The Ephrin B2 Receptor Tyrosine Kinase Is a Regulator of Proto-oncogene MYC and Molecular Programs Central to Barrett's Neoplasia.

BACKGROUND & AIMS: Mechanisms contributing to the onset and progression of Barrett's (BE)-associated esophageal adenocarcinoma (EAC) remain elusive. Here, we interrogated the major signaling pathways deregulated early in the development of Barrett's neoplasia. METHODS: Whole-transcriptome RNA sequencing analysis was performed in primary BE, EAC, normal esophageal squamous, and gastric biopsy tissues (n = 89). Select pathway components were confirmed by quantitative polymerase chain reaction in an independent cohort of premalignant and malignant biopsy tissues (n = 885). Functional impact of selected pathway was interrogated using transcriptomic, proteomic, and pharmacogenetic analyses in mammalian esophageal organotypic and patient-derived BE/EAC cell line models, in vitro and/or in vivo. RESULTS: The vast majority of primary BE/EAC tissues and cell line models showed hyperactivation of EphB2 signaling. Transcriptomic/proteomic analyses identified EphB2 as an endogenous binding partner of MYC binding protein 2, and an upstream regulator of c-MYC. Knockdown of EphB2 significantly impeded the viability/proliferation of EAC and BE cells in vitro/in vivo. Activation of EphB2 in normal esophageal squamous 3-dimensional organotypes disrupted epithelial maturation and promoted columnar differentiation programs, notably including MYC. EphB2 and MYC showed selective induction in esophageal submucosal glands with acinar ductal metaplasia, and in a porcine model of BE-like esophageal submucosal gland spheroids. Clinically approved inhibitors of MEK, a protein kinase that regulates MYC, effectively suppressed EAC tumor growth in vivo. CONCLUSIONS: The EphB2 signaling is frequently hyperactivated across the BE-EAC continuum. EphB2 is an upstream regulator of MYC, and activation of EphB2-MYC axis likely precedes BE development. Targeting EphB2/MYC could be a promising therapeutic strategy for this often refractory and aggressive cancer.

Loss of CD40 attenuates experimental diabetes-induced retinal inflammation but does not protect mice from electroretinogram defects.

Chronic low grade inflammation is considered to contribute to the development of experimental diabetic retinopathy (DR). We recently demonstrated that lack of CD40 in mice ameliorates the upregulation of inflammatory molecules in the diabetic retina and prevented capillary degeneration, a hallmark of experimental diabetic retinopathy. Herein, we investigated the contribution of CD40 to diabetes-induced reductions in retinal function via the electroretinogram (ERG) to determine if inflammation plays a role in the development of ERG defects associated with diabetes. We demonstrate that diabetic CD40-/- mice are not protected from reduction to the ERG b-wave despite failing to upregulate inflammatory molecules in the retina. Our data therefore supports the hypothesis that retinal dysfunction found in diabetics occurs independent of the induction of inflammatory processes.

Identification of Signaling Pathways by Which CD40 Stimulates Autophagy and Antimicrobial Activity against Toxoplasma gondii in Macrophages.

CD40 is an important stimulator of autophagy and autophagic killing of Toxoplasma gondii in host cells. In contrast to autophagy induced by nutrient deprivation or pattern recognition receptors, less is known about the effects of cell-mediated immunity on Beclin 1 and ULK1, key regulators of autophagy. Here we studied the molecular mechanisms by which CD40 stimulates autophagy in macrophages. CD40 ligation caused biphasic Jun N-terminal protein kinase (JNK) phosphorylation. The second phase of JNK phosphorylation was dependent on autocrine production of tumor necrosis factor alpha (TNF-α). TNF-α and JNK signaling were required for the CD40-induced increase in autophagy. JNK signaling downstream of CD40 caused Ser-87 phosphorylation of Bcl-2 and dissociation between Bcl-2 and Beclin 1, an event known to stimulate the autophagic function of Beclin 1. However, TNF-α alone was unable to stimulate autophagy. CD40 also stimulated autophagy via a pathway that included calcium/calmodulin-dependent kinase kinase ß (CaMKKß), AMP-activated protein kinase (AMPK), and ULK1. CD40 caused AMPK phosphorylation at its activating site, Thr-172. This effect was mediated by CaMKKß and was not impaired by neutralization of TNF-α. CD40 triggered AMPK-dependent Ser-555 phosphorylation of ULK1. CaMKKß, AMPK, and ULK1 were required for CD40-induced increase in autophagy. CD40-mediated autophagic killing of Toxoplasma gondii is known to require TNF-α. Knockdown of JNK, CaMKKß, AMPK, or ULK1 prevented T. gondii killing in CD40-activated macrophages. The second phase of JNK phosphorylation-Bcl-2 phosphorylation-Bcl-2-Beclin 1 dissociation and AMPK phosphorylation-ULK1 phosphorylation occurred simultaneously at ∼4 h post-CD40 stimulation. Thus, CaMKKß and TNF-α are upstream molecules by which CD40 acts on ULK1 and Beclin 1 to stimulate autophagy and killing of T. gondii.

