Olink proteomics and lipidomics analysis of serum from patients infected with non-tuberculous mycobacteria and Mycobacterium tuberculosis.

BACKGROUND: Non-tuberculous mycobacterial (NTM) and Mycobacterium tuberculosis (MTB) infections are difficult to diagnose and treat, significantly burdening global health. The host immune status is generally believed to be associated with the onset and progression of NTM and MTB infections, but its specific impact remains unclear. METHODS: In the present study, proteomics and lipidomics analysis of serum from normal controls (n = 26) and patients with MTB (n = 26), rapidly growing NTM (RGM, n = 15), and slowly growing NTM (SGM, n = 21) were conducted using the Olink technique based on a highly sensitive and specific neighborhood extension assay and the lipidomics technique. RESULTS: IFN-γ, CXCL9, CXCL10, CXCL11, and CXCL13, etc. were simultaneously upregulated in MTB, RGM, and SGM, while lipids FAHFA 22:3, FAHFA 26:4, FAHFA 24:4, FAHFA 20:5, FAHFA 18:2 simultaneously downregulated. IL8, CCL3, CXCL5, and MCP-2, etc. were simultaneously upregulated in RGM and SGM compared to MTB, as well as PCs, LPCs, PEs, and LPEs. Compared with RGM, IL7, CD27, CCL17, CXCL12, and LPC 28:7-SN2 were downregulated in SGM. Pathway analyses revealed that tuberculosis, sphingolipid signaling pathway, and adipocytokine signaling pathway were regulated at the protein level and metabolite level. Diagnostic panels comprising immune-associated proteins and lipids greatly enhance diagnostic specificity and sensitivity. CONCLUSION: This integrated multi-omics analysis provides a more comprehensive understanding of the molecular landscape of NTM and MTB, which may provide molecular targets for specialized therapies.

The TBAg/PHA ratio in T-SPOT.TB assay has high prospective value in the diagnosis of active tuberculosis: a multicenter study in China.

BACKGROUND: The positive rate of pathogenic examination about tuberculosis is low. It is still difficult to achieve early diagnosis for some TB patients. The value of Interferon-gamma release assays (IGRA) in the diagnosis of active tuberculosis remains controversial. The purpose of this multicenter prospective study was to verify and validate the role of TBAg/PHA ratio (TB-specific antigen to phytohaemagglutinin) of T-SPOT.TB assay in diagnosing ATB. METHODS: We prospectively enrolled 2390 suspected pulmonary tuberculosis patients with positive T-SPOT assay results from three tertiary hospitals. RESULTS: A total of 1549 ATB (active tuberculosis) patients (including 1091 confirmed and 458 probable ATB) and 724 non-tuberculosis (non-TB) patients with positive T-SPOT results were included. The results of this study showed that ESAT-6 and CFP-10 in the T-SPOT.TB assay were significantly higher in the ATB group compared with the non-TB group, while PHA was lower in the ATB group. Results of ESAT-6, CFP-10 and PHA show a certain diagnostic performance, but moderate sensitivity and specificity. The TBAg/PHA ratio, a further calculation of ESAT-6, CFP-10 and PHA in T-SPOT.TB assay showed improved performance in the diagnosis of active Tuberculosis. If using the threshold value of 0.2004, the specificity and sensitivity of TBAg/PHA ratio in distinguishing ATB from non-TB were 92.3% and 74.4%, PPV was 95.4, PLR was 9.6. CONCLUSION: By recalculating the results of T-SPOT.TB Assay, the TBAg/PHA ratio shows high prospect value in the diagnosis of active tuberculosis in high prediction areas.

Clofazimine for the treatment of multidrug-resistant tuberculosis: prospective, multicenter, randomized controlled study in China.

BACKGROUND: Clofazimine (Cfz) has shown activity against Mycobacterium tuberculosis, including multidrug-resistant (MDR) strains in vitro and in animal studies. Here we evaluate the clinical efficacy and tolerability of using Cfz to treat MDR tuberculosis in China. METHODS: We enrolled 105 patients who had sputum culture-positive MDR tuberculosis in 6 major tuberculosis specialty hospitals in China. Patients were randomly assigned to either the Cfz therapy group (n = 53) or control group (n = 52). Patients in the 2 groups were given 21 months of individual-based chemotherapy regimens based on medication history and drug susceptibility test results. The Cfz therapy group regimens incorporated 100 mg of Cfz once daily for 21 months. RESULTS: Three patients in each group discontinued therapy because of side effects or other reasons. Sputum culture conversion to negative was earlier in patients who received Cfz compared with controls (P = .042 by log-rank test). Chest computed tomography showed cavitary changes in 46 patients in the Cfz therapy group and 45 in the control group. Cavity closure was earlier in patient who received Cfz compared with controls (P = .047 by log-rank test). The treatment success rate in the Cfz group was 73.6%, higher than that in control group (53.8%; P = .035). Side effects in skin only occurred in the Cfz group. The rates of skin discoloration and ichthyosis were 94.3% and 47.2%, respectively. CONCLUSIONS: Using Cfz to treat MDR tuberculosis promotes cavity closure, accelerates sputum culture conversion, and improves treatment success rates.

