Increased Th17 activation and gut microbiota diversity are associated with pembrolizumab-triggered tuberculosis.

INTRODUCTION: A hypersensitivity response akin to immune reconstitution inflammatory syndrome (IRIS) has been proposed as a mechanism responsible for anti-PD-1 therapy-induced tuberculosis. IRIS is associated with enhanced activation of IL-17A-expressing CD4 + T cells (Th17). Gut microbiota is thought to be linked to pulmonary inflammation through the gut-lung axis. MATERIALS AND METHODS: We used ImmuCellAI to investigate the T cell population in lung cancer and tuberculosis samples. Then, we applied flow cytometry to monitor the expression levels of the Th17 cell activation marker CD38 in the peripheral blood of a patient experiencing adverse events, including tuberculosis, in response to pembrolizumab. The gut microbiome was examined by 16S rRNA sequencing to examine the alterations caused by pembrolizumab. RESULTS: The percentage of Th17 cells was increased in both lung cancer and tuberculosis. FACS analysis showed that pembrolizumab induced substantial CD38 expression in Th17 cells. The patient's fecal samples showed that the diversity of the gut microbiota was significantly increased in response to the pembrolizumab cycle. One enriched genus was Prevotella, which has previously been linked to lung inflammation and Th17 immune activation. DISCUSSION: The observed Th17 activation in our patient was consistent with a role of Th17-mediated IRIS in pembrolizumab-triggered tuberculosis. Pembrolizumab might trigger airway inflammation with a Th17 phenotype through microbiota interactions in the gut-lung axis.

Association of Overlapped and Un-overlapped Comorbidities with COVID-19 Severity and Treatment Outcomes: A Retrospective Cohort Study from Nine Provinces in China.

OBJECTIVE: Several COVID-19 patients have overlapping comorbidities. The independent role of each component contributing to the risk of COVID-19 is unknown, and how some non-cardiometabolic comorbidities affect the risk of COVID-19 remains unclear. METHODS: A retrospective follow-up design was adopted. A total of 1,160 laboratory-confirmed patients were enrolled from nine provinces in China. Data on comorbidities were obtained from the patients' medical records. Multivariable logistic regression models were used to estimate the odds ratio ( OR) and 95% confidence interval (95% CI) of the associations between comorbidities (cardiometabolic or non-cardiometabolic diseases), clinical severity, and treatment outcomes of COVID-19. RESULTS: Overall, 158 (13.6%) patients were diagnosed with severe illness and 32 (2.7%) had unfavorable outcomes. Hypertension (2.87, 1.30-6.32), type 2 diabetes (T2DM) (3.57, 2.32-5.49), cardiovascular disease (CVD) (3.78, 1.81-7.89), fatty liver disease (7.53, 1.96-28.96), hyperlipidemia (2.15, 1.26-3.67), other lung diseases (6.00, 3.01-11.96), and electrolyte imbalance (10.40, 3.00-26.10) were independently linked to increased odds of being severely ill. T2DM (6.07, 2.89-12.75), CVD (8.47, 6.03-11.89), and electrolyte imbalance (19.44, 11.47-32.96) were also strong predictors of unfavorable outcomes. Women with comorbidities were more likely to have severe disease on admission (5.46, 3.25-9.19), while men with comorbidities were more likely to have unfavorable treatment outcomes (6.58, 1.46-29.64) within two weeks. CONCLUSION: Besides hypertension, diabetes, and CVD, fatty liver disease, hyperlipidemia, other lung diseases, and electrolyte imbalance were independent risk factors for COVID-19 severity and poor treatment outcome. Women with comorbidities were more likely to have severe disease, while men with comorbidities were more likely to have unfavorable treatment outcomes.

On-line immobilized trypsin microreactor for evaluating inhibitory activity of phenolic acids by capillary electrophoresis and molecular docking.

