Establishment of novel anti-TIM-3 antibodies interfering with its binding to ligands.

The T cell immunoglobulin and mucin-domain containing-3 (TIM-3) receptor has gained significant attention as a promising target for cancer immunotherapy. The inhibitory effect of T cells by TIM-3 is mediated through the interaction between TIM-3 and its ligands. Ligand-blocking anti-TIM-3 antibodies possess the potential to reactivate antigen-specific T cells and augment anti-tumor immunity. However, the precise ligand-receptor interactions disrupted by the administration of TIM-3 blocking Abs have yet to be fully elucidated. In this study, we have developed a panel of monoclonal antibodies targeting human TIM-3, namely MsT001, MsT065, MsT229, and MsT286. They exhibited high sensitivities (10 pg/mL) and affinities (3.70 × 10-9 to 4.61 × 10-11 M) for TIM-3. The TIM-3 antibodies recognized distinct epitopes, including linear epitopes (MsT001 and MsT065), and a conformational epitope (MsT229 and MsT286). Additionally, the MsT229 and MsT286 displayed reactivity towards cynomolgus TIM-3. The interactions between TIM-3/Gal-9, TIM-3/HMGB-1, and TIM-3/CEACAM-1 disrupt the binding of MsT229 and MsT286, while leaving the binding of MsT001 and MsT065 unaffected. The inhibitory effect on the interaction between Gal-9 and TIM-3 was found to be dose-dependently in the presence of either MsT229 or MsT286. The findings suggested that the involvement of conformational epitopes in TIM-3 is crucial for its interaction with ligands, and we successfully generated novel anti-TIM-3 Abs that exhibit inhibitory potential. In conclusion, our finding offers valuable insights -on the comprehension and targeting of human TIM-3.

T-cell subsets and cytokines are indicative of neoadjuvant chemoimmunotherapy responses in NSCLC.

PURPOSE: Neoadjuvant PD-1 blockade combined with chemotherapy is a promising treatment for resectable non-small cell lung cancer (NSCLC), yet the immunological mechanisms contributing to tumor regression and biomarkers corresponding to different pathological responses remain unclear. METHODS: Using dynamic and paired blood samples from NSCLC patients receiving neoadjuvant chemoimmunotherapy, we analyzed the frequencies of CD8 + T-cell and Treg subsets and their dynamic changes during neoadjuvant treatment through flow cytometry. Cytokine profiles and function-related gene expression of CD8 + T cells and Tregs were analyzed through flow cytometry and mRNA-seq. Infiltrating T-cell subsets in resected tissues from patients with different pathological responses were analyzed through multiplex immunofluorescence. RESULTS: Forty-two NSCLC patients receiving neoadjuvant chemoimmunotherapy were enrolled and then underwent surgical resection and pathological evaluation. Nineteen patients had pCR (45%), 7 patients had MPR (17%), and 16 patients had non-MPR (38%). In patients with pCR, the frequencies of CD137 + CD8 + T cells (P = 0.0475), PD-1 + Ki-67 + CD8 + T cells (P = 0.0261) and Tregs (P = 0.0317) were significantly different from those of non-pCR patients before treatment. pCR patients usually had low frequencies of CD137 + CD8 + T cells, PD-1 + Ki-67 + CD8 + T cells and Tregs, and their AUCs were higher than that of tissue PD-L1 expression. Neoadjuvant chemoimmunotherapy markedly improved CD8 + T-cell proliferation and activation, especially in pCR patients, as the frequencies of CD137 + CD8 + (P = 0.0136) and Ki-67 + CD8 + (P = 0.0391) T cells were significantly increased. The blood levels of cytokines such as IL-2 (P = 0.0391) and CXCL10 (P = 0.0195) were also significantly increased in the pCR group, which is consistent with the high density of activated cytotoxic T cells at the tumor site (P < 0.0001). CONCLUSION: Neoadjuvant chemoimmunotherapy drives CD8 + T cells toward a proliferative and active profile. The frequencies of CD137 + CD8 + T cells, PD-1 + Ki-67 + CD8 + T cells and Tregs at baseline might predict the response to neoadjuvant chemoimmunotherapy in NSCLC patients. The increase in IL-2 and CXCL10 might reflect the chemotaxis and enrichment of cytotoxic T cells at the tumor site and a better response to neoadjuvant chemoimmunotherapy.

