Decidualization-derived cAMP promotes decidual NK cells to be angiogenic phenotype.

BACKGROUND: During early pregnancy, a large number of CD56bright natural killer cells (NKs) are accumulated in the decidua; unlike peripheral and cord blood NK cells (pNK and cNK), these decidual NK cells (dNK) display a great capacity to secrete a series of angiogenic/vascular factors, which are essential for placental development. However, the mechanism underlying the formation of dNK cells with an angiogenic phenotype remains unclear. METHODS: First, we compared the difference between dNK and cNK/pNK cells in terms of the expression of CD56 and VEGF, and the regulation of the tube formation. The effect of cAMP on the differentiation of NK cells was evaluated by its analog and inhibitor stimulation. We further analyzed the differences in the phenotype of dNK cells and the expression of VEGF in dNK cells from normal pregnancies and miscarriages. RESULTS: Different from cNK and pNK, dNK cells displayed high expression of CD56 and VEGF. And dNK cells showed a higher capacity of inducing tube formation of HUVEC by trophoblast. Meanwhile, we observed that cAMP-analogue increased the percentage of CD56bright NK population in cNK cells with up-regulated VEGF secretion and tube formation of HUVEC by trophoblast, which could be inhibited by pretreatment with VEGFR neutralizing antibody. Similar changes occurred when co-culturing cNK cells with DSCs but not ESCs. Interestingly, the inhibitor of cAMP signaling (Metadoxine, META) could significantly inhibit the upregulation of VEGF in cNK cells by DSCs. Furthermore, DSCs could secret much more cAMP than ESCs. Notably, decreased CD56bright NK population and VEGF secretion by dNK were related to pregnancy loss. CONCLUSIONS: These findings suggest that dNK cells display an angiogenic phenotype that can be induced by decidualized cAMP signaling. Our study indicates the significance of decidualization-derived cAMP in regulating angiogenesis of decidual NKs and reveals complex crosstalk between different cell types in a critical period during early pregnancy.

TIGIT Signaling Pathway Regulates Natural Killer Cell Function in Chronic Hepatitis B Virus Infection.

Background and Objective: Persistent infection of hepatitis B virus (HBV) and liver damage in immune active chronic hepatitis B (CHB) could be partly due to the overreaction of natural killer (NK) cells, including pro-inflammatory cytokine secretion and cytotoxicity. An immunosuppressive receptor, T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT) is specifically expressed in NK cells. This study aims to investigate the role of the TIGIT signaling pathway in regulating NK cell functions in patients with CHB. Method: We comparatively assessed the expression of TIGIT in NK cells of patients with immune active CHB (CHB-IA), carriers of immune control chronic HBV (CHB-IC), and healthy controls (HCs), and then explored mechanisms of the TIGIT signaling pathway in regulating NK cell-mediated liver injury by different molecular assessments. Result: The expression of TIGIT in NK cells was enhanced in CHB-IC but was reduced in CHB-IA compared with the HC group. In patients with CHB-IA, the expression of TIGIT was inversely correlated with intensity of the liver damage. Moreover, TIGIT-NK cells show higher IFN-γ secretion capability, degranulation activity, and cytotoxicity but lower apoptosis than TIGIT+ NK cells. Blockade of the TIGIT pathway with anti-TIGIT antibody increased NK cell function, while activation of the TIGIT pathway with TIGIT Fc and CD155 Fc chimera protein down-regulated NK cell function. Conclusion: Our data showed that the TIGIT signaling pathway participates in NK cell impairment, which could be used as a new therapeutic target to protect patients with chronic HBV infection from severe liver injury.

Detection of IgM and IgG antibodies in patients with coronavirus disease 2019.

