PHE1-based IgG-like antibody platform provides a novel strategy for enhanced T-cell immunotherapy.

Introduction: Bispecific antibodies (BsAbs) can simultaneously target two epitopes of different antigenic targets, bringing possibilities for diversity in antibody drug design and are promising tools for the treatment of cancers and other diseases. T-cell engaging bsAb is an important application of the bispecific antibody, which could promote T cell-mediated tumor cell killing by targeting tumor-associated antigen (TAA) and CD3 at the same time. Methods: This study comprised antibodies purification, Elisa assay for antigen binding, cytotoxicity assays, T cell activation by flow cytometry in vitro and xenogenic tumor model in vivo. Results: We present a novel bsAb platform named PHE-Ig technique to promote cognate heavy chain (HC)-light chain (LC) pairing by replacing the CH1/CL regions of different monoclonal antibodies (mAbs) with the natural A and B chains of PHE1 fragment of Integrin ß2 based on the knob-in-hole (KIH) technology. We had also verified that PHE-Ig technology can be effectively used as a platform to synthesize different desired bsAbs for T-cell immunotherapy. Especially, BCMA×CD3 PHE-Ig bsAbs exhibited robust anti-multiple myeloma (MM) activity in vitro and in vivo. Discussion: Moreover, PHE1 domain was further shortened with D14G and R41S mutations, named PHE-S, and the PHE-S-based BCMA×CD3 bsAbs also showed anti BCMA+ tumor effect in vitro and in vivo, bringing more possibilities for the development and optimization of different bsAbs. To sum up, PHE1-based IgG-like antibody platform for bsAb construction provides a novel strategy for enhanced T-cell immunotherapy.

The role of PALLD-STAT3 interaction in megakaryocyte differentiation and thrombocytopenia treatment.

Impaired differentiation of megakaryocytes constitutes the principal etiology of thrombocytopenia. The signal transducer and activator of transcription 3 (STAT3) is a crucial transcription factor in regulating megakaryocyte differentiation, yet the precise mechanism of its activation remains unclear. PALLD, an actin-associated protein, has been increasingly recognized for its essential functions in multiple biological processes. This study revealed that megakaryocyte/plateletspecific knockout of PALLD in mice exhibited thrombocytopenia due to diminished platelet biogenesis. In megakaryocytes, PALLD deficiency led to impaired proplatelet formation and polyploidization, ultimately weakening their differentiation for platelet production. Mechanistic studies demonstrated that PALLD bound to STAT3 and interacted with its DNA-binding domain (DBD) and Src homology 2 (SH2) domain via Immunoglobulin domain 3 (Ig3). Moreover, the absence of PALLD attenuated STAT3 Y705 phosphorylation and impeded STAT3 nuclear translocation. Based on the PALLD-STAT3 binding sequence, we designed a peptide C-P3, which can facilitate megakaryocyte differentiation and accelerate platelet production in vivo. In conclusion, this study highlights the pivotal role of PALLD in megakaryocyte differentiation and proposes a novel approach for treating thrombocytopenia by targeting the PALLD-STAT3 interaction.

Branched-chain amino acids promote thrombocytopoiesis by activating mTOR signaling.

BACKGROUND: Megakaryocyte differentiation and platelet production disorders are the main causes of thrombocythemia and thrombocytopenia and lead to thrombosis or hemorrhage. Branched-chain amino acids (BCAAs) are essential nutrients that regulate important metabolic signals. BCAA administration could also increase platelet activation and promote the risk of thrombosis. OBJECTIVES: To unveil the role of BCAAs in thrombocytopoiesis. METHODS: BCAA-fed mice and megakaryocyte/platelet-specific branched-chain α-keto acid dehydrogenase E1α subunit-deficient mice were used to study the role of BCAAs in thrombocytopoiesis. RESULTS: In this study, we found that BCAA diet could facilitate megakaryocyte differentiation and platelet production. Meanwhile, megakaryocyte/platelet-specific branched-chain α-keto acid dehydrogenase E1α subunit-deficient mice developed thrombocythemia, which was mainly caused by the excessive differentiation of megakaryocytes and proplatelet biogenesis. Moreover, the use of BT2, the agonist of BCAA catabolism, could affect proplatelet formation (PPF) and megakaryocyte polyploidization, as well as ameliorating the thrombocythemia of BCAA-fed mice. CONCLUSION: We found that deficiency in BCAA catabolism led to the activation of p70S6K/mammalian target of rapamycin (mTOR) signaling, megakaryocyte over differentiation, and the acceleration of PPF. Activating BCAA metabolism with BT2 could inhibit mTOR signaling, reduce PPF, and ameliorate thrombocythemia in BCAA-fed mice. Therefore, this study reveals a novel role of BCAAs in megakaryocyte differentiation and platelet production, suggesting that targeting BCAA-mediated p70S6K/mTOR signaling may be a potential strategy for the treatment of thrombocytopenia or thrombocythemia.