Efficacy, safety and tolerability of linezolid for the treatment of XDR-TB: a study in China.

Linezolid may be effective in treating multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis. We conducted a prospective, multicentre, randomised study to further evaluate the efficacy, safety and tolerability of linezolid in patients with extensively drug-resistant tuberculosis in China. 65 patients who had culture-positive sputum for extensively drug-resistant tuberculosis were randomly assigned to a linezolid therapy group or a control group. Patients in the two groups adopted a 2-year individually based chemotherapy regimen. The linezolid therapy group was given linezolid at a start dose of 1200 mg per day for a period of 4-6 weeks and this was then followed by a dose of 300-600 mg per day. The proportion of sputum culture conversions in the linezolid therapy group was 78.8% by 24 months, significantly higher than that in the control group (37.6%, p<0.001). The treatment success rate in linezolid therapy group was 69.7%, significantly higher than that in the control group (34.4%, p=0.004). 27 (81.8%) patients had clinically significant adverse events in the linezolid group, of whom 25 (93%) patients had events that were possibly or probably related to linezolid. Most adverse events resolved after reducing the dosage of linezolid or temporarily discontinuing linezolid. Linezolid containing chemotherapy for treatment of extensively drug-resistant tuberculosis may significantly promote cavity closure, increase sputum culture-conversion rate and improve treatment success rate.

[A cohort study on the outcome of multidrug-resistant tuberculosis in elderly patients].

OBJECTIVE: To investigate the clinical curative effect and outcomes of multidrug-resistant tuberculosis (MDR-TB) in elderly patients. METHODS: Fifty-nine elderly patients with MDR-TB were enrolled from Shanghai Pulmonary Hospital from January 2007 to January 2010, and 80 younger patients with MDR-TB during the same period served as the control group. Clinical characteristics, outcomes and adverse effects of treatment were reviewed, and the data of the 2 groups were compared using cohort analysis. Comparisons of categorical variables were performed using the Pearson Chi-square tests, and differences between groups were compared by Fisher's exact test. RESULTS: Compared with those of the younger patients, the clinical symptoms of the elderly patients showed no significant differences except shortness of breath. The proportion of retreated patients in the elderly patients (96.6%, 57/59) was significantly higher than that in the younger group (86.3%, 69/80) (χ² = 4.299, P < 0.05). The proportion of the patients with lesions involving 5-6 lung fields in the elderly group was 57.6% (34/59), higher than that in the younger group (22.5%, 18/80) (χ² = 17.894, P < 0.01). The treatment success rate in the elderly MDR-TB group was 28.2% (17/59), lower than that in the younger group (51.3%, 41/80) (χ² = 7.029, P < 0.01). The death rate in the elderly group was 17.0% (10/59), significantly higher than that in the younger group (3.4%, 3/80) (χ² = 6.837-17.894, P < 0.05). The incidence of liver injury in the elderly group was higher than that in the younger group (23.7%, 14/59 vs 10.0%, 8/80,χ² = 4.804, P < 0.05), and so was that of kidney dysfunction (10.2%, 6/59 vs 1.3%, 1/80, χ² = 5.649, P < 0.05). CONCLUSIONS: In elderly patients with MDR-TB, the pulmonary lesions were extensive, the number of retreated cases was higher, and the incidence of adverse drug reactions was also higher. The treatment success rate was lower while the mortality rate was higher for elderly patients with MDR-TB.

Metabolic Profiles of Clinical Isolates of Drug-Susceptible and Multidrug-Resistant Mycobacterium tuberculosis: A Metabolomics-Based Study.