OBJECTIVES: This study aimed to determine the IgM and IgG responses against severe acute respiratory syndrome coronavirus (SARS-CoV)-2 in coronavirus disease 2019 (COVID-19) patients with varying illness severities. METHODS: IgM and IgG antibody levels were assessed via chemiluminescence immunoassay in 338 COVID-19 patients. RESULTS: IgM levels increased during the first week after SARS-CoV-2 infection, peaked 2 weeks and then reduced to near-background levels in most patients. IgG was detectable after 1 week and was maintained at a high level for a long period. The positive rates of IgM and/or IgG antibody detections were not significantly different among the mild, severe and critical disease groups. Severe and critical cases had higher IgM levels than mild cases, whereas the IgG level in critical cases was lower than those in both mild and severe cases. This might be because of the high disease activity and/or a compromised immune response in critical cases. The IgM antibody levels were slightly higher in deceased patients than recovered patients, but IgG levels in these groups did not significantly differ. A longitudinal detection of antibodies revealed that IgM levels decreased rapidly in recovered patients, whereas in deceased cases, either IgM levels remained high or both IgM and IgG were undetectable during the disease course. CONCLUSION: Quantitative detection of IgM and IgG antibodies against SARS-CoV-2 quantitatively has potential significance for evaluating the severity and prognosis of COVID-19.

Diagnostic Performance of a 5-Marker Predictive Model for Differential Diagnosis Between Intestinal Tuberculosis and Crohn's Disease.

Background: The differentiation between intestinal tuberculosis (ITB) and Crohn's disease (CD) is a challenge. The aim of this study was to investigate a predictive model for differential diagnosis between ITB and CD. Methods: A total of 268 patients who were suspected of having ITB or CD were prospectively recruited between January 2013 and September 2016. The clinical, laboratory, radiological, endoscopic, and histological features were investigated and subjected to univariate and multivariate analyses. The final predictive model was developed based on the regression coefficients of multivariate logistic regression. To validate the model, the same regression equation was tested on the other group. Results: A total of 239 patients had a final diagnosis, including 86 ITB and 153 CD. Five variables (perianal disease, pulmonary involvement, longitudinal ulcer, left colon, and ratio of tuberculosis-specific antigen to phytohaemagglutinin) were selected for the predictive model to discriminate between ITB and CD. In the predictive model of the training data set, the area under the receiver operating characteristic (ROC) curve, sensitivity, specificity, and accuracy, with a cutoff level of 0.29, were 0.975 (95% confidence interval [CI], 0.939-0.993), 96.7%, 90.7%, and 92.8%, respectively. Application of the predictive model to the validation data set showed similar performance in distinguishing ITB from CD. The area under the ROC curve, sensitivity, specificity, and accuracy were 0.950 (95% CI, 0.871-0.987), 88.5%, 93.5%, and 91.7%, respectively. Conclusions: This 5-marker predictive model could be conveniently used by clinicians to draw a reliable differential diagnosis between ITB and CD in clinical practice. 10.1093/ibd/izy154_video1izy154.video15790725497001.

Establishment of the Reference Intervals of Lymphocyte Function in Healthy Adults Based on IFN-γ Secretion Assay upon Phorbol-12-Myristate-13-Acetate/Ionomycin Stimulation.

The function of lymphocytes is the key to reflect the immune status of hosts. Evaluation of lymphocyte function is a useful tool to monitor the effect of immunosuppressive treatment and predict the prognosis of immune-mediated diseases (e.g., cancer, autoimmune diseases, and infectious diseases). As the lymphocytes have various activities, such as activation, cytotoxicity, and cytokine secretion, it is a challenge to evaluate the function of lymphocytes in clinical practice and the reference intervals (RIs) of lymphocyte function are rarely reported. The present study showed that the secretion of IFN-γ was well correlated with the activation, chemotaxis, and cytotoxicity of CD4+, CD8+ T cells, and NK cells, which suggests that IFN-γ production can be used as a symbol of lymphocyte function. We therefore created a simple method to detect the function of CD4+, CD8+ T cells, and NK cells simultaneously according to IFN-γ secretion by using whole blood instead of peripheral blood mononuclear cells. We further established the RIs of lymphocyte function (CD4+ T cells: 15.31-34.98%; CD8+ T cells: 26.11-66.59%; NK cells: 39.43-70.79%) in healthy adults. This method showed good reproducibility for the evaluation of lymphocyte function. The established RIs were suitable for use in other centers based on the validation data. We also validated the RIs in individuals with different immune status, and the results showed that kidney transplant recipients and infants (0-1 year) had a decreased lymphocyte function, whereas T cells in systemic lupus erythematosus patients exhibited an opposite trend. Overall, we have successfully established the RIs of lymphocyte function in healthy adults in a simple way, which might be of important clinical value in the diagnosis, monitoring, and prognosis of immune-related diseases.

