Sensitive and effective method with 96-well plate for determination of levamlodipine in human plasma using LC-MS/MS.

we developed an effective protein precipitation method for determination of levamlodipine in human plasma using LC-MS/MS. Sample extraction was carried out by using liquid-liquid extraction in 96-well plate format. (S)-Amlodipine-d4 was used as internal standard (IS). The chromatographic separation was achieved using Philomen Chiral MX (2) column (3 µm, 2.1 × 100 mm). Mobile phase A was comprised of Acetonitrile (ACN), Mono ethanol amine (MEA) and Iso-Propyl alcohol (IPA) (1000:1:10, v/v/v), Mobile phase B was IPA-ACN (2:1, v/v). The flow rate was 0.4 mL/min. The total run time of each sample was 4.0 min with gradient elution. LC-MS/MS spectra were generated in positive ion mode, and multiple reaction monitoring (MRM) was used to detect the following transitions: m/z 409.20 â†’ 238.15 for levamlodipine and 415.25 â†’ 240.20 for (S)-Amlodipine-d4 (the IS). The method was linear from 50 to 10000 pg/mL（R2＝0.9988489）,and the lower limit of quantification (LLOQ) was 50 pg/mL. This method was applied to a bioequivalence study of levamlodipine.

Osa-miR1320 targets the ERF transcription factor OsERF096 to regulate cold tolerance via JA-mediated signaling.

MicroRNAs play key roles in abiotic stress response. Rice (Oryza sativa L.) miR1320 is a species-specific miRNA that contributes to miR168-regulated immunity. However, it is still unknown whether miR1320 is involved in rice response to abiotic stress. In this study, we illustrated that the miR1320 precursor generated two mature miR1320s, miR1320-3p, and miR1320-5p, and they both displayed decreased expression under cold stress. Genetic evidence showed that miR1320 overexpression resulted in increased cold tolerance, while miR1320 knock down (KD) reduced cold tolerance. Furthermore, an APETALA2/ethylene-responsive factor (ERF) transcription factor OsERF096 was identified as a target of miR1320 via 5'-RACE and dual luciferase assays. OsERF096 expression was altered by miR1320 overexpression and KD and exhibited an opposite pattern to that of miR1320 in different tissues and under cold stress. Consistently, OsERF096 negatively regulated cold stress tolerance. Furthermore, we suggested that OsERF096 could bind to the GCC and DRE cis-elements and act as a transcriptional activator in the nucleus. Based on RNA-sequencing and targeted metabolomics assays, we found that OsERF096 modified hormone content and signaling pathways. Finally, phenotypic and reverse transcription-quantitative PCR assays showed that jasmonic acid (JA) methyl ester application recovered the cold-sensitive phenotype and JA-activated expression of three Dehydration Responsive Element Binding genes in the OsERF096-OE line. Taken together, our results strongly suggest that the miR1320-OsERF096 module regulates cold tolerance by repressing the JA-mediated cold signaling pathway.

Predicted Near-Future Oceanic Warming Enhances Mercury Toxicity in Marine Copepods.

The effects of acute mercury exposure (118 µg/L) on the marine copepod Tigriopus japonicus were examined at 22 and 25 °C for 24 h and compared with controls. Mercury accumulation and seven genes related to antioxidant/stress responses were analyzed after exposure. The 24-h LC50 value decreased in the warmer environment and mercury accumulation was elevated. Under both temperatures, mercury significantly affected the expression of all analyzed genes and probably caused oxidative stress. Intriguingly, at the same mercury concentration, most genes were upregulated at the higher relative to the lower temperature, and the copepods likely initiated more compensatory reactions to counteract increased mercury toxicity associated with the warmer temperature. Overall, this study suggests a molecular mechanism by which marine copepods could respond to future oceanic warming and mercury pollution.

N-myristoyltransferase proteins in breast cancer: prognostic relevance and validation as a new drug target.

