Mechano-regulation of Peptide-MHC Class I Conformations Determines TCR Antigen Recognition.

TCRs recognize cognate pMHCs to initiate T cell signaling and adaptive immunity. Mechanical force strengthens TCR-pMHC interactions to elicit agonist-specific catch bonds to trigger TCR signaling, but the underlying dynamic structural mechanism is unclear. We combined steered molecular dynamics (SMD) simulation, single-molecule biophysical approaches, and functional assays to collectively demonstrate that mechanical force induces conformational changes in pMHCs to enhance pre-existing contacts and activates new interactions at the TCR-pMHC binding interface to resist bond dissociation under force, resulting in TCR-pMHC catch bonds and T cell activation. Intriguingly, cancer-associated somatic mutations in HLA-A2 that may restrict these conformational changes suppressed TCR-pMHC catch bonds. Structural analysis also indicated that HLA polymorphism might alter the equilibrium of these conformational changes. Our findings not only reveal critical roles of force-induced conformational changes in pMHCs for activating TCR-pMHC catch bonds but also have implications for T cell-based immunotherapy.

NKG2D discriminates diverse ligands through selectively mechano-regulated ligand conformational changes.

Stimulatory immune receptor NKG2D binds diverse ligands to elicit differential anti-tumor and anti-virus immune responses. Two conflicting degeneracy recognition models based on static crystal structures and in-solution binding affinities have been considered for almost two decades. Whether and how NKG2D recognizes and discriminates diverse ligands still remain unclear. Using live-cell-based single-molecule biomechanical assay, we characterized the in situ binding kinetics of NKG2D interacting with different ligands in the absence or presence of mechanical force. We found that mechanical force application selectively prolonged NKG2D interaction lifetimes with the ligands MICA and MICB, but not with ULBPs, and that force-strengthened binding is much more pronounced for MICA than for other ligands. We also integrated steered molecular dynamics simulations and mutagenesis to reveal force-induced rotational conformational changes of MICA, involving formation of additional hydrogen bonds on its binding interface with NKG2D, impeding MICA dissociation under force. We further provided a kinetic triggering model to reveal that force-dependent affinity determines NKG2D ligand discrimination and its downstream NK cell activation. Together, our results demonstrate that NKG2D has a discrimination power to recognize different ligands, which depends on selective mechanical force-induced ligand conformational changes.

[Tissue distribution of Qingfei Paidu Decoction based on HPLC-MS/MS].

The tissue distribution of Qingfei Paidu Decoction was studied by HPLC-MS/MS in vivo. Hypersil GOLD C_(18) column(2.1 mm×50 mm, 1.9 µm) was used for gradient elution with acetonitrile as the mobile phase A and 0.1% formic acid solution as the mobile phase B. High-resolution liquid chromatography-mass spectrometry in both positive and negative ion scanning mode and multiple response monitoring(MRM) mode was employed to analyze the behaviors of the active components of Qingfei Paidu Decoction in diffe-rent tissues. The results showed that 19, 9, 17, 14, 22, 19, 24, and 2 compounds were detected in plasma, heart, liver, spleen, lung, kidney, large intestine, and brain, respectively. The compounds belonged to 8 groups, covering 14 herbs in the prescription. After administration with Qingfei Paidu Decoction, the compounds were rapidly distributed in various tissues, especially in the lung, liver, large intestine, and kidney. The majority of the compounds displayed secondary distribution. This study comprehensively analyzed the distribution rules of the main active components in Qingfei Paidu Decoction and provided a basis for the clinical application.

[Determination of neohesperidin and naringin in Qingfei Paidu Granules by RP-HPLC and their transfer rates in preparation process].