Characteristics of diarrheagenic Escherichia coli among children under 5 years of age with acute diarrhea: a hospital based study.

BACKGROUND: Diarrhea is the leading infectious cause of childhood morbidity and mortality. Among bacterial agents, diarrheagenic Escherichia coli (DEC) is the major causal agent of childhood diarrhea in developing countries, particularly in children under the age of 5 years. Here, we performed a hospital-based prospective study to explore the pathotype distribution, epidemiological characteristics and antibiotic resistance patterns of DEC from < 5-year-old diarrheal children. METHODS: Between August 2015 and September 2016, 684 stool samples were collected from children (< 5 years old) with acute diarrhea. All samples were cultured and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and biochemical tests. PCR was used for subtyping, and enteropathogenic E. coli (EPEC) isolates were identified simultaneously with serology. Furthermore, antimicrobial sensitivity tests and sequencing of antibiotic resistance-related genes were conducted. RESULTS: DEC strains were identified in 7.9% of the 684 stool samples. Among them, the most commonly detected pathotype was EPEC (50.0% of DEC), of which 77.8% were classified as atypical EPEC (aEPEC). Age and seasonal distribution revealed that DEC tended to infect younger children and to occur in summer/autumn periods. Multidrug-resistant DEC isolates were 66.7%; resistance rates to ampicillin, co-trimoxazole, cefazolin, cefuroxime, cefotaxime, and ciprofloxacin were ≥ 50%. Among 5 carbapenem-resistant DEC, 60.0% were positive for carbapenemase genes (2 blaNDM-1 and 1 blaKPC-2). Among 30 cephalosporin-resistant DEC, 93.3% were positive for extended-spectrum ß-lactamase (ESBL) genes, with blaTEM-1 and blaCTX-M-55 being the most common types. However, no gyrA or gyrB genes were detected in 16 quinolone-resistant isolates. Notably, aEPEC, which has not received much attention before, also exhibited high rates of drug resistance (81.0%, 66.7%, and 14.3% for ampicillin, co-trimoxazole , and carbapenem resistance, respectively). CONCLUSIONS: EPEC was the most frequent DEC pathotype in acute diarrheal children, with aEPEC emerging as a dominant diarrheal agent in central China. Most DEC strains were multidrug-resistant, making even ciprofloxacin unsuitable for empiric treatment against DEC infection. Among carbapenem-resistant DEC strains, those harboring blaNDM-1 and blaKPC-2 were the main causal agents. blaTEM-1 and blaCTX-M-55 were the major genetic determinants associated with high levels of cephalosporin resistance.

Use of TBAg/PHA ratio in distinguishing tuberculoma from cancer in solitary pulmonary nodule or mass.

INTRODUCTION: Differentiation of tuberculoma from cancer in solitary pulmonary nodule or mass still remains a major challenge in diagnostic laboratories. OBJECTIVES: The objective of this study is to determine the performance of T-SPOT.TB assay in discriminating these 2 diseases. METHODS: We prospectively enrolled 331 patients with a solitary pulmonary nodule or mass on computed tomography scans. Conventional tests and T-SPOT.TB assay were simultaneously performed in all participants. RESULTS: Our results showed that the performance of directly using T-SPOT.TB results in distinguishing tuberculoma from cancer in solitary pulmonary nodule or mass was not satisfactory because of moderate sensitivity and specificity. However, a further calculation of the ratio of TB-specific antigen (TBAg) to phytohemagglutinin (PHA) (TBAg/PHA ratio) of T-SPOT.TB assay may lead to improvement in distinguishing these 2 diseases. If using the threshold value of 0.236, the sensitivity and specificity of the TBAg/PHA ratio in distinguishing tuberculoma from cancer in solitary pulmonary nodule or mass were, respectively, 80.6% and 93.3%. The area under the curve (AUC) of the receiver operating characteristic curve was 0.921 (95% confidence interval, 0.875-0.967). Furthermore, the TBAg/PHA ratio may also be used to distinguish tuberculoma from other benign diseases (AUC: 0.909, sensitivity: 85.07%, specificity: 90%). CONCLUSIONS: Calculation of the TBAg/PHA ratio might provide a useful non-invasive tool for distinguishing tuberculoma from cancer in patients with a solitary pulmonary nodule or mass in TB-endemic countries.

