POLE2 knockdown suppresses lymphoma progression via downregulating Wnt/ß-catenin signaling pathway.

Lymphoma is the most common malignant tumor arising from immune system. Recently, DNA polymerase epsilon subunit 2 (POLE2) was identified to be a tumor promotor in a variety of malignant tumors. However, the biological role of POLE2 in lymphoma is still largely unclear. In our present study, the expression patterns of POLE2 in lymphoma tissues were identified by immunohistochemistry (IHC) staining of human tissue microarray. Cell viability was determined by CCK-8 assay. Cell apoptosis and cycle distribution were evaluated by Annexin V and PI staining, respectively. Cell migration was analyzed by transwell assay. Tumor growth in vivo was observed by a xenograft model of mice. The potential signaling was explored by human phospho-kinase array and immunoblotting. POLE2 was significantly upregulated in human lymphoma tissues and cells. POLE2 knockdown attenuated the proliferation, migration capabilities of lymphoma cells, as well as induced cell apoptosis and cycle arrest. Moreover, POLE2 depletion impaired the tumor growth in mice. Furthermore, POLE2 knockdown apparently inhibited the activation of ß-Catenin and downregulated the expression of Wnt/ß-Catenin signaling-related proteins. POLE2 knockdown suppressed the proliferation and migration of lymphoma cells by inhibiting Wnt/ß-Catenin signaling pathway. POLE2 may serve as a novel therapeutic target for lymphoma.

Prediction and analyses of HLA-II restricted Mycobacterium tuberculosis CD4+ T cell epitopes in the Chinese population.

The Bacillus Calmette-Guérin (BCG) vaccine has been used to prevent tuberculosis (TB), but it cannot prevent adults against TB. The Mycobacterium tuberculosis Beijing strain is the most popular strain in China, but no vaccine is designed for the Beijing strain. It is vital to design a multiepitopes-based vaccine against the Beijing strain for the Chinese population. The bioinformatics tools were used to predict CD4+ T-cell epitopes in five protective antigens based on the Chinese population-specific alleles. The antigenicity, allergenicity, toxicity, IFN-γ level, population coverage, and three-dimensional structure were predicted using Vaxijen, AllerTOP, ToxinPred, IFN-γ epitope server, IEDB, and I-TASSER, respectively. One-hundred one promiscuous epitopes were obtained from Rv1813c, Rv2608, Rv3131, and Rv3628 proteins. After screening with antigenicity, allergenicity, toxicity, and IFN-γ level, seven epitopes from Rv2608 and Rv3131 proteins were selected to be vaccine candidates. Further study determined their three-dimensional structure and the coverage in the Chinese population as high as 99%. Our study predicted seven CD4+ T-cell dominant epitopes from the proteins Rv2608 and Rv3131 of M. tuberculosis Beijing strain for the first time, which may provide a basis for improving the design of multiepitopes-based vaccines for TB.

G-CSF-primed haplo-identical HSCT with intensive immunosuppressive and myelosuppressive treatments does not increase the risk of pre-engraftment bloodstream infection: a multicenter case-control study.

A multicenter retrospective study in 131 patients (44 females/87 males) with hematological disorders who underwent G-CSF-primed/haplo-identical (Haplo-ID) (n = 76) or HLA-identical (HLA-ID) HSCT (n = 55) from February 2013 to February 2016 was conducted to compare the incidence and risk factors for pre-engraftment bloodstream infection (PE-BSI). In the Haplo-ID group, 71/76 patients with high-risk (n = 28) or relapsed/refractory hematological malignancies (n = 43) received FA5-BUCY conditioning (NCT02328950). All received trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis. Blood cultures and catheter tip cultures were obtained to confirm the BSI. PE-BSI was detected in 24/131 HSCT patients (18/76 in Haplo-ID and 6/55 in HLA-ID) after 28 febrile neutropenic episodes. Among 28 isolates for the 24 patients, 21 (75%) were Gneg bacteria, 6 (21.4%) Gpos and 1 (3.6%) fungi. Bacteria sources were central venous line infection (7/29.2%), gastroenteritis (6/25%), lower respiratory tract infection (LRTI; 5/20.8%), perianal skin infection (4/16.7%), and unknown (2/8.3%). The duration of neutropenia (P = 0.046) and previous Gneg bacteremia (P = 0.037) were important risk factors by univariate analysis, while the type of HSCT was not. A trend of TMP-SMX-resistant BSI in both groups may be due to routine antibacterial prophylaxis strategies. Our data show that G-CSF-primed Haplo-ID HSCT did not increase the risk of PE-BSI, even with intensive immunosuppressive treatments.