Background: Mycobacterium tuberculosis (MTB) is a global and highly deleterious pathogen that creates an enormous pressure on global public health. Although several effective drugs have been used to treat tuberculosis, the emergence of multidrug-resistant Mycobacterium tuberculosis (MDR-MTB) has further increased the public health burden. The aim of this study was to describe in depth the metabolic changes in clinical isolates of drug-susceptible Mycobacterium tuberculosis (DS-MTB) and MDR-MTB and to provide clues to the mechanisms of drug resistance based on metabolic pathways. Methods: Based on the minimum inhibition concentration (MIC) of multiple anti-tuberculosis drugs, two clinical isolates were selected, one DS-MTB isolate (isoniazid MIC=0.06 mg/L, rifampin MIC=0.25 mg/L) and one MDR-MTB isolate (isoniazid MIC=4 mg/L, rifampin MIC=8 mg/L). Through high-throughput metabolomics, the metabolic profiles of the DS-MTB isolate and the MDR-MTB isolate and their cultured supernatants were revealed. Results: Compared with the DS-MTB isolate, 128 metabolites were significantly altered in the MDR-MTB isolate and 66 metabolites were significantly altered in the cultured supernatant. The differential metabolites were significantly enriched in pyrimidine metabolism, purine metabolism, nicotinate and nicotinamide metabolism, arginine acid metabolism, and phenylalanine metabolism. Furthermore, metabolomics analysis of the bacterial cultured supernatants showed a significant increase in 10 amino acids (L-citrulline, L-glutamic acid, L-aspartic acid, L-norleucine, L-phenylalanine, L-methionine, L-tyrosine, D-tryptophan, valylproline, and D-methionine) and a significant decrease in 2 amino acids (L-lysine and L-arginine) in MDR-MTB isolate. Conclusion: The present study provided a metabolite alteration profile as well as a cultured supernatant metabolite alteration profile of MDR-MTB clinical isolate, providing clues to the potential metabolic pathways and mechanisms of multidrug resistance.

Correlation of drug exposure and bacterial susceptibility with treatment response for Mycobacterium avium complex lung disease: protocol for a prospective observational cohort study.

INTRODUCTION: The burden of Mycobacterium avium complex (MAC) lung disease is increasing globally and treatment outcome is in general poor. Therapeutic drug monitoring has the potential to improve treatment outcome by ensuring adequate drug exposure. However, very limited population-based studies exist for MAC lung disease. This study aims to describe the distribution of drug exposure for key antimycobacterial drugs at population level, and to analyse them in relationship to treatment outcome in patients with MAC lung disease. METHODS AND ANALYSIS: A prospective cohort aiming to include 100 adult patients diagnosed with and treated for MAC lung disease will be conducted in Shanghai Pulmonary Hospital, China. Blood samples will be collected after 1 month MAC treatment for measurement of macrolides, rifamycin, ethambutol, amikacin and/or fluoroquinolones, using a validated liquid-chromatography tandem mass spectrometry method. Respiratory samples will be collected at inclusion and once every 3 months for mycobacterial culture until treatment completion. Minimum inhibitory concentration (MIC) determination will be performed using a commercial broth microdilution plate. In addition to mycobacterial culture, disease severity and clinical improvement will be assessed from the perspective of lung function, radiological presentation and self-reported quality of life. Whole genome sequencing will be performed for any longitudinal isolates with significant change of MIC to explore the emergence of drug resistance-conferring mutations. The relationship between drug exposure and treatment outcome will be analysed and potential confounders will be considered for adjustment in multivariable models. Meanwhile, the associations between drug exposure in relation to MIC and markers of treatment response will be explored using Cox proportional hazards or binary logistic regression models, as appropriate. ETHICS AND DISSEMINATION: This study has been approved by the ethics committee of Shanghai Pulmonary Hospital (No. K22-149Z). Written and oral informed consent will be obtained from all participants. The study results will be submitted to a peer-reviewed journal. TRIAL REGISTERATION NUMBER: NCT05824988.

Efficacy and safety of an innovative short-course regimen containing clofazimine for treatment of drug-susceptible tuberculosis: a clinical trial.

In preclinical studies, a new antituberculosis drug regimen markedly reduced the time required to achieve relapse-free cure. This study aimed to preliminarily evaluate the efficacy and safety of this four-month regimen, consisting of clofazimine, prothionamide, pyrazinamide and ethambutol, with a standard six-month regimen in patients with drug-susceptible tuberculosis. An open-label pilot randomized clinical trial was conducted among the patients with newly diagnosed bacteriologically-confirmed pulmonary tuberculosis. The primary efficacy end-point was sputum culture negative conversion. Totally, 93 patients were included in the modified intention-to-treat population. The rates of sputum culture conversion were 65.2% (30/46) and 87.2% (41/47) for short-course and standard regimen group, respectively. There was no difference on two-month culture conversion rates, time to culture conversion, nor early bactericidal activity (P > 0.05). However, patients on short-course regimen were observed with lower rates of radiological improvement or recovery and sustained treatment success, which was mainly attributed to higher percent of patients permanently changed assigned regimen (32.1% vs. 12.3%, P = 0.012). The main cause for it was drug-induced hepatitis (16/17). Although lowering the dose of prothionamide was approved, the alternative option of changing assigned regimen was chosen in this study. While in per-protocol population, sputum culture conversion rates were 87.0% (20/23) and 94.4% (34/36) for the respective groups. Overall, the short-course regimen appeared to have inferior efficacy and higher incidence of hepatitis but desired efficacy in per-protocol population. It provides the first proof-of-concept in humans of the capacity of the short-course approach to identify drug regimens that can shorten the treatment time for tuberculosis.