Long-term clinical and echocardiographic outcomes of extensive septal myectomy for hypertrophic obstructive cardiomyopathy in Chinese patients.

BACKGROUND: There has been limited data addressing outcomes of extensive septal myectomy in Chinese patients with hypertrophic obstructive cardiomyopathy (HOCM). In this study, the objective was to evaluate the clinical and echocardiographic outcomes of extensive septal myectomy in a relative large number of Chinese HOCM patients over long-term follow-up. METHODS: We retrospectively studied 139 consecutive HOCM patients (age 43 ± 15 years, 37 % male) who underwent extensive left ventricular septal myectomy. During the perioperative period, all patients were examined by echocardiography. All-cause death and cardiac death were considered as primary endpoints during follow-up. Perioperative data was obtained by retrospective review of institutional surgical databases. Follow-up data of echocardiography and clinical status was recorded through outpatient interview. RESULTS: Perioperative events consisted of arrhythmia, retraction injury to aortic valve leaflets, pleural effusion, and hemodialysis and the use of intra-aortic balloon pump. There was no in-hospital mortality. The follow-up period averaged 5.6 ± 0.9 years and overall survivals were 100.0, 99.3, 99.3, 98.5 and 97.8 % at 1, 2, 3, 4 and 5 years, respectively. Left ventricular outflow tract (LVOT) gradient decreased form preoperative 84 ± 17 mmHg to 12 ± 3 mmHg at 2.5 years after surgery and it further reduced to 6 ± 3 mmHg at 5 years after surgery (P < 0.05). Compared with the preoperative levels, interventricualr septal thickness decreased by 32 % while diastole left ventricular inner diameter approximately increased by 10 % and ejection fraction (EF) was significantly elevated during follow-up (P < 0.05). By echocardiography detection, mitral regurgitation was ameliorated for HOCM patients after surgery. There was significant improvement in New York Heart Association (NYHA) class. The proportion of NYHA III and IV decreased from preoperative 58 to 19 % at 2.5 years after surgery and it reduced to 11 % at 5 years after operation. CONCLUSION: Extensive septal myectomy offers minimal operative risk and provides long-term relief for LVOT obstruction in Chinese HOCM patients.

[Investigation of the arsenic levels in ecosystem aspect in water type of endemic arsenicosis area in Datong City].

OBJECTIVE: To investigate the arsenic levels in endemic arsenism in Datong City, Shanxi Province. METHODS: A total of 85 inhabitants from one village in endemic arsenism area in Datong City, Shanxi Province were collected as research subjects. The People's Republic of China health industry standard for endemic arsenism was used to identify and diagnosis the patients. Daily drinking water and soil were collected and detected by atomic fluorescence spectrometry. The content of vegetables were detected by inductively coupled plasma mass spectrometry (ICP-MS). RESULTS: In the study, 85 samples were collected. Arsenic concentration in the daily drinking water were 14.41 - 90.34 µg/L, and the median value was 43.88 µg/L. The arsenic concentration of vegetables were 0.001 - 0.771 mg/kg, and 43.04% of samples, were higher than the maximal permissible limit of As in food. The results that the arsenic concentration of vegetables constant changes in the leaf vegetables > tubers > fruit vegetables. The health risk of intaking arsenic pollution in vegetables up to 71.77%. The arsenic levels in village of four directions were not exceeded the Chinese standards. CONCLUSIONS: Arsenic concentration in drinking water and vegetables are high in waterborn endemic arsenicosis area of Shanxi province. Arsenic in drinking water has been considered as a primary cause of arsenism, but direct intake of arsenic from vegetables can not be ignored.