Human umbilical cord mesenchymal stem cells alleviated TNBS-induced colitis in mice by restoring the balance of intestinal microbes and immunoregulation.

AIMS: Human umbilical cord mesenchymal stem cells (HUMSCs) have been documented to be effective for several immune disorders including inflammatory bowel diseases (IBD). However, it remains unclear how HUMSCs function in regulating immune responses and intestinal flora in the trinitrobenzene sulfonic acid (TNBS)-induced IBD model. MATERIALS AND METHODS: We assessed the regulatory effects of HUMSCs on the gut microbiota, T lymphocyte subpopulations and related immune cytokines in the TNBS-induced IBD model. The mice were divided into the normal, TNBS, and HUMSC-treated groups. The effect of HUMSCs was evaluated by Hematoxylin and Eosin (H&E) staining, fluorescence-activated cell sorting (FACS), and enzyme-linked immunosorbent assay (ELISA) analyses. Metagenomics Illumina sequencing was conducted for fecal samples. KEY FINDINGS: We demonstrated that the disease symptoms and pathological changes in the colon tissues of TNBS-induced colitis mice were dramatically ameliorated by HUMSCs, which improved the gut microbiota and rebalanced the immune system, increasing the abundance of healthy bacteria (such as Lactobacillus murinus and Lactobacillus johnsonii), the Firmicutes/Bacteroidetes ratio, and the proportion of Tregs; the Th1/Th17 ratio was decreased. Consistently, the expression levels of IFN-γ and IL-17 were significantly decreased, and transforming growth factor-ß1 (TGF-ß1) levels were significantly increased in the plasma of colitis mice HUMSC injection. SIGNIFICANCE: Our experiment revealed that HUMSCs mitigate acute colitis by regulating the rebalance of Th1/Th17/Treg cells and related cytokines and remodeling the gut microbiota, providing potential future therapeutic targets in IBD.

Immunoassay with Novel Paired Antibodies for Detection of Lipoarabinomannan in the Pleural Fluid and Plasma of Patients with Tuberculous Pleurisy.

Tuberculous pleurisy (TP) is one of the most common forms of extrapulmonary tuberculosis, but its diagnosis is challenging. Lipoarabinomannan (LAM) antigen is a biomarker for Mycobacterium tuberculosis (Mtb) infection. LAM detection has potential as an auxiliary diagnostic method for TP. We have successfully generated five rabbit anti-LAM monoclonal antibodies (BJRbL01, BJRbL03, BJRbL20, BJRbL52, and BJRbL76). Here, anti-LAM antibodies were tested to detect LAM in the pleural fluid and plasma of patients with TP by sandwich enzyme-linked immunosorbent assays (ELISAs). The results revealed that all of the anti-LAM antibodies were successfully used as capture and detection antibodies in sandwich ELISAs. The BJRbL01/BJRbL01-Bio pair showed better performance than the other antibody pairs for detecting mycobacterial clinical isolates and had a limit of detection of 62.5 pg/mL for purified LAM. LAM levels were significantly higher in the pleural fluid and plasma of patients with TP than in those of patients with malignant pleural effusion or the plasma of non-TB, and LAM levels in the pleural fluid and plasma were positively correlated. Moreover, LAM levels in the pleural fluid sample were significantly higher in confirmed TP patients than in clinically diagnosed TP patients. Our studies provide novel LAM detection choices in the pleural fluid and plasma of TP patients and indicate that LAM detection assay has an auxiliary diagnostic value for TP, which may help to improve the diagnosis of TP.

Costimulatory capacity of CD137 mAbs on T cells depends on elaborate CRD structures but not on blocking ligand-receptor binding.

CD137 is mainly a costimulatory receptor of CD8+ T cells. Two representative CD137 antibodies, utomilumab, and urelumab, show different costimulatory capacities in clinical trials. Balancing the antitumor effect and systemic toxicity of T cells activated by CD137 signaling is a challenge that requires clinical consideration. In this study, a panel of specific anti-human CD137 monoclonal antibodies (mAbs) were prepared and their affinities, isotypes, CD137-CRD (cysteine-rich domain) binding regions, cross-reactivity to mouse and rhesus CD137, inhibition of ligand-receptor binding and costimulatory activities were analyzed. The results showed that anti-human CD137 mAbs had high cross-reactivity with rhesus CD137. MAbs fell into three clusters according to their different binding regions of the CD137 extracellular domain. They bound to CRDI+CRDII, CRDIII or CRDIV+STP. CRDIII-binding mAbs had the strongest blocking activity. Highly costimulatory CD137 mAbs showed stronger abilities to promote CD8+ T-cell proliferation. However, the costimulatory capacity of mAbs on T cells was not closely related to their ability to block CD137L-CD137 binding and may be controlled by more elaborate CRD conformational structures. This study provides additional information for the development of next-generation CD137 mAbs to meet clinical needs.