The present study established an RP-HPLC method for simultaneous determination of two active components in Qingfei Paidu Granules and investigated the transfer rates of neohesperidin and naringin in the preparation process to provide references for improving the quality control standard and production of Qingfei Paidu Granules.RP-HPLC was performed on a YMC Triart C_(18) column(4.6 mm×150 mm, 5 µm)with column temperature of 30 ℃, acetonitrile(A) and 0.2% phosphoric acid solution(B) as mobile phases for gradient elution at a flow rate of 1.0 mL·min~(-1) and detection wavelength of 284 nm.Good linearity was observed for naringin at 0.10-1.0 µg(R~2=0.999 9) and neohesperidin at 0.12-1.2 µg(R~2=0.999 9).The average recovery of naringin was 99.52% with an RSD of 1.2%, and that of neohesperidin was 100.8% with an RSD of 1.2%.The transfer rates of naringin and neohesperidin between medicinal materials, extracts, concentrates, and granules were measured by this method.The average transfer rate of naringin from medicinal materials to granules was 54.89%±4.38%, and that of neohesperidin was 57.63%±5.88%.The process from medicinal materials to extracts was presumedly the key link affecting the whole preparation process.The established method is simple and sensitive and can be adopted for the quality control of Qingfei Paidu Granules.Meanwhile, it can be used to investigate the transfer rate of neohesperidin and naringin in the preparation of Qingfei Paidu Granules, and further improve the quality control standard of Aurantii Fructus Immaturus in Qingfei Paidu Granules.

Measurement of the Lid Margin Thickness in Meibomian Gland Dysfunction with Vernier Micrometer.

INTRODUCTION: To investigate the lid margin thickness (LMT) from the posterior lash line to the mucocutaneous junction at the middle position in adults with and without meibomian gland dysfunction (MGD) by vernier micrometer (VM). METHODS: This is a cross-sectional, observational study. A hundred eyes from 100 volunteers aged 20 to 79, including 56 normal participants and 44 participants with MGD, were recruited. Measurements of the LMT by VM were performed by the same person. RESULTS: The mean age of 56 normal subjects (24 males and 32 females) and 44 MGD subjects (16 males and 28 females) was 40.0 ± 13.2 years and 42.7 ± 17.1 years, respectively. There was a significant difference in the upper LMT between normal and MGD subjects (1.36 ± 0.25 vs. 1.60 ± 0.27 mm, P < 0.001), but not in the lower LMT (1.0 ± 0.23 vs. 1.10 ± 0.28 mm, P = 0.07). In both normal and MGD subjects, the upper or lower LMT was significantly positively correlated with age (P < 0.05), and the upper LMT was greater than the lower LMT (P < 0.001). In addition, the lower LMT in MGD subjects was significantly positively correlated with meibum expressibility (rs = 0.35, P = 0.02). CONCLUSIONS: The LMT was closely related to age and could be an important indicator for detecting MGD. Furthermore, we found that the upper LMT was greater than the lower LMT, and the lower LMT in MGD subjects seemed to be related to meibum expressibility.

Surface-Anchored Nanogel Coating Endows Stem Cells with Stress Resistance and Reparative Potency via Turning Down the Cytokine-Receptor Binding Pathways.

Stem cell-based therapy has great potential in regenerative medicine. However, the survival and engraftment rates of transplanted stem cells in disease regions are poor and limit the effectiveness of cell therapy due to the fragility of stem cells. Here, an approach involving a single-cell coating of surface-anchored nanogel to regulate stem cell fate with anti-apoptosis capacity in the hypoxic and ischemic environment of infarcted hearts is developed for the first time. A polysialic acid-based system is used to anchor microbial transglutaminase to the external surface of the cell membrane, where it catalyzes the crosslinking of gelatin. The single-cell coating with surface-anchored nanogel endows mesenchymal stem cells (MSCs) with stress resistance by blocking the activity of apoptotic cytokines including the binding of tumor necrosis factor α (TNFα) to tumor necrosis factor receptor, which in turn maintains mitochondrial integrity, function and protects MSCs from TNFα-induces apoptosis. The administration of surface engineered MSCs to hearts results in significant improvements in engraftment, cardiac function, infarct size, and vascularity compared with using uncoated MSCs in treating myocardial infarction. The surface-anchored, biocompatible cell surface engineering with nanogel armor provides a new way to produce robust therapeutic stem cells and may explore immense potentials in cell-based therapy.

ARIH1 signaling promotes anti-tumor immunity by targeting PD-L1 for proteasomal degradation.