AGK Potentiates Arterial Thrombosis by Affecting Talin-1 and αIIbß3-Mediated Bidirectional Signaling Pathway.

BACKGROUND: AGK (acylglycerol kinase) was first identified as a mitochondrial transmembrane protein that exhibits a lipid kinase function. Recent studies have established that AGK promotes cancer growth and metastasis, enhances glycolytic metabolism and function fitness of CD8+ T cells, or regulates megakaryocyte differentiation. However, the role of AGK in platelet activation and arterial thrombosis remains to be elaborated. METHODS: We performed hematologic analysis using automated hematology analyzer and investigated platelets morphology by transmission electron microscope. We explored the role of AGK in platelet activation and arterial thrombosis utilizing transgenic mice, platelet functional experiments in vitro, and thrombosis models in vivo. We revealed the regulation effect of AGK on Talin-1 by coimmunoprecipitation, mass spectrometry, immunofluorescence, and Western blot. We tested the role of AGK on lipid synthesis of phosphatidic acid/lysophosphatidic acid and thrombin generation by specific Elisa kits. RESULTS: In this study, we found that AGK depletion or AGK mutation had no effect on the platelet average volumes, the platelet microstructures, or the expression levels of the major platelet membrane receptors. However, AGK deficiency or AGK mutation conspicuously decreased multiple aspects of platelet activation, including agonists-induced platelet aggregation, granules secretion, JON/A binding, spreading on Fg (fibrinogen), and clot retraction. AGK deficiency or AGK mutation also obviously delayed arterial thrombus formation but had no effect on tail bleeding time and platelet procoagulant function. Mechanistic investigation revealed that AGK may promote Talin-1Ser425 phosphorylation and affect the αIIbß3-mediated bidirectional signaling pathway. However, AGK does not affect lipid synthesis of phosphatidic acid/lysophosphatidic acid in platelets. CONCLUSIONS: AGK, through its kinase activity, potentiates platelet activation and arterial thrombosis by promoting Talin-1 Ser425 phosphorylation and affecting the αIIbß3-mediated bidirectional signaling pathway.

Correlation between endoscopic resection outcomes and endosonographic findings in gastric tumors with muscularis propria origin.

Endoscopic resection is an effective treatment for subepithelial tumors arising from the muscularis propria layer of the stomach. However, the invasion pattern revealed by the pathological examination of tumor specimens is often not consistent with the findings of preprocedural endoscopic ultrasounds (EUS). We compared the real growing patterns of tumors, as evaluated on histopathological examination, with their EUS images, and analyzed the outcomes of endoscopic resections in relation to the EUS findings. From January 2006 to June 2015, 32 patients underwent endoscopic resection for gastric tumors originating from the muscularis propria at our hospital. We divided the patients into 3 groups according to the location of the tumor as diagnosed using pre procedural EUS: submucosa (group I, n = 5), muscularis propria (group II, n = 14), and tumors extending into the outer cavity (group III, n = 13). Histopathological examination revealed 15 patients with gastrointestinal stromal tumors (GISTs), 14 with leiomyomas, and 3 with schwannomas. Accuracy of EUS in evaluating tumor invasion was 56%. Some tumors in groups I and II was removed by endoscopic submucosal dissection only. Muscular dissection was needed in 10 patients (71%) in group II and 9 patients (69%) in group III. Four patients (31%) in group III were found to have subserosal tumors. The complete resection rate was 88% (23 patients) among patients who underwent endoscopic submucosal dissection and endoscopic muscular dissection, and 67% (4 patients) among patients who underwent endoscopic subserosal dissection (ESSD). The tumor was completely removed in 12 patients (86%) in group II and 10 patients (77%) in group III. EUS accurately predicts the layer of the subepithelial tumor in the stomach; however, the pattern of invasion of surrounding structures is difficult to evaluate using EUS.

Photoaging and release profile of acrylonitrile butadiene styrene microplastics under simulated solar radiation in water.