Association between response to anti-PD-1 treatment and blood soluble PD-L1 and IL-8 changes in patients with NSCLC.

In this study, we explored the dynamic changes in blood sPD-L1 and its clinical value during anti-PD-1 immunotherapy in non-small cell lung cancer (NSCLC) patients. First, we established a sandwich ELISA for functional sPD-L1 that can bind to PD-1 and has biological functions. By monitoring functional sPD-L1 in 39 NSCLC patients treated with anti-PD-1 antibodies, we found a positive correlation between baseline sPD-L1 and tissue PD-L1 (P = 0.0376, r = 0.3581), with patients with lymph node metastasis having higher sPD-L1 levels (P = 0.0037) than those without lymph node metastasis. Although baseline functional sPD-L1 and PFS did not correlate significantly in this study, changes in sPD-L1 in patients with different clinical responses showed different trends. Blood sPD-L1 increased in 93% of patients after two cycles of anti-PD-1 treatment (P = 0.0054); sPD-L1 in nonresponsive patients continued to increase (P = 0.0181), but sPD-L1 started to decline in responsive patients. Blood IL-8 levels were associated with tumor load, and when combined with IL-8, the evaluation accuracy of sPD-L1 improved to 86.4%. This study preliminarily shows that the combination of sPD-L1 and IL-8 is a convenient and effective method for monitoring and evaluating the effectiveness of anti-PD-1 immunotherapy in NSCLC patients.

Enhanced Serum IgG Detection Potential Using 38KD-MPT32-MPT64, CFP10-Mtb81-EspC Fusion Protein and Lipoarabinomannan (LAM) for Human Tuberculosis.

For the rapid, reliable, and cost-effective methods of tuberculosis (TB) auxiliary diagnosis, antibody (Ab) detection to multiple antigens of Mycobacterium tuberculosis (Mtb) has great potential; however, this methodology requires optimization. We constructed 38KD-MPT32-MPT64, CFP10-Mtb81-EspC, and Ag85B-HBHA fusion proteins and evaluated the serum Ab response to these fusion proteins and to lipoarabinomannan (LAM) by ELISA in 50 TB patients and 17 non-TB subjects. IgG responses to the three fusion proteins and to LAM were significantly higher in TB patients, especially in Xpert Mtb-positive TB patients (TB-Xpert+), than in non-TB subjects. Only the anti-38KD-MPT32-MPT64 Ab showed higher levels in the Xpert Mtb-negative TB patients (TB-Xpert-) than in the non-TB, and only the anti-LAM Ab showed higher levels in the TB-Xpert+ group than in the TB-Xpert- group. Anti-Ag85B-HBHA Ab-positive samples could be accurately identified using 38KD-MPT32-MPT64. The combination of 38KD-MPT32-MPT64, CFP10-Mtb81-EspC, and LAM conferred definite complementarity for the serum IgG detection of TB, with relatively high sensitivity (74.0%) and specificity (88.2%). These data suggest that the combination of 38KD-MPT32-MPT64, CFP10-Mtb81-EspC, and LAM antigens provided a basis for IgG detection and for evaluation of the humoral immune response in patients with TB.

Soluble programmed cell death-ligand 1 as a new potential biomarker associated with acute coronary syndrome.