Cytokine and soluble adhesion molecule profiles and biomarkers for treatment monitoring in Re-treated smear-positive patients with pulmonary tuberculosis.

Relapse of pulmonary tuberculosis (PTB) is associated with a failure of the host immune system to control the invading Mycobacterium tuberculosis. Severe immunodeficiency or immune disorders may be the main reason for TB recurrence. This study aimed to quantify serum inflammatory cytokine and soluble adhesion molecule levels in Re-treated smear-positive PTB patients before and after re-anti-TB drug therapy. Serum samples were collected from 30 healthy controls and 215 Treated active PTB patients at baseline and 2, 4, and 6 months post-re-treatment. Levels of 18 serum cytokines and soluble adhesion molecules were measured by a high-throughput Cytometric Bead Array. At baseline, IL-1, IL-2, IL-12P70, and soluble CD62E levels were significantly higher in PTB patients than those in the healthy controls (p < 0.05); IL-4, IL-5, IL-7, IL-8, IL-10, IL-17, IL-21, soluble CD54, MIG, and TGF-ß levels in PTB patients were significantly lower than those in the healthy controls (p < 0.05), of which TGF-ß, IL-7, IL-8, IL-10, soluble CD54, and MIG were most notably (p < 0.0005). After re-treatment, IFN-γ, IL-2, IL-7, and soluble CD54 levels and IL-2/IL-10 and IFN-γ/IL-10 ratios showed an upward trend during the re-treatment period. They were more sensitive than other cytokines and adhesion molecules and could be effective as serum indicators for re-treatment response. The immune response was imbalance in treated smear-positive PTB patients: Th1 response was elevated, but Th2 and Th17 responses were reduced. Systematic and comprehensive understanding of the cytokine and soluble adhesion molecule profiles provides a theoretical basis for immuno-diagnosis, immunotherapy, and immuno-monitoring of Re-treated PTB patients.

Immunogenicity and therapeutic effects of a Mycobacterium tuberculosis rv2190c DNA vaccine in mice.

BACKGROUND: Tuberculosis (TB) is a major global public health problem. New treatment methods on TB are urgently demanded. In this study, Mycobacterium tuberculosis (MTB) rv2190c DNA vaccine was prepared and its immunogenicity and therapeutic effects were evaluated. RESULTS: Non-infected mice immunized with rv2190c DNA or ag85a DNA showed higher levels of interferon-gamma (IFN-γ) in stimulated spleen lymphocyte culture supernatants, and had more Th1 cells and an elevatory ratio of Th1/Th2 immune cells in whole blood, indicating that Th1-type immune response was predominant. Compared with the saline group, ag85a DNA group and rv2190c DNA group in the infected mice decreased the lung colony-forming units (CFUs) by 0.533 and 0.283 log10, and spleen CFUs by 0.425 and 0.321 log10 respectively, and pathological lesion. CONCLUSIONS: The rv2190c DNA had some immunotherapeutic effect on TB.

The Construction and Identification of Induced Pluripotent Stem Cells Derived from Acute Myelogenous Leukemia Cells.