Cancer expression of PD-L1 suppresses anti-tumor immunity. PD-L1 has emerged as a remarkable therapeutic target. However, the regulation of PD-L1 degradation is not understood. Here, we identify several compounds as inducers of PD-L1 degradation using a high-throughput drug screen. We find EGFR inhibitors promote PD-L1 ubiquitination and proteasomal degradation following GSK3α-mediated phosphorylation of Ser279/Ser283. We identify ARIH1 as the E3 ubiquitin ligase responsible for targeting PD-L1 to degradation. Overexpression of ARIH1 suppresses tumor growth and promotes cytotoxic T cell activation in wild-type, but not in immunocompromised mice, highlighting the role of ARIH1 in anti-tumor immunity. Moreover, combining EGFR inhibitor ES-072 with anti-CTLA4 immunotherapy results in an additive effect on both tumor growth and cytotoxic T cell activation. Our results delineate a mechanism of PD-L1 degradation and cancer escape from immunity via EGFR-GSK3α-ARIH1 signaling and suggest GSK3α and ARIH1 might be potential drug targets to boost anti-tumor immunity and enhance immunotherapies.

Effect of the Meibomian Gland Squeezer for Treatment of Meibomian Gland Dysfunction.

PURPOSE: To investigate the effect of the meibomian gland squeezer for treatment of meibomian gland dysfunction (MGD). METHODS: Seventy patients (140 eyes) with MGD were randomly divided into 2 groups: 36 patients who were treated by the meibomian gland squeezer as the treatment group and 34 patients were selected as the control group. Patients were evaluated at baseline, and 2-week and 1-month visits for subjective symptoms, objective signs and pain assessments, including ocular symptom scores, Ocular Surface Disease Index, tear breakup time, corneal fluorescein staining, Schirmer scores with no anesthetic (Schirmer I test), meibum quality, meibum expressibility, and Numeric Rating Scale-11. RESULTS: Sixty-five patients were followed in the study, and mean (±SD) age was 57.0 (±12.6) years. Compared with baseline, the 2 groups had varying degrees of improvement in ocular symptom scores and Ocular Surface Disease Index at the 2-week and 1-month visits; there was a statistically significant difference between groups (P < 0.001). At the 1-month visit, the treatment group showed a greater improvement in the breakup time (3.8 ± 1.6 vs. 1.8 ± 1.0 seconds, P < 0.001), corneal fluorescein staining (-2.1 ± 2.13 vs. -0.9 ± 1.3, P = 0.03), Schirmer I test (5.3 ± 2.9 vs. 2.3 ± 2.8 mm, P < 0.001), meibum quality (-7.5 ± 2.9 vs. -5.3 ± 2.4, P = 0.004), and meibum expressibility (-1.2 ± 0.8 vs. -0.7 ± 0.4, P = 0.007). In the treatment group, the mean (±SD) of total pain scores was 2.4 ± 1.0, which indicated that mild pain was still predominant under topical anesthesia. CONCLUSIONS: The meibomian gland squeezer may be safe, effective, and helpful for treatment of MGD and may offer an attractive treatment option for some patients with MGD, although it can cause mild pain or discomfort.

The Expression of Notch/Notch Ligand, IL-35, IL-17, and Th17/Treg in Preeclampsia.

UNLABELLED: The aim of this study was to examine the interaction of Notch/Notch ligand with Th17/Treg, cytokines IL-35 and IL-17 in cases of preeclampsia (PE). METHODS: Peripheral blood was obtained from 42 PE patients and 22 health pregnant women. The mRNA expressions of Notch/Notch ligand, Treg transcription factor FoxP3 and Th17 transcription factor RORγt, EBI3 and P35 (IL-35 two subunits), and IL-17 were determined by qPCR. The serum levels of IL-17 and IL-35 were measured by ELISA. RESULTS: It was observed that the expressions of Foxp3, EBI3, and P35 in PE patients were lower compared with normal pregnancy, whereas the RORγt expression was significantly increased. The results also demonstrated that PE patients exhibited decreased levels of Treg-related cytokine IL-35, whereas IL-17 was significantly increased. PE patients expressed higher levels of Notch receptor (1-4) and Notch ligand of DLL4, whereas Notch ligand of Jagged-1, -2 was much lower. Furthermore, the levels of FoxP3 T cells correlated positively with Jagged-2. In addition, there were positive correlations between the mRNA level of IL-17 and DLL4. CONCLUSION: Our results indicated that maternal immunological changes may reverse maternal tolerance in PE, and this phenomenon may due to the Th17/Treg imbalance affected by Notch/Notch ligand.

[Expression of microRNA-155 and regulative T cell in sepsis patients and their relationship].