Microplastics (MPs) pollution has become a major environmental problem and poses a risk to a variety of organisms. In this study, the photoaging behavior of acrylonitrile butadiene styrene microplastics (ABS-MP) in aqueous environment was investigated under simulated solar irradiation. Results showed that the long chains of ABS-MP broke under the light irradiation, and its thermal stability was reduced. ABS-MP was oxidized during photoaging and produced a large number of oxygen-containing functional groups. Structure destruction of ABS-MP decreased the formation of environmentally persistent free radicals (EPFRs) and further photoirradiation generated secondary EPFRs. Nuclear magnetic resonance (NMR) analysis of the aged leachates confirmed that ABS-MP was oxidized and some small molecular fragments were dropped during photoaging. Meanwhile, C-Br bond broke of additive tetrabromobisphenol A (TBBPA) resulting in more bromine released into water and Sb(III) of additive Sb2O3 was oxidized to Sb(V) during photoaging. These findings illustrate the necessity of considering the aging of MPs in natural environment, expand the understanding of the potential harm and fate of MPs in aqueous environment, which is important for the management of MPs.

Endoscopic subserosal dissection for gastric tumors: 18 cases in a single center.

BACKGROUND: Endoscopic submucosal dissection has technical limitations for the resection of gastric epithelial tumors with severe fibrosis and mixed or exophytic gastric subepithelial tumors (SETs). AIMS: To analyze the safety and effectiveness of endoscopic subserosal dissection (ESSD) for tumors growing in the subserosal space. METHODS: This observation study enrolled 18 patients who were diagnosed with gastric tumors and underwent ESSD at the Presbyterian Medical Center from 2010 to March 2019. RESULTS: A total of 18 patients were treated by ESSD. The mean age of the patients was 61.7 years. The mean length of the long axis of the tumor was 18.0 ± 5.0 mm. The mean operation time was 44 (range 11-167) min. The pathologic diagnosis was leiomyoma in nine cases (50.0%), gastrointestinal stromal tumor in six (33.3%), low-grade adenoma with severe fibrosis in two (11.1%), and schwannoma in one (5.6%). There were three cases of perforation, which were treated conservatively. The complete resection rate was 94%, and local or systemic tumor recurrence was not seen in a mean 70 months of follow-up. CONCLUSION: ESSD can be used for epithelial tumors with severe fibrosis and mixed or exophytic gastric SETs in the lesser curvature, cardia, or fundus that cannot be treated using standard methods.

Serum procalcitonin as an independent diagnostic markers of bacteremia in febrile patients with hematologic malignancies.

BACKGROUND: Serum procalcitonin (PCT) and C-reactive protein (CRP) are biomarkers of infection. In patients with hematologic disorders with or without hematopoietic stem cell transplantation (HSCT), it is difficult to distinguish bloodstream infections from aseptic causes of febrile episodes. The objective of this study was to investigate diagnostic values of PCT and CRP in predicting systemic bacterial infection in patients with hematologic malignancies. METHODS: Clinical and laboratory data of 614 febrile episode cases from 511 patients were analyzed. Febrile episodes were classified into four groups: (1) culture-positive bacterial infection by Gram-positive cocci (GPC), (2) culture-positive bacterial infection by Gram-negative bacilli (GNB), (3) fungal infection, and (4) viral infection or a noninfectious etiology. RESULTS: Of 614 febrile cases, systemic bacterial infections were confirmed in 99 (16.1%) febrile episodes, including 38 (6.2%) GPC and 61 (9.9%) GNB infections. PCT levels were significantly higher in GNB infectious episodes than those in febrile episodes caused by fungal infection (0.58 ng/mL (95% CI: 0.26-1.61) vs. 0.22 ng/mL (0.16-0.38), P = 0.047). Bacterial infectious episodes showed higher PCT and CRP levels than non-bacterial events (PCT: 0.49 (0.26-0.93) ng/mL vs. 0.20 (0.18-0.22) ng/mL, P < 0.001; CRP: 76.6 (50.5-92.8) mg/L vs. 58.0 (51.1-66.5) mg/L, P = 0.036). For non-neutropenic febrile episodes, both PCT and CRP discriminated bacteremia from non-bacteremia. However, in neutropenic febrile episodes, PCT only distinguished bacteremia from non-bacteremia. In non-neutropenic episode, both PCT and CRP showed good diagnostic accuracy (AUC: 0.757 vs. 0.763). In febrile neutropenia, only PCT discriminated bacteremia from non-bacterial infection (AUC: 0.624) whereas CRP could not detect bacteremia (AUC: 0.500, 95% CI: 0.439-0.561, P > 0.05). CONCLUSIONS: In this single-center observational study, PCT was more valuable than CRP for discriminating between bacteremia and non-bacteremia independent of neutropenia or HSCT.

Intrachromosomal amplification of chromosome 21 in Korean pediatric patients with B-cell precursor acute lymphoblastic leukemia in a single institution.