Factors Influencing False-Negative Results of QuantiFERON-TB Gold In-Tube (QFT-GIT) in Active Tuberculosis and the Desirability of Resetting Cutoffs for Different Populations: A Retrospective Study.

Objectives The value of QuantiFERON-TB Gold In-Tube (QFT-GIT) in the diagnosis of TB varies by population, comorbidities, and other factors. In this study, we aimed to investigate factors that influence false-negative results of QFT-GIT test in the diagnosis of TB as well as the impact of different cutoffs on the diagnostic value. Methods A total of 3562 patients who underwent QFT-GIT tests at Shanghai Pulmonary Hospital were enrolled retrospectively between May 2016 and May 2017. False-negative and false-positive results were analyzed using different clinical stratifications. The optimal cutoff values were established under different clinical conditions. Results Positive QFT-GIT results greatly shortened the time taken to diagnose smear-negative TB. The factors of age, smear and culture results, site of TB, comorbidity with tumors, white blood cell count, neutrophil count, and CD4/CD8 ratio were significantly correlated with false-negative QFT-GIT results (p < 0.05). Personalized cutoff values were established according to different influencing factors. The results showed high consistency between the smear-negative and total populations. Conclusion QFT-GIT can facilitate the early diagnosis of smear-negative TB. The diagnostic performance of the QFT-GIT test in the diagnosis of active TB was shown to be affected by many clinical factors. Personalized cutoff values may have superior value in the identification of active tuberculosis under different conditions.

Extensively drug-resistant tuberculosis at a tuberculosis specialist hospital in Shanghai, China: clinical characteristics and treatment outcomes.

BACKGROUND: Extensively drug-resistant tuberculosis (XDR-TB) has recently emerged as a global public health problem. The objective of this study was to investigate the clinical characteristics, management and outcomes of human immunodeficiency virus-negative patients with XDR-TB at a specialist TB hospital in Shanghai, China. METHODS: From July 2007 to June 2009 we analyzed TB patients with culture-proven XDR-TB at a specialist TB hospital in Shanghai. RESULTS: Among 1156 TB cases, all culture-positive for Mycobacterium tuberculosis complex, 494 cases (42.7%) were classified as MDR-TB; 126 cases (10.9%) were XDR-TB. At least 3 lung fields were involved in 90.5% of XDR-TB patients and in 80.7% of other MDR-TB patients (p = 0.008). Of the XDR-TB cases, 40.5% were complicated by diabetes and other diseases, significantly higher compared with the other MDR-TB cases (p = 0.002). The rates of resistance to all drugs except isoniazid and rifampicin were significantly higher in patients with XDR-TB than in patients with other MDR-TB (p < 0.001). Treatment failure was more common in patients with XDR-TB than in those with other MDR-TB (p < 0.001), whereas the mortality and default rates did not differ significantly. CONCLUSIONS: The prevalence of XDR-TB is high in some areas of China. The clinical treatment outcome for XDR-TB is usually very poor.

[The changes and the significance of cellular immune function in peripheral blood of patients with multidrug-resistant and extensively drug-resistant pulmonary tuberculosis].