OBJECTIVE: To investigate the adjustment effect of microRNA-155 on CD4(+)CD25(+) regulative T cell (Treg) in peripheral blood of sepsis patients, and to elucidate the role of miR-155 in the pathogenesis of sepsis. METHODS: A retrospective study was conducted. 60 sepsis patients (mild n=20, moderate n=20, and severe n=20) from emergency room or intensive care unit (ICU) of the Fourth Hospital of Jiangsu University were enrolled. 20 healthy volunteers were enrolled as controls. Real-time fluorescent quantitation polymerase chain reaction (qRT-PCR) was used to detect the levels of miR-155 and Foxp3 mRNA expressions in peripheral blood. CD4(+)CD25(+) Treg cells in peripheral blood were identified by flow cytometry. Peripheral interleukin-10 (IL-10) was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: The expressions of miR-155, Treg, Foxp3 mRNA and the level of IL-10 were higher in the patients with sepsis than those in healthy control group [Treg: (2.89±1.13)% vs. (2.32±0.91)%, t=10.540, P=0.002; miR-155: 1.19±0.48 vs. 0.80±0.33, t=8.605, P=0.006; Foxp3 mRNA: 0.18±0.08 vs. 0.13±0.03, t=6.862, P=0.008; IL-10: 56.89±17.28 ng/L vs. 33.24±11.93 ng/L, t=12.742, P=0.001]. These variables were elevated gradually with the elevation of acute physiology and chronic health evaluation II (APACHEII) score. The expressions of Treg, miR-155, Foxp3 mRNA and the level of IL-10 were (3.05±1.21)%, 1.36±0.79, 0.21±0.10, (62.82±21.38) ng/L in severe sepsis group; they were (2.86±0.88)%, 1.25±0.56, 0.17±0.08, (56.38±19.65) ng/L in moderate group; they were (2.61±0.87)%, 0.94±0.52, 0.15±0.05, (45.43±14.40) ng/L in mild group. The values showed significant statistical difference among the mild, moderate and severe sepsis groups (all P<0.01). Above values were significantly higher in non-survival group than those in survival group [Treg: (3.46±1.53)% vs. (2.85±1.03)%, t=14.250, P=0.005; miR-155: 1.41±0.85 vs. 1.16±0.76, t=11.875, P=0.006; Foxp3 mRNA: 0.24±0.11 vs. 0.17±0.09, t=8.795,P=0.001; IL-10: 65.47±23.58 ng/L vs. 51.70±16.86 ng/L, t=16.313, P=0.001]. The expression of miR-155 was positively correlated with the expression of CD4(+)CD25(+) Treg and Foxp3 mRNA (r 1=0.635, P 1=0.007; r 2=0.671, P 2=0.005). CONCLUSIONS: The result of this study suggest that miR-155 is involved in the cell proliferation regulation of CD4(+)CD25(+) Treg cells, and play some role in the immunological dissonance in sepsis.

Effects of phacoemulsification on intraocular pressure and anterior chamber depth.

The aim of this study was to investigate the effects of phacoemulsification with intraocular lens (IOL) implantation on intraocular pressure (IOP) and anterior chamber depth (ACD) in patients with cataract or cataract associated with primary angle closure (PAC). A total of 361 patients (481 affected eyes) with senile cataract (cataract group) and 44 patients (52 affected eyes) with cataract associated with PAC (cataract with PAC group) underwent phacoemulsification with IOL implantation from July 2005 to May 2007 and were followed up for 3 to 25 months. There was a significant difference between pre-operative and post-operative IOPs (t=9.270, P<0.01) in the cataract group and in the cataract with PAC group (t=3.29, P<0.01). No significant differences were identified in pre-operative IOP (t=-2.437, P>0.05) and the IOP three months after surgery (t=2.154, P>0.05) between the two groups. There was a significant difference between the pre-operative and post-operative ACDs (t=7.781, P<0.01) in the cataract group and in the cataract with PAC group (t=4.528, P<0.01). A significant difference in ACD between the two groups (t=8.325, P<0.01) existed prior to surgery but following surgery, the ACDs of the two groups were not significantly different (t=2.86, P>0.05). Phacoemulsification with IOL implantation has IOP-lowering effects on cataract and cataract with PAC patients. The International Society of Geography and Epidemiology of Ophthalmology classification method for angle closure glaucoma was adopted in our study. Furhter studies are required to prove the safety and mechanism of lowering IOP impact of phacoemulsifation towards PAC glaucoma (PACG).