The performance of the TBAg/PHA ratio in the diagnosis of active TB disease in immunocompromised patients.

OBJECTIVES: The results of the T-SPOT.TB (T-SPOT) assay are reduced in immunocompromised patients with active tuberculosis (ATB), and it is difficult using T-SPOT results to distinguish ATB from latent tuberculosis infection (LTBI) in this condition. The aim of this study was to determine the performance of the TBAg/PHA ratio in T-SPOT assay in the diagnosis of ATB in immunocompromised patients. METHODS: One hundred and forty three immunocompromised ATB patients and 124 LTBI individuals were diagnosed according to conventional tests and T-SPOT assay. RESULTS: The results of T-SPOT assay are of no value in the diagnosis of ATB in immunocompromised patients. However, the number of phytohaemagglutinin (PHA) spot-forming cells (sfc) in T-SPOT assay was substantially decreased in immunocompromised ATB patients compared with that in LTBI individuals. Receiver operating characteristic (ROC) analysis revealed that a further calculation of the TBAg/PHA ratio (the larger of the ESAT-6/PHA and CFP-10/PHA) showed a better performance in distinguishing these two diseases. Using the threshold value of 0.316, the sensitivity and specificity for distinguishing immunocompromised ATB patients from LTBI individuals were respectively 79.21 and 94.05%. CONCLUSIONS: Our findings suggest that the TBAg/PHA ratio might have some significance for the diagnosis of TB disease in immunocompromised patients.

TIGIT signalling pathway negatively regulates CD4+ T-cell responses in systemic lupus erythematosus.

B-lymphocyte hyperactivity in systemic lupus erythematosus (SLE) is T-cell-dependent, and CD4+ T-cell activation is essential to SLE pathogenesis. However, the mechanism of the deregulation of CD4+ T cells in SLE is largely unknown. T-cell immunoglobulin and ITIM domain (TIGIT) is a new inhibitory receptor preferentially expressed on activated CD4+ T cells. Here, we address the role of TIGIT in the pathogenesis of SLE. Our results showed that TIGIT expression on CD4+ T cells was significantly elevated in patients with SLE and highly correlated with the activity of the disease. TIGIT+ CD4+ T cells from both healthy individuals and patients with SLE had a more activated phenotype than TIGIT- CD4+ T cells. In contrast, the activation, proliferation and cytokine production potential of TIGIT+ CD4+ T cells were significantly lower than those of TIGIT- CD4+ T cells. Furthermore, activation of the TIGIT pathway by using CD155 could substantially down-regulate the activities of CD4+ T cells from SLE patients in vitro, and in vivo administration of CD155 resulted in a delayed development of SLE in MRL/lpr mice. TIGIT is a powerful negative regulator of CD4+ T cells in SLE, which suggests that the TIGIT signalling pathway may be used as a potential therapeutic target for treating this disease.

Establishing the reference intervals of NK cell functions in healthy adults.

Natural killer (NK) cells play a key role in host defense against microbial pathogens. Establishing the reference intervals (RIs) of NK cell functions would be valuable in assessing the immune status of hosts. We evaluated the NK cell activity in healthy adults. We further established and validated the RIs of representative NK cell functions. Flow cytometry was used to evaluate the cytokine production and CD107a degranulation of NK cells. Levels of soluble IFN-γ in the culture supernatants were evaluated by ELISA. Our results demonstrated that the intracellular IFN-γ production of NK cells was positively correlated with CD107a expression and soluble IFN-γ levels. There were no significant differences in NK cell functions between different age and gender groups. The mean values and RIs of representative NK cell functions are as following: IFN-γ(+) NK cells (%): 28.09 (11.3-51.95); CD107a(+) NK cells (%): 17.90 (9.852-27.56); soluble IFN-γ (pg/ml): 330.4 (41.38-717.8). In addition, the intracellular IFN-γ production and degranulation activity of NK cells in patients with colorectal cancer were significantly lower than that in healthy adults. Our study has established the RIs of NK cell functions in healthy adults, which might be used for monitoring the immune status of the hosts.