Background: Soluble programmed cell death-ligand 1 (sPD-L1) has been well documented to activate immunosuppression and is considered an essential predictor of negative clinical outcomes for several malignances and inflammatory conditions. However, the clinical significance of sPD-L1 in the peripheral blood of patients with coronary artery disease (CAD) remains unclear. The aim of this study was to assess the correlations of sPD-L1 with clinical features in CAD patients and evaluate the diagnostic value of this protein in CAD. Methods: A total of 111 CAD patients and 97 healthy volunteers who served as healthy controls (HCs) were consecutively enrolled. Plasma levels of sPD-L1 were measured with an amplified enzyme-linked immunosorbent assay (ELISA), and hs-CRP was measured with a C-reactive protein assay kit. The levels of other inflammatory cytokines were assessed in 88 CAD patients and 47 HCs by a multiparameter immunoluminescence flow cytometry detection technique. A logistic regression model was used to assess the independent association of sPD-L1 with acute coronary syndrome (ACS). The correlation between sPD-L1 and inflammatory cytokines in ACS was also assessed. Results: Plasma levels of sPD-L1 were significantly increased in CAD patients, especially those with ACS. Univariate logistic regression analysis revealed that sPD-L1 (OR: 3.382, 95% CI: 2.249-5.084, p < 0.001), BMI, hypertension, diabetes, dyslipidemia, previous MI, and the levels of HDL-C, LDL-C and hs-CRP were significantly associated with ACS. sPD-L1 (OR: 3.336, 95% CI: 1.084-6.167, p = 0.001) was found to be independently and significantly associated with ACS in the subsequent multivariable logistic regression analysis. Additionally, elevated plasma sPD-L1 levels were associated with increased interleukin-6 and interleukin-8 levels in ACS patients. Receiver operating characteristic (ROC) analysis showed that the AUC of sPD-L1 for diagnosing ACS was 0.778, with a sensitivity of 73.9% and a specificity of 73.4%, which was comparable with that of the inflammatory biomarker hs-CRP. Conclusion: The plasma sPD-L1 level reflects the severity of CAD, is associated with inflammatory responses and is a potential new biomarker for the diagnosis of ACS.

Pathological complete response to neoadjuvant chemoimmunotherapy correlates with peripheral blood immune cell subsets and metastatic status of mediastinal lymph nodes (N2 lymph nodes) in non-small cell lung cancer.

BACKGROUND: Neoadjuvant chemo-immunotherapy has got clinical benefits in parts of resectable non-small cell lung cancer (NSCLC) patients. The factors affecting the pathological response of NSCLC remain controversial. METHODS: A retrospective study of 59 patients with resectable stage IIA-IIIB NSCLC who were treated with neoadjuvant chemo-immunotherapy was performed. The clinical characteristics were analyzed in the pathological complete response (pCR) group and the non-pCR group. The immune cell subsets in peripheral blood were detected by flow cytometry. RESULTS: By analyzing the correlation between pathological response and clinical characteristics, we found that patients with N2 metastases were less effective in neoadjuvant chemo-immunotherapy (P = 0.001). Programmed death-ligand 1 (PD-L1) expression and treatment cycle were not related to pathological response (P > 0.05). Lower levels of total T cells, Th cells, and higher levels of NK cells in baseline were associated with pCR (P < 0.05). And during neoadjuvant chemo-immunotherapy, total T cells and activated T cells were significantly increased in patients with pCR (P < 0.05). CONCLUSION: The peripheral blood immune cell subsets and lymph node status were closely related to pathological response in patients with neoadjuvant chemo-immunotherapy. No significant correlation was found between pathologic response and PD-L1 expression.

Elevated Natural Killer Cell-Mediated Cytotoxicity Is Associated with Cavity Formation in Pulmonary Tuberculosis Patients.

Cavitation is a major pathological feature of pulmonary tuberculosis (TB). The study is aimed at investigating the mechanism of natural killer (NK) cells participating the cavity formation during Mycobacterium tuberculosis (MTB) infection. Human peripheral blood samples were donated by pulmonary TB patients with cavity or not. Real-time quantitative PCR and enzyme-linked immunosorbent assay were performed to analyze the expression of cytokines secreted by NK cells. And the cytotoxicity of NK cells was compared between two groups. Our data showed that NK cells were more abundant in cohorts of cavity. Increased abundance of granzyme A and granzyme B was observed in culture supernatants of NK cells isolated from cavitary TB patients, which also resulted in a higher level of nonviable MTB-infected monocytes. Our data firstly demonstrates that NK cells participate in cavity formation in pulmonary TB patients. The elevated level and increased cytotoxicity of NK cells accelerate the cavitary formulation.

Heterogeneity and prognosis of programmed cell death-ligand 1 expression in the circulating tumor cells of non-small cell lung cancer.