OBJECTIVE: The present study aimed to establish an induced pluripotent stem cell (iPSC) line from acute myelogenous leukemia (AML) cells in vitro and identify their biological characteristics. METHODS: Cells from the AML-infiltrated skin from an M6 patient were infected with a lentivirus carrying OCT4, SOX2, KLF4 and C-MYC to induce iPSCs. The characteristics of the iPSCs were confirmed by alkaline phosphatase (ALP) staining. The proliferation ability of iPSCs was detected with a CCK-8 assay. The expression of pluripotency markers was measured by immunostaining, and the expression of stem cell-related genes was detected by qRT-PCR; distortion during the induction process was detected by karyotype analysis; the differentiation potential of iPSCs was determined by embryoid body-formation and teratoma-formation assays. ALP staining confirmed that these cells exhibited positive staining and had the characteristics of iPSCs. RESULTS: The CCK-8 assay showed that the iPSCs had the ability to proliferate. Immunostaining demonstrated that iPSC clones showed positive expression of NANOG, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. qRT-PCR results revealed that the mRNA expression of Nanog, Lin28, Cripto, FOX3, DNMT3b, DPPA2, and DPPA4 significantly increased in iPSCs. Karyotype analysis found no chromosome aberration in the iPSCs. The results of the embryoid body-formation and teratoma-formation assays indicated that the iPSCs had the potential to differentiate into all three germ layers. CONCLUSION: Our study provided evidence that an iPSC line derived from AML cells was successfully established.

Effects of Blood Purification on Serum Levels of Inflammatory Cytokines and Cardiac Function in a Rat Model of Sepsis.

OBJECTIVE: The study aimed to explore the effects of blood purification (BP) on serum levels of inflammatory cytokines and cardiac function in a rat model of sepsis. METHODS: A rat model of sepsis was established by cecal ligation and puncture. All rats were divided into the normal control, sham operation, model, sham treatment, and BP treatment groups. Cardiac functions, inflammatory cytokines, myocardial enzymes, pathological score of cardiac muscle tissue, and myocardial apoptosis of rats in each group were compared. RESULTS: Sepsis rats had higher serum levels of inflammatory cytokines and lower cardiac function than those in the normal control and sham operation groups. Compared with the model and sham treatment groups, improved cardiac functions, decreased inflammatory cytokines, myocardial enzymes, pathological score, and myocardial apoptosis and mortality were observed in the BP treatment group. CONCLUSION: BP may reduce serum levels of inflammatory cytokines and improve cardiac function of sepsis rats.

Evaluation of a Whole Blood Chemiluminescent Immunoassay of Interferon-gamma Inducible Protein 10 (IP-10) for Diagnosis of Tuberculosis Patients.

BACKGROUND: The rapid diagnosis of bacterium-negative pulmonary TB and extra pulmonary TB is a significant problem in clinical practice. METHODS: 466 individuals were prospectively enrolled, 247 patients with TB, 147 with non-tuberculous pulmonary disease, and 65 healthy controls. Whole blood was stimulated in vitro with rCFP10/ESAT6 antigen of Mycobacterium tuberculosis. A chemiluminescence immunoassay was used to detect the stimulated release of interferon-gamma inducible protein 10 (IP-10), and a receiver operating characteristic (ROC) curve was drawn to determine cutoff value for diagnosing TB and to evaluate the diagnostic efficacy of the IP-10 assay. RESULTS: The antigen-specific release of IP-10 in the TB group was significantly higher than in the non-tuberculous pulmonary disease group (p < 0.001) and the healthy control group (p < 0.001). The IP-10 test correctly detected more cases of pulmonary TB and extra-pulmonary TB than the acid-fast staining microscopy examination (AFB). The sensitivity, specificity, and diagnostic efficiency for IP-10 were 75.3%, 84.9%, and 79.7%, respectively. Patients with TB and cancer comorbidity produced significantly higher levels of the TB-specific IP-10 release compared to the other groups (p < 0.05), whereas respiratory disease patients produced lower levels than the healthy controls (p > 0.05). CONCLUSIONS: Our findings indicate high sensitivity and specificity of IP-10 as a novel biomarker for TB in China.