CD40 promotes the development of early diabetic retinopathy in mice.

AIMS/HYPOTHESIS: Microangiopathy is a leading complication of diabetes that commonly affects the retina. Degenerate capillaries are a central feature of diabetic retinopathy. An inflammatory process has been linked to the development of diabetic retinopathy but its regulation is incompletely understood. Cluster of differentiation (CD) 40 is a member of the TNF receptor superfamily that promotes the development of certain inflammatory disorders. The role of CD40 in diabetic microangiopathy is unknown. METHODS: B6 and Cd40−/− mice were administered streptozotocin to induce diabetes. Leucostasis was assessed using fluorescein isothiocyanate-conjugated concanavalin A. Retinal Icam1 and Cd40 mRNA levels were examined using real-time PCR. Protein nitration was assessed by immunohistochemistry. Histopathology was examined in the retinal vasculature. CD40 expression was assessed by flow cytometry and immunohistochemistry. Intercellular adhesion molecule 1 (ICAM-1) and nitric oxide synthase 2 (NOS2) were examined by immunoblot and/or flow cytometry. Nitric oxide production was examined by immunoblot and Griess reaction. RESULTS: In mouse models of diabetes, Cd40−/− mice exhibited reduced retinal leucostasis and did not develop capillary degeneration in comparison with B6 mice. Diabetic Cd40−/− mice had diminished ICAM-1 upregulation and decreased protein nitration. Cd40 mRNA levels were increased in the retinas of diabetic B6 mice compared with non-diabetic controls. CD40 expression increased in retinal Müller cells, endothelial cells and microglia of diabetic animals. CD40 stimulation upregulated ICAM-1 in retinal endothelial cells and Müller cells. CD40 ligation upregulated NOS2 and nitric oxide production by Müller cells. CONCLUSIONS/INTERPRETATION: CD40-deficient mice were protected fromthe development of diabetic retinopathy. These mice exhibited diminished inflammatory responses linked to diabetic retinopathy. CD40 stimulation of retinal cells triggered these pro-inflammatory responses.

Analysis of hope level and its influencing factors in patients with decompensated cirrhosis.

AIM: The aim of this study was to examine hope level and its influencing factors in patients with decompensated liver cirrhosis. DESIGN: A prospective observational study. METHODS: We selected 93 patients with decompensated liver cirrhosis from a Chinese university hospital based on the inclusion and exclusion criteria. A general information questionnaire and Herth Hope Index were used, and multiple linear regression identified factors associated with the patients' hope level. RESULTS: The participants' average hope level was 32.01 ± 6.14 (moderate). The hope score's highest and lowest dimensions were "interconnectedness" (11.29 ± 2.17) and "temporality and future" (10.12 ± 2.28), respectively. Multiple linear regression showed that education level and monthly per capita income were independent influencing factors (p < .05). These variables explained 38.3% of the variation in hope. CONCLUSION: The participants' hope level was not optimal. Thus, medical staff should pay special attention to patients with low education level and poor economic status, and guide them to adopt a positive attitude.

Diagnostic Value of Echocardiography Combined with Serum h-FABP and cTnI in Myocardial Infarction and Its Evaluation Value in Left Ventricular Function.