OBJECTIVE: To explore the expressions and the significance of CD(3)(+)CD(16)(+)CD(56)(+) NKT cells, CD(3)(-)CD(16)(+)CD(56)(+) NK cells and T lymphocyte subsets in peripheral blood of patients with multi-drug resistant (MDR-TB) and extensively drug-resistant (XDR-TB) pulmonary tuberculosis. METHODS: The data of 316 patients with pulmonary tuberculosis hospitalized in Shanghai Pulmonary Hospital from January 2008 to June 2009 were retrospectively analyzed, of whom 119 were newly diagnosed, and 197 were retreated patients. There were 204 males and 112 females, aged from 17 - 88 years, mean (44 ± 16) years. According to the results of drug-resistance, these patients were divided into a MDR group, an XDR group and a sensitive group. There were 146 patients in the MDR group, with 102 males and 44 females, aged from 19 - 84 years, mean (42 ± 16) years. There were 77 patients in the XDR group, with 42 males and 35 females, aged from 18 - 88 years, mean (50 ± 16) years. There were 93 patients in the susceptible group, with 60 males and 33 females, aged from 17 - 83 years, mean (43 ± 19) years. According to the distribution of cavitation in lung fields, these patients were also divided into 1 - 2 lung field affected group (n = 132), 3 - 4 lung field affected group (n = 49) and 5 - 6 lung field affected group (n = 9). The frequencies of NKT cells, NK cells and T cells from whole blood were tested by flow cytometry. Rank test (SAS software) was used for statistic analyses. RESULTS: The expression rate of NKT cells and NK cells was the highest in the XDR group [11% (6% - 16%) and 7% (4% - 12%)], as compared to the MDR group [8% (5% - 14%) and 6% (4% - 11%)], and the susceptible group [7% (4% - 11%) and 5% (3% - 9%)], the difference being statistically significant (H = 6.478 - 8.369, P < 0.05). The expression rate of the NKT (14 ± 9)% and NK cells (11 ± 7)% in males of the XDR group was significantly higher than that in females [NKT (9 ± 5)% and NK cell (6 ± 4)%], while CD(4) (38 ± 10)% and CD(4)/CD(8) (1.9 ± 1.3) were significantly lower than those of the females [CD(4) (44 ± 10)% and CD(4)/CD(8)(2.2 ± 0.7)], the difference being statistically significant (z = -2.91 - -2.79, P < 0.05, P < 0.01). The expression rate of CD(4) (42 ± 9)% was the highest, but CD(8) (22 ± 8)% was the lowest in the 1 - 2 lung field group. While in the 5 - 6 lung field group, the expression rate of CD(4) (36 ± 11)% was the lowest, CD(8) (28 ± 12)% was the highest, and CD(4)/CD(8) (1.5 ± 0.8) was the lowest, the difference being statistically significant (H = 8.404 - 16.175, P < 0.01). CONCLUSIONS: With the increasing level of drug resistance, the expression rate of NKT cells and NK cells increased, while the expression of T cell subsets did not change. The value of CD(4) and CD(4)/CD(8) in peripheral blood decreased, but CD(8) increased as the extent of cavitation increased in these patients. The impairment of cellular immune function in XDR-TB was more prominent in male patients.

[The correlation between polymorphisms of genes with susceptibility to tuberculosis and the clinical characteristics of tuberculosis in 459 Han patients].

OBJECTIVE: To study the correlation between polymorphisms of genes with susceptibility to tuberculosis and the clinical characteristics of tuberculosis in Han population. METHODS: Four hundred and fifty-nine tuberculosis inpatients of Han population in Shanghai Pulmonary Hospital from Jan 2007 to Dec 2008 were recruited. The clinical characteristics of tuberculosis (gender, fever, extent of lesions, cavity formation, hemoptysis, initial treatment and retreatment) were observed. The polymorphisms of VDR gene (variants in FokI and TaqI), NRAMP1 gene (variants in INT4, D543N and 3 UTR), MBL gene (variants in HL, YX and QP) and IFNG gene (variants in 874AT) were genotyped by a variety of SNP genotyping techniques. The correlation between polymorphisms of genes with susceptibility to tuberculosis and the clinical characteristics of the disease was analyzed by ANOVAs. RESULTS: The frequency of CC, CT and TT variants of FokI in VDR gene in cases with fever were 54.7% (29/53), 13.2% (7/53) and 32.1% (17/53), respectively, compared to 40.6% (52/128), 30.5% (39/128) and 28.9% (37/128) in cases without fever, the difference being significant (χ² = 6.183, P < 0.05). In patients with CT variants, 15.2% (7/46) had fever, while in patients with non-CT variants, 34.1% (46/135) had fever (χ² = 5.891, P < 0.05), suggesting that patients with CT variants were less likely to have fever. The frequencies of TT + TC and CC variants of QP in the MBL gene in initial treatment cases were 28.3% (60/212) and 71.7% (152/212), respectively, compared to 19.1% (41/215) and 80.9% (174/215) in retreatment cases, the difference being significant (χ² = 5.038, P < 0.05). No significant correlation was observed between the other variants and the clinical characteristics of tuberculosis (χ² = 0.001 - 2.732, P > 0.05). CONCLUSIONS: The polymorphisms of FokI in VDR gene was associated with fever among the clinical characteristics of tuberculosis, and patients with CT variants might be protected from fever. The polymorphisms of QP in MBL gene might be associated with recurrence of tuberculosis.

In vitro Synergism of Six Antituberculosis Agents Against Drug-Resistant Mycobacterium tuberculosis Isolated from Retreatment Tuberculosis Patients.