Phenolic acids, which are important aromatic secondary metabolites, are widely distributed in plant foods. In this study, a simple, economical and fast on-line immobilized trypsin microreactor was developed for evaluating the inhibitory activity of phenolic acids by capillary electrophoresis. The Michaelis-Menten constant (Km) of immobilized trypsin was determined as 0.99 mM, and the half-maximal inhibitory concentration (IC50) and inhibition constant (Ki) of benzamidine were measured as 3.39 and 1.68 mM, respectively. Then, the developed strategy was applied to investigate the inhibitory activity of six phenolic acids on trypsin. The results showed that gallic acid, caffeic acid and ferulic acid had high inhibitory activity at concentration of 150 µM. Molecular docking results illustrated that gallic acid, caffeic acid and ferulic acid can interact indirectly with the catalytic and substrate-binding sites of trypsin. The developed strategy is an effective tool for evaluating inhibitory activity of phenolic acids on trypsin.

Cupric ion functionalized polydopamine coated magnetic microspheres as solid-phase adsorbent for the extraction of purines in plasma.

In this paper, Cu2+ functionalized Fe3O4@polydopamine core-shell (Fe3O4@PDA@Cu2+) magnetic microspheres were prepared by the chelation between Cu2+ and catechol of polydopamine surface. The synthetic magnetic adsorbent was characterized by Fourier transform infrared spectroscopy, energy dispersive X-ray spectroscopy, vibrating sample magnetometry, thermogravimetric analysis, scanning electron microscope and transmission electron microscopy. Four purines include guanine, adenine, hypoxanthine and xanthine were selected as model compounds to evaluate the applicability of this adsorbent. Several parameters that effected the extraction efficiency, such as extraction time, adsorbent amount, solution pH, ionic strength, eluent type, concentration of eluent and eluent time, were investigated. Under the optimized conditions, good linearity was obtained with correlation coefficients between 0.9983 and 0.9999 for the four analytes, and their LOD and LOQ were 0.42-2.15 ng/mL and 1.41-6.50 ng/mL, respectively. Meanwhile, the RSDs of intra-day and inter-day precision were in the range of 1.43%-5.55% and 4.56%-7.01%, respectively. The spiked recoveries of four purines in real sample were 70.01%-102.42%, indicating this proposed method might have potential applications for the analysis of purines in real samples. In addition, the developed method was used to monitor the concentrations of adenine in rat plasma at different time points after intragastric administration.

Extraction of nucleobases, nucleosides and nucleotides by employing a magnetized graphene oxide functionalized with hydrophilic phytic acid and titanium(IV) ions.

A magnetite@graphene oxide nanocomposite was first coated with polyethylenimine and then modified with phytic acid and titanium(IV) ions. The high loading with Ti(IV) and the good hydrophilicity of PEI and PA result in a material that can be applied to the efficient extraction of highly polar nucleobases, nucleosides and nucleotides. The physicochemical properties of the composite were investigated by scanning electron microscopy, transmission electron microscopy, energy dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, water contact angle measurements, thermogravimetric analysis, and vibrating sample magnetometry. A series of parameters that affect extraction and elution under the conditions of immobilized metal affinity chromatography (IMAC) and hydrophilic interaction liquid chromatography (HILIC) were examined. The analytes were eluted from the nanocomposites using 10 mM trisodium phosphate as the elution solution in the IMAC mode, and 50% methanol-water as elution solution in the HILIC mode. Figures of merit include (a) an intra-day precision of 0.1-1.0% in the IMAC mode; (b) an intra-day precision of 0.4%-0.8% in the HILIC mode; (c) detection limits between 1.8-2.8 ng mL-1 in the IMAC mode; and (d) detection limits of 4.0-10.5 ng mL-1 in the HILIC mode. The method was applied to the extraction of the nucleotides cytidine-5'-monophosphate (CMP), uridine-5'-monophosphate (UMP), guanosine-5'-monophosphate (GMP), and adenosine-5'-monophosphate (AMP), and the nucleobases and nucleosides hypoxanthine, adenosine, cytosine, inosine and cytidine from Cordyceps sinensis, Lentinus edodes and plasma samples. Graphical abstract Schematic presentation of the workflow for the extraction of nucleobases, nucleosides and nucleotides using phytic acid-Ti(IV) functionalized magnetite@graphene oxide nanocomposites under two distinct modes.