BACKGROUND: Intrachromosomal amplification of chromosome 21 (iAMP21), defined as the presence of three or more RUNX1 signals on one marker chromosome, is a distinct cytogenetic subgroup of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) that is known to have a poor prognosis when treated with standard therapy. The aim of this study was to evaluate the clinical characteristics of Korean children with iAMP21. METHODS: The cytogenetic data from BCP-ALL children were reviewed. The ETV6/RUNX1 ES Dual Color Probe was used for fluorescence in situ hybridization (FISH). RESULTS: In total, 295 children were included. Of these, 10 patients (3.4%) had iAMP21. The median age of iAMP21 patients was 9 years, and the median value of white blood cell count was 5.09×109/L. Slow early treatment response was observed more in iAMP21 patients. Patients with iAMP21 had a higher incidence of relapse and worse survival rates. In patients with iAMP21, the estimated 10-year cumulative incidence of relapse was 53.3%. The estimated 10-year event-free survival and overall survival rate were 46.7% and 64.8%, respectively. Most cases of leukemic relapse developed in the late period (median, 43 mo). In multivariate analysis, high risk group was the only factor that had a significant impact on death. CONCLUSION: The existence of iAMP21 was related to delayed treatment response and was likely to affect increased relapse and death in the late period. Further studies are needed to reveal its effect on BCP-ALL treatment outcomes and its role as an independent prognostic factor.

Tetrahydrobiopterin reverse left ventricular hypertrophy and diastolic dysfunction through the PI3K/p-Akt pathway in spontaneously hypertensive rats.

Hypertension induced hypertrophy and diastolic dysfunction and is associated with cardiac oxidation and reduced NO production. We hypothesized that tetrahydrobiopterin (BH4) can regulate the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway and reverse cardiac hypertrophy and diastolic dysfunction in spontaneously hypertensive rats. Ten-week-old male spontaneously hypertensive rats (SHR) and age-matched normotensive control Wistar-Kyoto (WKY) rats were divided into five groups, WKY, WKY + BH4, SHR, SHR + BH4 and SHR + VAL. In SHR, diastolic dysfunction was accompanied by concentric hypertrophy, cardiac oxidation, and reduced cardiac BH4 and NO production. Four-week BH4 and valsartan administration reversed hypertrophy and improved diastolic function. BH4 and valsartan blunted the expression of hypertrophy markers α-skeletal actin (α-SA) and ß-myosin heavy chain (ß-MHC). Only BH4 reduced hypertension and induced myocardial fibrosis and expression of transforming growth factor-ß1 (TGF-ß1). BH4 reduced cardiac oxidant stress and increased NO production. Exogenous BH4 increased phosphorylated Akt levels and increased Bcl-2 expression. In conclusion, less BH4 and reduced NO increases myocardial hypertrophy and cardiac oxidative stress, which exacerbates diastolic dysfunction. Exogenous BH4 ameliorates cardiac hypertrophy and diastolic dysfunction through the PI3K/p-Akt pathway. BH4 may be a potent therapy for hypertension with diastolic dysfunction.

[Effect of Astragali Radix in improving early renal damage in metabolic syndrome rats through ACE2/Mas pathway].

To study the expression of angiotensin converting enzyme 2 (ACE2) and angiotensin (Ang) 1-7 specific receptor Mas protain in renal blood vessels of metabolic syndrome ( MS) rats and its anti-oxidative effect. A total of 80 male SD rats were divided into four groups: the normal control group (NC, the same volume of normal saline), the MS group (high fat diet), the MS + Astragali Radix group (MS + HQ, 6 g x kg(-1) x d(-1) in gavage) and the MS + Valsartan group (MS + XST, 30 mg x kg(-1) x d(-1) in gavage). After four weeks of intervention, their general indexes, biochemical indexes and blood pressure were measured; plasma and renal tissue Ang II, malondialdehyde (MDA) and superoxide demutase (SOD) levels were measured with radioimmunoassay. The protein expressions of Mas receptor, AT1R, ACE and ACE2 were detected by western blot analysis. According to the result, compared with the NC group, the MS group and the MS + HQ group showed significant increases in systolic and diastolic pressures, body weight, fasting glucose, fasting insulin, triglycerides, free fatty acid and Ang II level of MS rats (P < 0.05). The MS + XST group showed notable decreases in systolic and diastolic pressures than that of the MS group. The MS group showed significant increases in the SOD activity and NO level and decrease in the MDA level after being intervened with Astragali Radix. ACE and AT1R protein expressions in renal tissues of the MS group were higher than that in the NC group, but with lower ACE2 and -Mas receptor expressions (all P < 0.05). Compared with the MS group, the MS + HQ group showed significant increase in Mas receptor expression in renal tissues, whereas the MS + XST group showed notable decrease in AT1R (all P < 0.05). In conclusion, Astragali Radix can increase the Mas receptor expressions in renal tissues, decrease ACE expression and change local Ang II, MDA, NO and SOD in kidneys, so as to protect early damages in renal tissues.