BACKGROUND: Retreatment tuberculosis (TB) has become a major source of drug-resistant TB. In contrast to the combination of isoniazid (INH) and rifampicin (RIF), that of pasiniazid (Pa) and rifabutin (RFB) or rifapentine (RFP) appears to have better activity in vitro against drug-resistant Mycobacterium tuberculosis (MTB), especially when combined with moxifloxacin (MXF). However, there has been limited study of potential synergism among Pa, RFB, RFP, and MXF, or simultaneous comparison with the standard INH and RIF combination. METHODS: In vitro synergism of four two-drug combinations (INH and RIF, Pa and RFB, Pa and RFP, MXF and Pa) and two three-drug combinations (MXF and Pa combined with RFB or RFP) was evaluated against 90 drug-resistant MTB strains isolated from retreatment TB patients by the checkerboard method. The fractional inhibitory concentration index (FICI) was calculated for each combination. RESULTS: The synergistic activity of the combination of Pa with RFB or RFP was higher than that of INH and RIF or MXF and Pa, and the synergistic activity of Pa in combination with RFP was even higher than that of RFB, although RFP yielded an MIC90 of 64 mg/liter, higher than that of RFB of 8 mg/liter against 90 drug-resistant MTB strains. Meanwhile, for three-drug combinations, the synergistic effects of MXF and Pa combined with RFB or RFP were similar. Further stratification analysis showed that, for XDR-MTB strains, the synergistic effect of the Pa and RFP combination was also better than those of other two-drug combinations. CONCLUSION: The combination of Pa with RFP shows better in vitro synergism than Pa with RFB and standard INH with RIF combinations, which can provide a reference for new regimens for retreatment TB patients.

A CD4+CD161+ T-Cell Subset Present in Unexposed Humans, Not Tb Patients, Are Fast Acting Cells That Inhibit the Growth of Intracellular Mycobacteria Involving CD161 Pathway, Perforin, and IFN-γ/Autophagy.

It remains undefined whether a subset of CD4+ T cells can function as fast-acting cells to control Mycobacterium tuberculosis (Mtb) infection. Here we show that the primary CD4+CD161+ T-cell subset, not CD4+CD161-, in unexposed healthy humans fast acted as unconventional T cells capable of inhibiting intracellular Mtb and BCG growth upon exposure to infected autologous and allogeneic macrophages or lung epithelial A549 cells. Such inhibition coincided with the ability of primary CD4+CD161+ T cells to rapidly express/secrete anti-TB cytokines including IFN-γ, TNF-α, IL-17, and perforin upon exposure to Mtb. Mechanistically, blockades of CD161 pathway, perforin or IFN-γ by blocking mAbs abrogated the ability of CD4+CD161+ T cells to inhibit intracellular mycobacterial growth. Pre-treatment of infected macrophages with inhibitors of autophagy also blocked the CD4+CD161+ T cell-mediated growth inhibition of mycobacteria. Furthermore, adoptive transfer of human CD4+CD161+ T cells conferred protective immunity against mycobacterial infection in SCID mice. Surprisingly, CD4+CD161+ T cells in TB patients exhibited a loss or reduction of their capabilities to produce perforin/IFN-γ and to inhibit intracellular growth of mycobacteria in infected macrophages. These immune dysfunctions were consistent with PD1/Tim3 up-regulation on CD4+CD161+ T cells in active tuberculosis patients, and the blockade of PD1/Tim3 on this subset cells enhanced the inhibition of intracellular mycobacteria survival. Thus, these findings suggest that a fast-acting primary CD4+CD161+T-cell subset in unexposed humans employs the CD161 pathway, perforin, and IFN-γ/autophagy to inhibit the growth of intracellular mycobacteria, thereby distinguishing them from the slow adaptive responses of conventional CD4+ T cells. The presence of fast-acting CD4+CD161+ T-cell that inhibit mycobacterial growth in unexposed humans but not TB patients also implicates the role of these cells in protective immunity against initial Mtb infection.

Characterization of antibody response against 16kD and 38kD of M. tuberculosis in the assisted diagnosis of active pulmonary tuberculosis.

BACKGROUND: In view of the inability of traditional etiological methods to diagnose pulmonary tuberculosis rapidly and effectively, the antibody responses against 38kD and 16kD-antigens of Mycobacterium tuberculosis (M. tuberculosis) were both detected in order to obtain a better serological detection method for M. tuberculosis. METHODS: M. tuberculosis-secreted protein 38kD and membrane protein 16kD were prokaryotically expressed and purified, and then used as detection antigens. A novel evolved immunoglobulin-binding molecule (NEIBM)-ELISA method was used to detect antibody levels against 38kD and 16kD in active tuberculosis patients (confirmed tuberculosis cases and clinically diagnosed cases), to explore the significance of these two antigens in serological detection of M. tuberculosis, and to study the diagnostic value of the combined detection of the two antigens in active pulmonary tuberculosis. RESULTS: The results showed that the positive detection rates of the 16kD antigen and 38kD antigen of M. tuberculosis were higher (about 44%) in the confirmed cases of tuberculosis, and there was no significant difference in the positive detection rates of the two antigens (P=0.786). The combined detection of these two antigens showed that the positive detection rate could be increased to 61.5%, which was significantly better than the detection effect of the two antigens alone. The positive detection rates of 16kD and 38kD antigens were 26-30% in clinically diagnosed tuberculosis cases, which were lower than those in confirmed tuberculosis cases, and there was no significant difference in the positive detection rates of the two antigens (P=0.242). The detection effect of the two combined antigens was better than that of the 16kD and 38kD antigens alone, but the detection rate was still lower than that of the confirmed tuberculosis cases. CONCLUSIONS: This study found that the detection effect of 16kD and 38kD antigens was similar in confirmed cases and clinically diagnosed cases of pulmonary tuberculosis, and that the detection effect needs to be further improved. The combined detection of the two antigens showed a significantly better detection effect than the two antigens alone, suggesting that the combined detection of multiple antigens can be used for serological diagnosis of M. tuberculosis infection in clinic.