[Prediction of Antigen Epitopes of Associated Protein Rv2004c Latent-infected by Mycobacterium Tuberculosis].

To screen new tuberculosis diagnostic antigens and vaccine candidates,we predicted the epitopes of Mycobacterium tuberculosis latent infection-associated protein Rv2004 cby means of bioinformatics.The homology between Rv2004 cprotein and human protein sequences was analyzed with BLAST method.The second structures,hydrophilicity,antigenicity,flexibility and surface probability of the protein were analyzed to predict B cell epitopes and T cell epitopes by Protean software of DNAStar software package.The Th epitopes were predicted by RANKPEP and SYFPEITHI supermotif method,the CTL epitopes were predicted by means of combination analyses of SYFPEITHI supermotif method,BIMAS quantitative motif method and NetCTL prediction method.The peptide sequences with higher scores were chosen as the candidate epitopes.Blast analysis showed that Rv2004 cprotein had low homology with human protein.This protein had abundant secondary structures through analysis of DNAStar software,the peptide segments with high index of hydrophilicity,antigenicity,surface probability and flexibility were widely distributed and were consistent with segments having beta turn or irregular coil.Ten candidates of B cell epitopes were predicted.The Th epitopes of Rv2004 cprotein were located after the 200 th amino acid.Of 37 Th cell epitopes predicted,there were more epitopes of HLA-DRB1*0401and HLA-DRB1*0701phenotypes,and the MHC restrictive types of some Th cell epitopes exist cross overlap.Of 10 CTL epitopes predicted,there were more number and higher score of HLA-A2 restricted epitopes.Therefore MycobacteriumtuberculosisRv2004 cprotein is a protein antigen with T cell and B cell epitopes,and is expected to be a new target protein candidate for tuberculosis diagnosis and vaccine.

Diagnostic value of enzyme-linked immunospot assay using CFP10/ESAT6 fusion protein as antigen in spinal tuberculosis.

OBJECTIVE: To establish a method of detecting spinal tuberculosis (TB) infection by enzyme-linked immunospot (ELlSPOT) assay and evaluate the value of CFP10/ESAT6 fusion protein for diagnosis of spinal TB. METHODS: Suspected spinal TB patients were prospectively recruited in two hospitals (First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine; Nanfang Hospital, Southern Medical University) from May 2012 to December 2013. Data on clinical characteristics of the patients and conventional laboratory results were collected. Compare and analyze the positive detection rate in spinal TB diagnosis by different methods including ELISPOT detection and conventional detection methods. RESULTS: 47 patients with spinal TB had available biopsy or surgical specimens for histopathological examination and 41 specimens had pathological features consistent with a diagnosis of TB infection. Among the spinal TB patients and non-TB disease patients,the overall sensitivity, specificity, positive predictive value, and negative predictive value of the ELISPOT assay in spinal TB diagnosis were 82.7%,87.2%,89.6%, and 79.1%,respectively; the 4 indexes of the PPD skin test were 61.5%, 46.2%, 60.4%, and 47.4%, respectively;those of the antibody detection were 55.8%, 61.5%, 65.9%, and 51.1%. The positive rate of ELISPOT was significantly higher than those of PPD skin test and antibody detection test (82.7% vs. 61.5%, Χ² =5.786, P=0.016; 82.7% vs. 55.8%, Χ² =8.847, P=0.003), but not significantly different from the positive rate of pathological examination (82.7% vs. 87.2%, Χ² =0.396, P=0.529). Moderate agreement was found between pathological examination and the ELISPOT assay (87.2%, Κ=0.498, P=0.001). CONCLUSION: With high sensitivity and specificity, the ELISPOT assay using CFP10/ESAT6 fusion protein as antigen is an effective technique for auxiliary diagnosis of spinal TB.

