910 (5 713, 16 074), 2 081 (1 547, 3 122) and 12 262 (7 694, 18 571) yuan, respectively.The direct medical expenses accounted for 89.4% of the direct economic burden, the direct economic burden accounted for 84.9% of the total economic burden, the total economic burden of 80.0% cases accounted for only about 20.0% of the compensation amount, and the total economic burden of only 2.3% cases accounted for more than 60.0% of the compensation amount.The direct, indirect and total economic burden of patients with inpatient only and outpatient then inpatient was higher than that of patients with outpatient only; the direct, indirect and total economic burden of patients with operation was higher than that of patients with non-operation; the direct and total economic burden of patients with unulcerated lymph node was higher than that of patients with ulcerated lymph node(all P values<0.05). Conclusion: The economic burden of BCG lymphadenitis cases in Shandong Province is influenced by the mode of diagnosis and treatment, with direct medical expenses as the predominant component.
BCG Vaccine ; Cost of Illness ; Financial Stress ; Humans ; Infant ; Lymphadenitis/epidemiology* ; Male ; Vaccination

To explore the protective immune effect induced by mucosal delivery heparin-binding hemagglutinin (HBHA)-a candidate vaccine antigen of Mycobacterium tuberculosis. Female C57BL/6 mice were between 6 and 8 weeks of age before experimental use. Thirty mice received different immunization strategies and were randomly divided into the control group, the early secreting antigen target-6 (ESAT-6) intranasal immunization group, the HBHA intranasal immunization group, the BCG priming PBS control group, or BCG priming HBHA boost group, 6 mice in each group. In order to analyzed the immune effect, the concentrations of plasma Interleukin-17A (IL-17A) and other cytokines were measured by ELISA. Quantitative real-time PCR analyses were performed to detect the relative quantity (RQ) mRNA of IL-17A in the lung. The lung tissue sections were stained to detect the formation of the tertiary lymphoid structures. The chemokines contributed to formation of the tertiary lymphoid structures were also measured. Flow cytometry was used to detect the frequency of Th1 and Th17 cells in the system. Sixty mice in the BCG priming PBS control group and the BCG priming HBHA boost group were sacrificed at different time points after infection to count the lung bacterial burden. The concentrations of plasma IL-17A and relative quantity of lung IL-17A mRNA were highest in the BCG priming HBHA boost group [(14.76±4.73) pg/mL,RQ (12.27±6.71)], which was significantly higher than the control group [(5.57±2.95) pg/mL,RQ (1.30±0.97)] (t=4.213, P<0.001; t=5.984, P<0.001), and also significantly higher than the BCG priming PBS control group [(6.81±2.18) pg/mL,RQ (1.44±1.16)] (t=3.646 P=0.001; t=6.185 P<0.001). Compared with the BCG priming PBS control group (0.38±0.38)% the frequency of spleen Th17 cells were also significantly increased (t=-0.280 , P=0.048) in the BCG-primary HBHA boost group (1.02±0.34)%. In addition, HBHA boosting could promote better formation of the tertiary lymphoid structures in the lung, and decrease the bacterial load on the early stage after BCG challenge. Collectively, mucosal delivery of HBHA can effectively enhance the protective effect after BCG vaccination, and it is a potential candidate vaccine component.
Animals ; Antigens, Bacterial ; Bacterial Proteins ; Female ; Humans ; Immunization, Secondary ; Interleukin-17 ; Lectins ; Mice ; Mice, Inbred C57BL ; Mycobacterium tuberculosis ; Tuberculosis/prevention & control* ; Tuberculosis Vaccines