The source of Mycobacterium tuberculosis-specific IFN-γ production in peripheral blood mononuclear cells of TB patients.

Mycobacterium tuberculosis (Mtb)-specific IFN-γ secretion plays important roles in anti-tuberculosis (TB) immunity. Mtb-specific IFN-γ response can be induced in HIV/TB co-infected patients with a low CD4 lymphocyte count; this suggests that the source of Mtb-specific IFN-γ production is not limited in CD4(+) T lymphocytes. Currently, the major sources of Mtb-specific IFN-γ production and the function and phenotype of Mtb-specific IFN-γ-producing cells still remain unclear. Thirty-nine participants (24 active TB patients, 10 HIV/TB co-infected patients, and 5 healthy volunteers) were recruited according to conventional tests and Mtb-specific IFN-γ ELISPOT assay. Multicolor flow cytometry was used to investigate the production of intracellular IFN-γ in peripheral blood mononuclear cells (PBMCs) after Mtb-specific antigen stimulation. Our results showed that CD4(+), CD8(+) T cells and NK cells are all major sources of Mtb-specific IFN-γ production in PBMCs of TB patients. Moreover, CD8(+) T cells are the highest number of Mtb-specific IFN-γ-producing cells in HIV/TB co-infected patients. Although the activity of NK cells is significantly reduced in TB patients when compared with healthy controls, Mtb-specific antigen stimulation induces a significant increase in NK cell activity. We also showed that CD45RO is the characteristic marker of Mtb-specific IFN-γ-producing T cells but not that of Mtb-specific IFN-γ-producing NK cells in peripheral blood. High expression of CD11a may be the characteristic feature of Mtb-specific IFN-γ-producing NK cells. This study put forward a new insight on the source of antigen-specific IFN-γ-production in PBMCs of TB patients.

TIGIT expression levels on human NK cells correlate with functional heterogeneity among healthy individuals.

Human NK cells display extensive phenotypic and functional heterogeneity among healthy individuals, but the mechanism responsible for this variation is still largely unknown. Here, we show that a novel immune receptor, T-cell immunoglobulin and ITIM domain (TIGIT), is expressed preferentially on human NK cells but shows wide variation in its expression levels among healthy individuals. We found that the TIGIT expression level is related to the phenotypic and functional heterogeneity of NK cells, and that NK cells from healthy individuals can be divided into three categories according to TIGIT expression. NK cells with low levels of TIGIT expression show higher cytokine secretion capability, degranulation activity, and cytotoxic potential than NK cells with high levels of TIGIT expression. Blockade of the TIGIT pathway significantly increased NK-cell function, particularly in NK cells with high levels of TIGIT expression. We further observed that the TIGIT expression level was inversely correlated with the IFN-γ secretion capability of NK cells in patients with cancers and autoimmune diseases. Importantly, we propose a novel mechanism that links TIGIT expression with NK-cell functional heterogeneity, and this mechanism might partially explain why individuals have different susceptibilities to infection, autoimmune disease, and cancer.

Tim-3 pathway affects NK cell impairment in patients with active tuberculosis.

Active tuberculosis (TB) patients show impaired NK cell function, and the underlying mechanism remains largely unknown. In this study, we confirmed the decrease in activation, cytokine secretion, and degranulation potential of NK cells in active TB patients. We further investigated whether coinhibitory receptor Tim-3 was involved with impairment of NK cells. Our results revealed that the expression of Tim-3 on NK cells was increased in active TB patients. Tim-3 expression was inversely correlated with IL-12-stimualted IFN-γ production. Moreover, blocking the Tim-3 pathway restored IFN-γ secretion and degranulation of NK cells. Blocking this pathway also increased NK cell cytotoxicity against K562 target cells, and improved the ability of NK cells to control Mtb growth in monocyte-derived macrophages. The Tim-3 expression on NK cells was also observed to be significantly decreased in TB patients post-treatment. In this study, we have identified that Tim-3 is involved with NK cell impairment in TB patients.