[Clinical, pathological and radiological characteristics of 83 cases of pleural tuberculoma].

OBJECTIVE: To investigate the clinical, pathological and radiological characteristics of pleural tuberculoma, so as to improve the understanding of this disease. METHODS: We retrospectively analyzed the clinical, laboratory, pathological and radiological data of 83 cases of pleural tuberculoma diagnosed by pathology and(or) bacteriology in Shanghai Pulmonary Hospital Affiliated to Tongji University. RESULTS: In the recruited 83 cases, there were 50 males and 33 females, aged from 7 to 85 years old, with a mean age of 37.8 years. Cough, fever and chest pain were common symptoms, but no significant symptoms were seen in 25 patients (31.3%). Some patients had positive physical signs, such as dullness to percussion and low breath sound. Pulmonary tuberculosis was also present in 36 patients (43.3%) with pleural tuberculoma. A history of tuberculous pleurisy was elicited in 80 patients, among whom 45 (56.3%) received delayed antituberculous treatment and 42 (52.5%) received nonstandard treatment. Forty-eight cases (60.0%) did not receive corticosteroids. Fifty-nine cases underwent CT-guided percutaneous biopsy, while 24 underwent thoracoscopic surgery, and tuberculosis was pathologically confirmed in 62 (74.7%). Pathological profiles included granuloma, coagulation or caseation necrosis, lymphocyte infiltration, epithelioid cells, inflammatory cells, histiocytes and scar tissue. Fifteen (18.1%) specimens from percutaneous biopsy were anti-fast smear positive, while Mycobacterium tuberculosis was obtained by culture in 21 (25.3%) cases. Chest X-ray showed that solitary lesions were seen in 68 cases, multiple foci in 15. The lesions of 46 cases (55.4%) occupied the lower right lobes. Round-like shadows were the most common signs, which were present in 63 cases (75.9%). CT examination demonstrated homogeneous density in 20, heterogeneous density in 40, calcification in 9, central attenuation in 34, and peripheral intensification in 28 cases. CONCLUSIONS: Pleural tuberculoma is an important sequelae of tuberculous pleurisy. Understanding its clinical, pathological and radiological characteristics is helpful for the differential diagnosis of pleural and lung diseases.

Effect of thymopentin combined with levofloxacin for retreatment smear positive pulmonary tuberculosis / 中国热带医学

@#Abstract: Objective　To analyze the effect of adjuvant to levofloxacin in the treatment of retreatment smear positive pulmonary tuberculosis, as well as its effect on respiratory function, immune function and inflammatory factors. Methods　One hundred cases of retreatment smear positive pulmonary tuberculosis patients admitted to Rudong County People's Hospital in Nantong city in Jiangsu province from 2017 to 2021 were randomly divided into a control group (n=50) and an observation group (n=50) according to random number table method. Both groups received conventional treatment (3 months of isoniazid, rifampicin, ethambutol, pyrazinamide / 6 months of isoniazid, rifampicin, ethambutol), with levofloxacin added to the control group, and thymopentin added to the observation group for the first three months in addition to routine treatment. The treatment effect of the two groups were compared. Results　The sputum smear conversion rate of the observation group was significantly higher than that of the control group after 3 months and 5 months of treatment (χ2=7.142, P<0.05; χ2=6.250, P<0.05). The cavity absorption time and lesion absorption time in the observation group were significantly lower than those in the control group (t=4.006, P<0.05; t=5.165, P<0.05). The turning time of bacteriological culture in the observation group was significantly lower than that in the control group (t=4.220,P<0.05). After 3 months of treatment, CD4+, CD3+, CD4+/CD8+ of the observation group were higher than those of the control group, the differences were statistically significant (t=8.885, P<0.05; t=6.274, P<0.05; t=4.357, P<0.05). After 3 months of treatment, the IFN-γ (interferon-γ) of the observation group was higher than that of the control group (t=8.892, P<0.05), whereas the , IL-10 (interleukin-10) was significantly lower than that in the control group (t=5.986, P<0.05). After 3 months of treatment, forced vital capacity (FVC), forced expiratory volume in one second (forced expiratory volume in one second, FEV 1) and the one-second rate (forced expiratory volume in one second / forced vital capacity, FEV1/FVC) in the observation group were significantly higher than those in the control group (t=11.223, P<0.05; t=10.128, P<0.05; t=4.464, P<0.05). There was no statistically significant difference in the incidence of adverse reactions between the two groups (χ2=0.378, P>0.05). Conclusions　Thymopentin combined with levofloxacin had a significant application effect in the treatment of retreatment smear positive pulmonary tuberculosis, s, which led to improved inflammatory reaction, respiratory function and immune function. Additionally, it can increase sputum smear conversion rate and accelerate patient recovery, improving overall treatment efficacy, with a relatively high clinical application value.