Diagnostic value of ELISPOT technique for osteoarticular tuberculosis.

BACKGROUND: Osteoarticular tuberculosis (TB) is a common severe form of extrapulmonary tuberculosis. Early di- agnosis and treatment can decrease the deformity of spine and limbs and joint dysfunction. METHODS: We compared and evaluated two commercially available rapid test kits based on the ELISPOT assay for the diagnosis of osteoarticular disease. RESULTS: The diagnostic sensitivity and specificity of the FS-SPOT assay (50.0% and 85.7%) were similar to those of the T-SPOT-TB assay (45.5% and 81.0%). When the two test wells in the T-SPOT-TB assay were both positive, the test wells in FS-SPOT assay were usually positive with the number of SFCs exceeding those in the negative control wells by more than 30. The sensitivities, specificities, PPV, NPV, and agreement of FS-SPOT assay results in 99 TB cases and 54 non-TB disease cases were 55.6%, 83.3%, 84.7%, 52.9%, and 66.0%, respectively. SFC counts from test wells in the TB group were significantly higher than those from the non-TB group (p < 0.001). CONCLUSIONS: Higher numbers of SFCs in the ELISPOT assay suggest higher risk of active TB. ELISPOT may be a diagnostic aide for active osteoarticular TB.

The advances in adjuvant therapy for tuberculosis with immunoregulatory compounds.

Tuberculosis (TB) is a chronic bacterial disease, as well as a complex immune disease. The occurrence, development, and prognosis of TB are not only related to the pathogenicity of Mycobacterium tuberculosis (Mtb), but also related to the patient's own immune state. The research and development of immunotherapy drugs can effectively regulate the body's anti-TB immune responses, inhibit or eliminate Mtb, alleviate pathological damage, and facilitate rehabilitation. This paper reviews the research progress of immunotherapeutic compounds for TB, including immunoregulatory compounds and repurposing drugs, and points out the existing problems and future research directions, which lays the foundation for studying new agents for host-directed therapies of TB.

Research Progress on Liposome Pulmonary Delivery of Mycobacterium tuberculosis Nucleic Acid Vaccine and Its Mechanism of Action.

Traditional vaccines have played an important role in the prevention and treatment of infectious diseases, but they still have problems such as low immunogenicity, poor stability, and difficulty in inducing lasting immune responses. In recent years, the nucleic acid vaccine has emerged as a relatively cheap and safe new vaccine. Compared with traditional vaccines, nucleic acid vaccine has some unique advantages, such as easy production and storage, scalability, and consistency between batches. However, the direct administration of naked nucleic acid vaccine is not ideal, and safer and more effective vaccine delivery systems are needed. With the rapid development of nanocarrier technology, the combination of gene therapy and nanodelivery systems has broadened the therapeutic application of molecular biology and the medical application of biological nanomaterials. Nanoparticles can be used as potential drug-delivery vehicles for the treatment of hereditary and infectious diseases. In addition, due to the advantages of lung immunity, such as rapid onset of action, good efficacy, and reduced adverse reactions, pulmonary delivery of nucleic acid vaccine has become a hot spot in the field of research. In recent years, lipid nanocarriers have become safe, efficient, and ideal materials for vaccine delivery due to their unique physical and chemical properties, which can effectively reduce the toxic side effects of drugs and achieve the effect of slow release and controlled release, and there have been a large number of studies using lipid nanocarriers to efficiently deliver target components into the body. Based on the delivery of tuberculosis (TB) nucleic acid vaccine by lipid carrier, this article systematically reviews the advantages and mechanism of liposomes as a nucleic acid vaccine delivery carrier, so as to lay a solid foundation for the faster and more effective development of new anti-TB vaccine delivery systems in the future.

The novel HLA-C*07:1089 allele, identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-C*07:1089 differs from HLA-C*07:02:01:01 by one nucleotide in exon 3.

The changes and its significance of peripheral blood NK cells in patients with tuberculous meningitis.