Due to tumor heterogeneity, the consistency of programmed cell death-ligand 1 (PD-L1) expression between circulating tumor cells (CTCs) and tissue is controversial. This study aimed to establish a method for detecting CTC PD-L1 expression and exploring the impact of the same on the prognosis of lung cancer. In 32 patients with non-small cell lung cancer, lung cancer cells in the blood were enriched using CD326 immunomagnetic beads. Goat anti-mouse polyclonal CD326 antibody stained the epithelial lung cancer cells and anti-PD-L1 antibody was used to detect the expression of CTC PD-L1. The DAKO Link 48 automatic staining device detected the expression in lung cancer tissue. The consistency of PD-L1 expression was analyzed in lung cancer tissue and CTCs. The effect of plasma interferon gamma, tumor necrosis factor alpha, and interleukin-2 on PD-L1 expression and prognosis was analyzed. The number of CTCs detected in patients was 1-36, with a median of 2. There was no significant difference in PD-L1 expression fractions between CTCs and paired tumor tissue (p>0.05). The correlation coefficient was 0.20. Regardless of lung cancer tissue or CTCs, there was no statistically significant difference in the blood cytokine levels between the two groups with positive or negative PD-L1 expression (p>0.05). There was no correlation between CTCs and PD-L1 in 23 untreated patients. The expression of PD-L1 in CTCs and lung cancer tissue is heterogeneous and unaffected by the peripheral cytokines' levels. PD-L1 expression has no correlation between CTCs and tissues and is not related to prognosis.

Generation of mycobacterial lipoarabinomannan-specific monoclonal antibodies and their ability to identify mycobacterium isolates.

BACKGROUND/PURPOSE: The World Health Organization has recommended commercial urine-sourced lipoarabinomannan (LAM) detection as a tool for screening HIV patients with suspected TB, but more sensitive immunodetection assays would help to identify HIV-negative TB patients. Here, we aimed to develop novel rabbit monoclonal antibodies (mAbs) against LAM for immunodetection purposes. METHODS: Rabbits were immunized with cell-wall components from the Mycobacterium tuberculosis (Mtb) H37Rv strain. An immune single-chain fragment variable (scFv) phage display library was generated. The scFv mAbs to LAM were identified through ELISA screening. The light and heavy chain variable region genes from the selected clones were sequenced. Vectors containing the full-length light and heavy chains were constructed and co-expressed in 293 T cells to generate whole IgG antibodies. The performances and binding characteristics of the mAbs against purified LAM from M.tb H37Rv, multiple mycobacteria species (M.tb H37Rv, M. bovis and non-tuberculous mycobacteria (NTM) strains), and mycobacteria clinical isolates (Mtb and NTM isolates) were determined using various immunoassay methods. RESULTS: We obtained five rabbit mAbs against LAM, four of which had high sensitivities (100 pg/ml) and affinities (1.16-1.73 × 10-9 M) toward LAM. They reacted with M.tb H37Rv, M. bovis, and slow-growing NTM, but not with rapid-growing NTM. Similar results were obtained with mycobacterium isolates, where 96% of the Mtb isolates and 90% of the M. avium-intracellulare isolates were successfully identified. CONCLUSION: The novel rabbit LAM-specific mAbs performed well at recognizing LAM from slow-growing pathogenic mycobacteria, which support their future clinical application.

Circulating CD137+ CD8+ T cells accumulate along with increased functional regulatory T cells and thoracic tumour burden in lung cancer patients.

CD137 is a promising target for immunostimulation strategies against cancer. Previous studies showed that CD137+ CD8+ T cells are enriched in antitumour effector T cells in both preclinical tumour models and cancer patients, but to date, such T cells in the blood of lung cancer patients have not been sufficiently investigated. In this study, circulating antigen-activated CD8+ T cell subsets, identified as CD137+ CD8+ or PD-1+ (programmed cell death protein 1) CD8+ , and regulatory T cells (Treg), identified as CD4+ CD25+ CD127low/- , in 40 untreated lung cancer patients and in 49 age- and sex-matched healthy controls (HCs) were assessed by flow cytometry. Results were evaluated for associations with lung cancer patient clinical characteristics. Correlations between antigen-activated CD8+ T cells and effector Treg (CTLA-4+ [cytotoxic T-lymphocyte antigen 4] CD4+ CD25+ CD127low/- ) were also investigated. Higher percentages of PD-1+ , CD137+ and PD-1+ CD137+ amongst CD8+ T cells were observed in lung cancer patients compared with HCs. The percentages of CD137+ CD8+ and PD-1+ CD137+ CD8+ T cell subsets amongst CD8+ T cells were positively correlated with thoracic tumour burden and were strongly positively correlated with the percentage of effector Treg subset. Smoking patients harboured higher percentages of the PD-1+ CD8+ T cell subset compared with non-smoking patients. This study demonstrated that circulating antigen-activated CD8+ T cells accumulated in lung cancer patients along with increased effector Treg and thoracic tumour burden. These findings aid a better understanding of immune-host interactions in lung cancer patients using peripheral blood, and further support immunotherapeutic intervention strategies using combination therapy for differential control of Treg and activation of tumour-specific effector T cells.