Basic Fibroblast Growth Factor Protects Astrocytes Against Ischemia/Reperfusion Injury by Upregulating the Caveolin-1/VEGF Signaling Pathway.

A previous in vivo study demonstrated that intracerebroventricular injection of basic fibroblast growth factor (bFGF) in middle cerebral artery occlusion rats increased the expression of caveolin-1 (cav-1) and vascular endothelial growth factor (VEGF) in cerebral ischemia penumbra. Because astrocytes are the largest population in the brain, the aim of this in vitro study was to investigate the influence of bFGF on cav-1 and VEGF expression in rat astrocytes following oxygen glucose deprivation/reoxygenation (OGD/R). For this, an ischemic model in vitro of oxygen glucose deprivation lasting for 6 h, followed by 24 h of reoxygenation was used. Primary astrocytes from newborn rats were pre-treated with siRNA targeting bFGF before OGD/R. Cell viability was measured by a CCK-8 assay. The protein and mRNA expressions of bFGF, cav-1, and VEGF were evaluated by western blotting, immunofluorescence staining, and reverse transcription-quantitative polymerase chain reaction. The results showed that OGD/R reduced cell viability, which was decreased further following bFGF knockdown; however, restoring bFGF improved cell survival. A cav-1 inhibitor abrogated the effect of bFGF on cell viability. The expression levels of bFGF mRNA, bFGF protein, cav-1 mRNA, cav-1 protein, and VEGF protein were higher in OGD/R astrocytes. bFGF knockdown markedly decreased the expression levels of cav-1 mRNA, cav-1 protein, and VEGF protein, which were effectively reversed by exogenous bFGF treatment. Moreover, exogenous bFGF treatment significantly increased the expression levels of cav-1 mRNA, cav-1 protein, and VEGF protein in OGD/R astrocytes; however, a cav-1 inhibitor abolished the effect of bFGF on VEGF protein expression. These results suggested that bFGF may protect astrocytes against ischemia/reperfusion injury by upregulating caveolin-1/VEGF signaling pathway.

Role of caveolin-1/vascular endothelial growth factor pathway in basic fibroblast growth factor-induced angiogenesis and neurogenesis after treadmill training following focal cerebral ischemia in rats.

Exercise is known to aid functional recovery following ischemia, though the mechanisms responsible for the beneficial effects of exercise on recovery from ischemic stroke are not fully understood. Basic fibroblast growth factor (bFGF) contributes to angiogenesis and promotes neurologic functional recovery after stroke. The present study aimed to investigate the possible mechanisms whereby treadmill exercise ameliorated impaired angiogenesis and neurogenesis following transient cerebral ischemia in middle cerebral artery occlusion (MCAO) rats. Treadmill exercise was started 2days after ischemia-reperfusion in MCAO rats and continued until 7 or 28days after MCAO, after which the animals were sacrificed. Changes in neurological deficit, infarction volume, neuronal morphology, expression levels of bFGF, caveolin-1, and vascular endothelial growth factor (VEGF), and angiogenesis and neurogenesis in the ischemic penumbra were examined by reverse transcription-polymerase chain reaction, western blots, and/or double immunofluorescence. The results suggested that treadmill exercise promoted the expression of bFGF, improved neurological recovery, and reduced infarct volume compared with non-exercised rats, and also enhanced the expression of caveolin-1, VEGF, VEGF receptor 2(FIK-1)/CD34, and Brdu/nestin staining. Small interfering RNA targeting bFGF blocked the protective effects of bFGF. In addition, 4weeks of post-stroke recovery still ameliorated ischemia-induced damage without bFGF shRNA. These findings suggest a novel mechanism underlying the beneficial effects of bFGF following stroke, and indicate that treadmill exercise may aid stroke recovery by regulating the caveolin-1/VEGF pathway in the ischemic zone.