Objective: Tuberculous meningitis (TBM) is the most severe form of tuberculosis (TB). The purpose of this study was to explore the relationship between the number of natural killer (NK) cells and adaptive immune status, and disease severity in TBM patients. Methods: We conducted a retrospective study on 244 TB patients and 146 healthy control subjects in the 8th Medical Center of the PLA General Hospital from March 2018 and August 2023. Results: The absolute count of NK cells in the peripheral blood of TBM patients was significantly lower than that in normal controls (NC), latent tuberculosis infection (LTBI), and non-severe TB (NSTB) patients (p < 0.05). The proportion of TBM patients (48.7%) with a lower absolute count of NK cells than the normal reference value was significantly higher than that in NC (5.2%) and LTBI groups (4.0%) (p < 0.05), and slightly higher than that in NSTB group (36.0%) (p > 0.05). The absolute counts of lymphocyte subsets in TBM combined with other active TB group, etiology (+) group, IGRA (-) group, and antibody (+) group were lower than that in simple TBM group, etiology (-) group, IGRA (+) group, and antibody (-) group, respectively. The CD3+ T, NK, and B cells in BMRC-stage III TBM patients were significantly lower than those in stage I and stage II patients (p < 0.05). The counts of CD3+ T, CD4+ T, and B cells in the etiology (+) group were significantly lower than those in the etiology (-) group (p < 0.05). Conclusion: The absolute counts of lymphocyte subsets in the peripheral blood of TBM patients were significantly decreased, especially in NK cells. The reduction of these immune cells was closely related to the disease severity and had a certain correlation with cellular and humoral immune responses. This study helps to better understand the immune mechanism of TBM and provides reliable indicators for evaluating the immune status of TBM patients in clinical practice.

Reactivation of cytomegalovirus and bloodstream infection and its impact on early survival after allogeneic haematopoietic stem cell transplantation: a multicentre retrospective study.

Cytomegalovirus reactivation (CMVr) and bloodstream infections (BSI) are the most common infectious complications in patients after allogeneic haematopoietic stem cell transplantation (allo-HSCT). Both are associated with great high morbidity whilst the BSI is the leading cause of mortality. This retrospective study evaluated the incidence of CMVr and BSI, identified associated risk factors, assessed their impact on survival in allo-HSCT recipients during the first 100 days after transplantation. The study comprised 500 allo-HSCT recipients who were CMV DNA-negative and CMV IgG-positive before allo-HSCT. Amongst them, 400 developed CMVr and 75 experienced BSI within 100 days after allo-HSCT. Multivariate regression revealed that graft failure and acute graft-versus-host disease were significant risk factors for poor prognosis, whereas CMVr or BSI alone were not. Amongst all 500 patients, 56 (14%) developed both CMVr and BSI in the 100 days after HSCT, showing significantly reduced 6-month overall survival (p = 0.003) and long-term survival (p = 0.002). Specifically, in the initial post-transplant phase (within 60 days), BSI significantly elevate mortality risk, However, patients who survive BSI during this critical period subsequently experience a lower mortality risk. Nevertheless, the presence of CMVr in patients with BSI considerably diminishes their long-term survival prospects. This study provides real-world data on the impact of CMVr and BSI following transplantation on survival, particularly in regions such as China, where the prevalence of CMV IgG-positivity is high. The findings underscore the necessity for devising and executing focused prevention and early management strategies for CMVr and BSI to enhance outcomes for allo-HSCT recipients.

SARS-CoV-2 variants and COVID-19 vaccines: Current challenges and future strategies.