[Early secretory antigenic target protein-6/culture filtrate protein-10 fusion protein-specific Th1 and Th2 response and its diagnostic value in tuberculous pleural effusion].

OBJECTIVE: To detect the Th1 and Th2 cell percentage in pleural effusion mononuclear cells (PEMCs) stimulated by early secretory antigenic target protein-6 (ESAT-6)/culture filtrate protein-10 (CFP-10) fusion protein (E/C) with flow cytometry (FCM), and therefore to explore the local antigen specific Th1 and Th2 response and its diagnostic value in tuberculous pleuritis. METHODS: Forty patients with tuberculous pleural effusion and 30 patients with malignant pleural effusion were included in this study from Sep.2008 to Mar.2009. PEMCs were isolated and cryopreserved. After resuscitation, the cells were cultured with E/C (simultaneously with positive control and negative control), and antigen-specific Th1 and Th2 cells were detected with intracellular cytokine staining of FCM. Normal distribution data using t test, abnormal distribution data using Wilcoxon test. RESULTS: In the TB group,the medians (quartile range) of Th1 cells and Th1/Th2 ratio among PEMCs stimulated by ESAT-6/CFP-10 fusion protein were 3.06% (1.59%-6.92%) and 17 (7.38-35.53), significantly higher than those of the negative control [0.38% (0.02%-1.80%) and 3.59 (0.49-25.09)], the differences being statistically significant (Z = -5.345 and 3.314, P < 0.01). The percentage of Th2 cells [(0.22 ± 0.19)%] was also increased compared with that of the negative control [(0.10 ± 0.08)%], the difference being statistically significant (t = 4.108, P < 0.01). In the malignant effusion group, the medians (quartile range) of Th1 percentage and Th1/Th2 ratio were 0.12% (0.05%-0.39%) and 1.05 (0.25-2.52), which were significantly different as compared with those of the TB group (Z = -6.624 and -5.536, P < 0.01). The Th2 percentage in the 2 groups were (0.22 ± 0.19)% and (0.15 ± 0.02)%, respectively (t = 1.954, P > 0.05). The receiver operating characteristic curve indicated that the area under the curve (AUC), sensitivity, and specificity were 0.937, 85.4%, and 90.6% respectively for Th1 to diagnose tuberculous pleurisy. For Th1/Th2, the AUC, sensitivity, and specificity were 0.883, 81.5%, and 90.6% respectively. CONCLUSIONS: The feature of ESAT-6/CFP-10 fusion protein-specific Th1 and Th2 response in tuberculous pleurisy was a mixed reaction of Th1 and Th2 with Th1 predominance. Th1 percentage and Th1/Th2 ratio could be diagnostic indexes for identifying tuberculous from malignant pleural effusions.

[Role of BCG-depleted immunodominant antigens derived from M. tuberculosis in serological test for tuberculosis].