Objective: To explore the value of color Doppler echocardiography (CDE) combined with serum heart-type fatty acid-binding protein (h-FABP) and cardiac troponin I (cTnI) in the diagnosis of myocardial infarction and its evaluation value in left ventricular function. Methods: A total of 44 patients with myocardial infarction who were treated in Cangzhou Central Hospital from October 2018 to February 2020 were included in the observation group, and 45 healthy subjects were included in the control group. The serum h-FABP and cTnI levels of the two groups were compared and analyzed. The coincidence rate of echocardiography plus serum h-FABP and cTnI for single diagnosis and combined diagnosis was analyzed. The left ventricular function indexes of patients with myocardial infarction in different cardiac function grades were compared, including left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular end-systolic volume (LVESV), left ventricular end-diastolic volume (LVEDV), stroke volume (SV), cardiac index (CI), and the ratio of peak velocity blood flow from left ventricular relaxation in early diastole to peak velocity flow in late diastole (E/A). The value of echocardiography combined with serum h-FABP and cTnI in the left ventricular function in patients with myocardial infarction was analyzed. Results: The levels of serum h-FABP and cTnI in the observation group were significantly higher than those in the control group (P < 0.05). CDE plus serum h-FABP and cTnI was associated with significantly higher sensitivity, specificity, and accuracy in diagnosing myocardial infarction versus single detection (P < 0.05). The LVEDV, SV, and CI parameters were similar in patients with different cardiac function grades (P > 0.05). Compared with cardiac function grades I and II, the level of LVEF in patients with myocardial infarction in grades III and IV of cardiac function decreased, while the levels of LVEDD, LVESD, LVESV, and E/A increased (P < 0.05). The levels of serum h-FABP and cTnI in patients with myocardial infarction increased with the increase of cardiac function grades (P < 0.05). Conclusion: Patients with myocardial infarction show high levels of h-FABP and cTnI, and CDE plus the detection of serum h-FABP and cTnI levels can significantly improve the detection accuracy and effectively evaluate the left ventricular function of patients with myocardial infarction, with a certain predictive value for cardiac function grading in myocardial infarction.

Evaluation of left ventricular function in patients with heart failure after myocardial infarction by real-time three-dimensional transesophageal echocardiography.

OBJECTIVE: To evaluate the left ventricular function in patients with heart failure (HF) after myocardial infarction (MI) by real-time three-dimensional transesophageal echocardiography (RT-3D-TEE) and explore its correlation with serum cTnI and H-FABP levels. METHODS: The data of 60 HF patients after MI from March 2019 to January 2021 were analyzed retrospectively and included in the research group. According to cardiac function grades, they were assigned to group A (20 cases), group B (20 cases), and group C (20 cases). During the same period, 50 healthy patients were included in the control group. The left atrial diameter (LAD), interventricular septum thickness (IVST), left ventricular posterior wall thickness (LVPWT), left ventricular end-systolic volume (LVESV), left ventricular end-diastolic volume (LVEDV), left ventricular stroke volume (LVSV), and left ventricular ejection fraction (LVEF) of participants were recorded and compared in the four groups. The serum levels H-FABP and cTnI were tested by ELISA. RESULTS: HF patients had poorer left heart structure and lower function and higher serum H-FABP and cTnI levels, as compared to the subjects in control group. Correlation analysis indicated that the cardiac function grade was positively correlated with LVEDV, LVESV, H-FABP, and cTnI, but negatively correlated with LVEF. The serum H-FABP and cTnI levels of HF patients were positively correlated with LVEDV and LVESV, but negatively correlated with LVEF. Logistic regression analysis revealed that cTnI and H-FABP were risk factors for HF, and LVEF was a protective factor for HF. CONCLUSION: Serum H-FABP and cTnI levels in HF patients are correlated with left ventricular function parameters, which presents a close relation to HF. RT-3D-TEE combined with the detection of serum H-FABP and cTnI yields important clinical significance for early diagnosis of HF.

Combined treatment with temozolomide and methoxyamine: blocking apurininc/pyrimidinic site repair coupled with targeting topoisomerase IIalpha.