The ongoing COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global threat. Despite strict control measures implemented worldwide and immunization using novel vaccines, the pandemic continues to rage due to emergence of several variants of SARS-CoV-2 with increased transmission and immune escape. The rapid spread of variants of concern (VOC) in the recent past has created a massive challenge for the control of COVID-19 pandemic via the currently used vaccines. Vaccines that are safe and effective against the current and future variants of SARS-CoV-2 are essential in controlling the COVID-19 pandemic. Rapid production and massive rollout of next-generation vaccines against the variants are key steps to control the COVID-19 pandemic and to help us return to normality. Coordinated surveillance of SARS-CoV-2, rapid redesign of new vaccines and extensive vaccination are needed to counter the current SARS-CoV-2 variants and prevent the emergence of new variants. In this article, we review the latest information on the VOCs and variants of interest (VOIs) and present the information on the clinical trials that are underway on evaluating the effectiveness of COVID-19 vaccines on VOCs. We also discuss the current challenges posed by the VOCs in controlling the COVID-19 pandemic and future strategies to overcome the threat posed by the highly virulent and rapidly transmissible variants of SARS-CoV2.

The COVID-19 is a contagious respiratory disease caused by a virus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that emerged in 2019. The COVID-19 has now spread to all part of the world and has become a global threat. Even after the strict control measures and immunization programs to prevent the disease, COVID-19 is still causing destruction due to appearance of new strains of SARS-CoV-2 that transmit faster and capable of escaping the immunity. The faster spread of the new strains of viruses that cause more severe disease is the biggest challenge to control the COVID-19 pandemic by using the presently available vaccines. To control the COVID-19 pandemic we urgently need safe and effective vaccines against the corona viral variants. This can be achieved by tracking the appearance of new viral types, design and rapid production and supply of new vaccines against the virus. This article presents the latest information on the new types of SARS-CoV-2, and on the status of vaccine trials and their effectiveness against these viruses. Similarly, the information on the challenges posed by the new viral strains in controlling the COVID-19 and future strategies to overcome the threat posed by corona viruses is also provided.

Mechanisms of ag85a/b DNA vaccine conferred immunotherapy and recovery from Mycobacterium tuberculosis-induced injury.

Our previous research developed a novel tuberculosis (TB) DNA vaccine ag85a/b that showed a significant therapeutic effect on the mouse tuberculosis model by intramuscular injection (IM) and electroporation (EP). However, the action mechanisms between these two vaccine immunization methods remain unclear. In a previous study, 96 Mycobacterium tuberculosis (MTB) H37 Rv-infected BALB/c mice were treated with phosphate-buffered saline, 10, 50, 100, and 200 µg ag85a/b DNA vaccine delivered by IM and EP three times at 2-week intervals, respectively. In this study, peripheral blood mononuclear cells (PBMCs) from three mice in each group were isolated to extract total RNA. The gene expression profiles were analyzed using gene microarray technology to obtain differentially expressed (DE) genes. Finally, DE genes were validated by real-time reverse transcription-quantitive polymerase chain reaction and the GEO database. After MTB infection, most of the upregulated DE genes were related to the digestion and absorption of nutrients or neuroendocrine (such as Iapp, Scg2, Chga, Amy2a5), and most of the downregulated DE genes were related to cellular structural and functional proteins, especially the structure and function proteins of the alveolar epithelial cell (such as Sftpc, Sftpd, Pdpn). Most of the abnormally upregulated or downregulated DE genes in the TB model group were recovered in the 100 and 200 µg ag85a/b DNA IM groups and four DNA EP groups. The pancreatic secretion pathway downregulated and the Rap1 signal pathway upregulated had particularly significant changes during the immunotherapy of the ag85a/b DNA vaccine on the mouse TB model. The action targets and mechanisms of IM and EP are highly consistent. Tuberculosis infection causes rapid catabolism and slow anabolism in mice. For the first time, we found that the effective dose of the ag85a/b DNA vaccine immunized whether by IM or EP could significantly up-regulate immune-related pathways and recover the metabolic disorder and the injury caused by MTB.

Verapamil Regulates the Macrophage Immunity to Mycobacterium tuberculosis through NF-κB Signaling.