Evaluation of thrombin inhibitory activity of catechins by online capillary electrophoresis-based immobilized enzyme microreactor and molecular docking.

An online capillary electrophoresis (CE)-based thrombin (THR) immobilized enzyme microreactor (IMER) method was established to screen THR inhibitors in this study. S-2366 was used as chromogenic substrate for determination of THR activity and other kinetic constants. After continuously run for 50 times, the prepared IMER could still remain 89% of the initial immobilized enzyme activity. The Michaelis-Menten constant (Km) of immobilized THR was measured as 0.514 mmol/L and the half-maximal inhibitory concentration (IC50) and inhibition constant (Ki) of argatroban on THR were determined as 78.07 and 26.53 nmol/L, respectively, which indicated that CE-based THR IMER was successfully established and could be applied to screen THR inhibitors. Then the prepared IMER was used to investigate the inhibitory potency on THR of four main catechins in green tea including epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG). The results showed that ECG and EGCG had good THR inhibition activity and their inhibition rates at concentration of 200 µmol/L were 53.2 ±â€¯3.8% and 55.8 ±â€¯2.6%, respectively, which was in consistent with the results of microplate reader assay. Additionally, molecular docking results showed that the benzopyran groups of ECG and EGCG were inserted into the THR active pocket and interacted with residues LYS60F, TRP60D, TRY60A, IEU99, GLY216, HIS57 and SER195, but EC and EGC did not. Therefore, the developed CE-based THR IMER is reliable method for measuring THR inhibitory activity of natural inhibitors.

Evaluation of interactions between RAW264.7 macrophages and small molecules by capillary electrophoresis.

In this study, the affinity interactions between RAW 264.7 macrophages and three small molecules including naringin, oleuropein and paeoniflorin were evaluated by affinity capillary electrophoresis (ACE), partial filling affinity capillary electrophoresis (PFACE) and frontal analysis capillary electrophoresis (FACE), respectively. The result indicated that ACE (varying concentrations of cell suspension were filled in the capillary as receptor) may not be suitable for the evaluation of interactions between cell and small molecules due to the high viscosity of cell suspension; PFACE can qualitatively evaluate the interaction, but the difference in viscosity between RAW264.7 suspension and buffer effects on the liner relationship between filling length and injection time, which makes the calculation of binding constant difficult. Furthermore, based on the PFACE results, naringin showed stronger interaction with macrophages than the other two molecules; taking advantage of the aggregation phenomenon of cell induced by electric field, FACE was successfully used to determine the stoichiometry (n = 5×109 ) and binding constant (Kb = 1×104 L/mol) of the interaction between RAW264.7 and naringin.

Evaluation of affinity interaction between small molecules and platelets by open tubular affinity capillary electrochromatography.

In this paper, an open tubular affinity capillary electrochromatography (OT-ACEC) was developed by physical adsorption of rabbit platelets on the inner surface of capillary. The interactions between small molecules include adenosine diphosphate (ADP) (positive control), protocatechuic acid (negative control) and seven natural products (salvianolic acid B, salvianic acid A sodium, hydroxysafflor yellow A, ferulic acid, chlorogenic acid, sinapic acid, caffeic acid) and platelets were evaluated by their retention factors and binding constants obtained based on peak-shift assay. Then, the activities of anti-platelet aggregation induced by thrombin (THR), ADP and arachidonic acid (AA) for those small molecules (except ADP) were evaluated by turbidimetric method. The results indicate that: (i) ADP, a platelet aggregation inducer, had strong interaction with platelet, while protocatechuic acid that had no inhibition on platelet aggregation behaved no specific interaction; (ii) there was a positive correlation between the anti-platelet aggregation activities of small molecules and their interactions with platelet, generally those compounds with higher binding constants with platelet exhibited higher activities. Therefore, the OT-ACEC method developed in the present study can be a potential method to evaluate affinity interactions between small molecules and platelets, so as to predict the biological activities such as anti-platelet aggregation for the small molecules.