OBJECTIVE: To explore the roles of BCG-depleted immunodominant antigens derived from M. tuberculosis in serological tests for tuberculosis (TB). METHODS: Four different combinations of current mainstream antigens used for serological diagnosis of TB were selected: Reagent A [Mycobacterium TB immunoglobulin G (IgG) antibody assay kit]; Reagent B (Mycobacterium TB detection kit); Reagent C (M. tuberculosis-specific antibody detection kit); Reagent D [Active TB antibody detection enzyme-linked immunosorbent assay (ELISA) system]. Immunological methods of Western blot, colloidal gold and ELISA were developed to test the antibodies in 109 patients with active tuberculosis (TB) and 97 healthy populations. They were divided into purified protein derivative of tuberculin (PPD) positive and negative groups. Bayesian statistical analysis was used to analyze the influences of variable combinations of different antigens on the detection accuracy of TB. RESULTS: For Reagent A, B, C, D, the detection rates of IgG antibodies in the patients with active TB were 80.0%, 66.7%, 80.7%, 56.0% versus 23.9%, 8.9%, 6.6% and 1.0% respectively in healthy populations. The TB antibody detection rates in four TB patient populations were all higher than that in healthy populations (χ(2) = 47.53, 51.59, 90.48, 69.68, all P < 0.01). The TB antibody detection rates of Reagents A and B increased with the intensity of positive reaction to PPD in healthy populations (χ(2) = 2.124, 2.220, all P < 0.05) while those of Reagents C, D in healthy populations were irrelevant to PPD reaction. (χ(2) = 0.122, 0.479, all P > 0.05). Reagent D has the highest accuracy. The immunoglobulin M (IgM) antibody detection rate of Reagent D was only 2.1% in the patients with active TB. CONCLUSIONS: The detecting sensitivity of TB IgG antibodies is associated with antigen selection. And it is also positively correlated with the number of combined antigens. High-sensitivity detection is often accompanied by a loss of specificity. With the BCG-depleted antigens derived from M. tuberculosis, the specificity of serological test for TB may significantly improve.

[Preparation of two types p53 recombinant adenovirus and quantitative exogenous expression of green fluorescence protein by flow cytometry].

BACKGROUND AND OBJECTIVE: The p53 as a transcription factor in cell stress was activated to regulate cell cycle and programmed cell death to inhibit tumor growth. Usually, p53 is kept in non-activated state through various mechanisms, including the action of p53 C-terminal negative regulatory sequences. The purpose of the study is to prepare the two types p53 recombinant adenoviruses that carry full-length p53 as well as deletion of negative regulatory sequences at p53 C-terminus and to detect exogenous GFP expression in human lung cancer cell infected-virus by FCM scatter plot. METHODS: Using pAdEasy-Track vector system the p53 recombinant plasmids was constructed and the homologous recombinants in E. coli was produced. The three kinds of recombinant adenovirus in L293 cells was generated, sequencing proved. Exogenous GFP expression in human lung cancer 801D cells infected-virus was detected by FCM scatter plot. RESULTS: p53 recombinant adenoviruses named Ad-p53(wtp), Ad-p53(del) and Ad-(empty carrier) were produced. Results of sequences indicate that the Ad-p53(del) was deletion of 111 bases before stop codon TGA and of 3 untranslated region at p53, the Ad-p53(wtp) no loss of any p53 base, the Ad-(empty carrier) no p53 sequence. FCM scatter plot indicate the percentage of 801D cells expressed GFP with three kinds of viral infection was almost same and was increased with the virus density. 801D contains ratio of cells with different fluorescence intensity. CONCLUSION: The preparation of recombinant adenovirus, Ad-p53(del), pA-p53(wtp) and Ad-(empty carrier). The cells expressed-GFP can be quantitatively detected by FCM scatter plot. It was provide that the reliability of the virus system and accurate method for selecting viruses density to infecting cells.

[Nitric oxide production and expression of cytokines by macrophages infected by M. tuberculosis H(37)R(v)].

OBJECTIVE: To study nitric oxide (NO) production and cytokine expression by macrophages infected by M. tuberculosis H(37)R(v), and to compare the difference between dead and live M. tuberculosis in the induction of immune responses, and thus to show if dead bacteria could be a possible candidate for new vaccines. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and ELISA were used to measure the production of NO and cytokines in macrophages infected by H(37)R(v). RESULTS: Macrophages infected by viable M. tuberculosis produced more NO, IL-1, IL-12, IL-18, TNF-alpha and inducible nitric oxide synthases (iNOS), as compared with macrophages infected by dead bacteria. The number of bacteria was also an important factor determining the production of NO and cytokines. CONCLUSIONS: Viable M. tuberculosis H(37)R(v) can induce the activation of macrophages and the production of more NO and cytokines which play important roles in the host immune response. Heat-killed M. tuberculosis H(37)R(v) failed to induce activation of macrophages and the production of NO and cytokines, which makes it unlikely to be a candidate for vaccine development.