BACKGROUND: Verapamil enhances the sensitivity of Mycobacterium tuberculosis to anti-tuberculosis (TB) drugs, promotes the macrophage anti-TB ability, and reduces drug resistance, but its mechanism is unclear. Herein, we have investigated the effect of verapamil on cytokine expression in mouse peritoneal macrophages. METHODS: Macrophages from mice infected with M. tuberculosis or S. aureus were cultured with verapamil, the cytokines were detected by enzyme-linked immunosorbent assay, and the RNA was measured with quantitative real-time polymerase chain reaction and agarose gel electrophoresis. The intracellular calcium signaling was measured by confocal microscopy. RESULTS: Significantly higher levels of NF-κB, IL-12, TNF-α, and IL-1ß were observed after TB infection. The levels of NF-κB and IL-12 increased when verapamil concentration was less than 50 µg/ml, but decreased when verapamil concentration was greater than 50µg/ml. With the increase in verapamil concentration, TNF-α and IL-1ß expressed by macrophages decreased. The L-type calcium channel transcription significantly increased in M. tuberculosis rather than S. aureus-infected macrophages. Furthermore, during bacillus Calmette-Guerin (BCG) infection, verapamil stimulated a sharp peak in calcium concentration in macrophages, while calcium concentration increased mildly and decreased smoothly over time in the absence of verapamil. CONCLUSION: Verapamil enhanced macrophage immunity via the NF-κB pathway, and its effects on cytokine expression may be achieved by its regulation of intracellular calcium signaling.

Preventive effects of Mycobacterium tuberculosis DNA vaccines on the mouse model with latent tuberculosis infection.

Background: About a quarter of the world's population with latent tuberculosis infection (LTBI) are the main source of active tuberculosis. Bacillus Calmette Guerin (BCG) cannot effectively control LTBI individuals from developing diseases. Latency-related antigens can induce T lymphocytes of LTBI individuals to produce higher IFN-γ levels than tuberculosis patients and normal subjects. Herein, we firstly compared the effects of M. tuberculosis (MTB) ag85ab and 7 latent DNA vaccines on clearing latent MTB and preventing its activation in the mouse LTBI model. Methods: A mouse LTBI model was established, and then immunized respectively with PBS, pVAX1 vector, Vaccae vaccine, ag85ab DNA and 7 kinds of latent DNAs (including rv1733c, rv2660c, rv1813c, rv2029c, rv2628, rv2659c and rv3407) for three times. The mice with LTBI were injected with hydroprednisone to activate the latent MTB. Then, the mice were sacrificed for the bacterial count, histopathological examination, and immunological evaluation. Results: Using chemotherapy made the MTB latent in the infected mice, and then using hormone treatment reactivated the latent MTB, indicating that the mouse LTBI model was successfully established. After the mouse LTBI model was immunized with the vaccines, the lung colony-forming units (CFUs) and lesion degree of mice in all vaccines group were significantly decreased than those in the PBS group and vector group (P<0.0001, P<0.05). These vaccines could induce antigen-specific cellular immune responses. The number of IFN-γ effector T cells spots secreted by spleen lymphocytes in the ag85ab DNA group was significantly increased than those in the control groups (P<0.05). In the splenocyte culture supernatant, IFN-γ and IL-2 levels in the ag85ab, rv2029c, and rv2659c DNA groups significantly increased (P<0.05), and IL-17A levels in ag85ab and rv2659c DNA groups also significantly increased (P<0.05). Compared with the PBS and vector groups, the proportion of CD4+CD25+FOXP3+ regulatory T cells in spleen lymphocytes of ag85ab, rv2660c, rv2029c, and rv3407 DNA groups were significantly reduced (P<0.05). Conclusions: MTB ag85ab and 7 kinds of latent DNA vaccines showed immune preventive efficacies on a mouse model of LTBI, especially the rv2659c, and rv1733c DNA. Our findings will provide candidates for the development of new multi-stage vaccines against TB.