PURPOSE: Methoxyamine has been shown to potentiate the cytotoxic effect of temozolomide both in vitro and in human tumor xenograft models. We postulate that the enhanced cytotoxicity is mediated by methoxyamine-bound apurininc/pyrimidinic (MX-AP) site, a key lesion formed by the combination of temozolomide and methoxyamine. When located within topoisomerase IIalpha (topo II) cleavage sites in DNA, MX-AP sites act as dual lethal targets, not only functionally disrupting the base excision repair (BER) pathway but also potentially poisoning topo II. EXPERIMENTAL DESIGN: Using oligonucleotide substrates, in which a position-specific MX-AP site is located within topo II cleavage sites, we examined the effect of MX-AP site on both AP endonuclease- and topo II-mediated DNA cleavage in vitro. RESULTS: MX-AP sites were refractory to the catalytic activity of AP endonuclease, indicating their ability to block BER. However, they were cleaved by either purified topo II or nuclear extracts from tumor cells expressing high levels of topo II, suggesting that MX-AP sites stimulate topo II-mediated DNA cleavages. In cells, treatment with temozolomide and methoxyamine increased the expression of topo II and enriched the formation of gammaH2AX foci, which were colocalized with up-regulated topo II, confirming that DNA double-strand breaks marked by gammaH2AX foci are associated with topo II in cells. CONCLUSIONS: Our findings identify a molecular mechanism of cell death whereby MX-AP sites that cumulated in cells due to resistance to BER potentially convert topo II into biotoxins, resulting in enzyme-mediated DNA scission and cell death.

CD40 in Retinal Müller Cells Induces P2X7-Dependent Cytokine Expression in Macrophages/Microglia in Diabetic Mice and Development of Early Experimental Diabetic Retinopathy.

Müller cells and macrophages/microglia are likely important for the development of diabetic retinopathy; however, the interplay between these cells in this disease is not well understood. An inflammatory process is linked to the onset of experimental diabetic retinopathy. CD40 deficiency impairs this process and prevents diabetic retinopathy. Using mice with CD40 expression restricted to Müller cells, we identified a mechanism by which Müller cells trigger proinflammatory cytokine expression in myeloid cells. During diabetes, mice with CD40 expressed in Müller cells upregulated retinal tumor necrosis factor-α (TNF-α), interleukin 1ß (IL-1ß), intracellular adhesion molecule 1 (ICAM-1), and nitric oxide synthase (NOS2), developed leukostasis and capillary degeneration. However, CD40 did not cause TNF-α or IL-1ß secretion in Müller cells. TNF-α was not detected in Müller cells from diabetic mice with CD40+ Müller cells. Rather, TNF-α was upregulated in macrophages/microglia. CD40 ligation in Müller cells triggered phospholipase C-dependent ATP release that caused P2X7-dependent production of TNF-α and IL-1ß by macrophages. P2X7-/- mice and mice treated with a P2X7 inhibitor were protected from diabetes-induced TNF-α, IL-1ß, ICAM-1, and NOS2 upregulation. Our studies indicate that CD40 in Müller cells is sufficient to upregulate retinal inflammatory markers and appears to promote experimental diabetic retinopathy and that Müller cells orchestrate inflammatory responses in myeloid cells through a CD40-ATP-P2X7 pathway.

N-benzoylstaurosporine (PKC412) inhibits Akt kinase inducing apoptosis in multiple myeloma cells.

Multiple myeloma is a clonal malignancy of plasma cells that invariably progresses to a chemoresistant state. The PI3K/Akt pathway mediates signals downstream of several growth factors involved in myeloma pathogenesis, and constitutive activation of Akt was observed in myeloma cells. We now report that a staurosporine derivative, N-benzoylated staurosporine or PKC412, induces cell death in myeloma cell lines (RPMI8226S, U266, MM1S and MM1R) with loss of mitochondrial membrane potential Delta psi m, caspase 3 and PARP cleavage. ZVAD.fmk, but not interleukin-6, rescued these cells from PKC412 effects. Upstream of the mitochondria, PKC412 inhibited Bad phosphorylation and attenuated Akt kinase activity by suppressing its phosphorylation on serine residue in its activation loop. Reduced phosphorylation of downstream Akt substrates GSK3 alpha/beta and FKHR was also noted. Stable transfection of 8226S cells with constitutively active Akt (8226S-myAkt) partially protected against PKC412 cytotoxicity. Primary myeloma cells isolated from refractory myeloma patients (n=4), were equally sensitive to PKC412 treatment. More importantly, PKC412 did not affect CFU-GM or BFU-E colony formation. In summary, our results demonstrate that PKC412 suppresses Akt kinase activation and induces apoptosis in myeloma cell lines, as well as primary resistant cells. PKC412 is an appropriate candidate for novel treatment protocols for multiple myeloma.