PURPOSE: N-myristoyltransferases 1 and 2 (NMT1 and NMT2) catalyze the addition of 14-carbon fatty acids to the N-terminus of proteins. Myristoylation regulates numerous membrane-bound signal transduction pathways important in cancer biology and the pan-NMT inhibitor PCLX-001 is approaching clinical development as a cancer therapy. The tissue distribution, relative abundances, and prognostic value of the two human NMTs remain poorly understood. METHODS: We generated and validated mutually exclusive monoclonal antibodies (mAbs) specific to human NMT1 and NMT2. These mAbs were used to perform immunohistochemical analysis of the abundance and distribution of NMT1 and NMT2 in normal breast epithelial samples and a large cohort of primary breast adenocarcinomas from the BCIRG001 clinical trial (n = 706). RESULTS: NMT1 protein was readily quantified in normal and most transformed breast epithelial tissue and was associated with higher overall histologic grade, higher Ki67, and lower hormone receptor expression. While NMT2 protein was readily detected in normal breast epithelial tissue, it was undetectable in the majority of breast cancers. Detectable NMT2 protein correlated with significantly poorer overall survival (hazard ratio 1.36; P = 0.029) and worse biological features including younger age, higher histologic grade, lower hormone receptor expression, higher Ki67, and p53 positivity. Treatment of cultured breast cancer cells with PCLX-001 reduced cell viability in vitro. Daily oral administration of PCLX-001 to immunodeficient mice bearing human MDA-MB-231 breast cancer xenografts produced significant dose-dependent tumor growth inhibition in vivo. CONCLUSIONS: These results support further evaluation of NMT immunohistochemistry for patient selection and clinical trials of NMT inhibition in breast cancer patients.

Tumor-induced inflammation in mammary adipose tissue stimulates a vicious cycle of autotaxin expression and breast cancer progression.

Compared to normal tissues, many cancer cells overexpress autotaxin (ATX). This secreted enzyme produces extracellular lysophosphatidate, which signals through 6 GPCRs to drive cancer progression. Our previous work showed that ATX inhibition decreases 4T1 breast tumor growth in BALB/c mice by 60% for about 11 d. However, 4T1 cells do not produce significant ATX. Instead, the ATX is produced by adjacent mammary adipose tissue. We investigated the molecular basis of this interaction in human and mouse breast tumors. Inflammatory mediators secreted by breast cancer cells increased ATX production in adipose tissue. The increased lysophosphatidate signaling further increased inflammatory mediator production in adipose tissue and tumors. Blocking ATX activity in mice bearing 4T1 tumors with 10 mg/kg/d ONO-8430506 (a competitive ATX inhibitor, IC90 = 100 nM; Ono Pharma Co., Ltd., Osaka, Japan) broke this vicious inflammatory cycle by decreasing 20 inflammatory mediators by 1.5-8-fold in cancer-inflamed adipose tissue. There was no significant decrease in inflammatory mediator levels in fat pads that did not bear tumors. ONO-8430506 also decreased plasma TNF-α and G-CSF cytokine levels by >70% and leukocyte infiltration in breast tumors and adjacent adipose tissue by >50%. Hence, blocking tumor-driven inflammation by ATX inhibition is effective in decreasing tumor growth in breast cancers where the cancer cells express negligible ATX.

Liver, biliary and pancreatic injuries in pancreaticobiliary maljunction model in cats.

BACKGROUND: Pancreaticobiliary maljunction is a high risk factor of pancreatitis and biliary tract cancer. How this maljunction affects the liver remains obscure. This study aimed to examine the effects of pancreaticobiliary maljunction on the liver, pancreas and gallbladder in a cat model. METHODS: A model of choledocho-pancreatic side-to-side ductal anastomosis was created in ten cats. Before the procedure, a small piece of tissue from the liver, pancreas and gallbladder was collected as a control. The common channel formation was checked by cholecystography. The livers, pancreases and gallbladders of these cats were harvested for histological examination. The expression of proliferating cell nuclear antigen in the gallbladder was examined with immunohistochemistry. RESULTS: Seven of the 10 cats survived for 6 months after surgery. The color of the liver was darker in the PBM model than the control specimen, with nodules on the surface. Histological examination showed ballooning changes and inflammatory infiltrations and the histopathological score increased significantly (P<0.05). Also, mitochondria swelling and lipid droplet in cytoplasm were observed under an electron microscope. The pancreas also appeared darker in the PBM model than the control specimen and dilated pancreatic ducts were found in three cats. Histopathological examination revealed vascular proliferation and inflammatory infiltration with numerous neutrophils. Gallbladder epithelial cells were featured by expanded endoplasmic reticulum, increased intercellular space and cellular nucleus deformation. The positive cells of proliferating cell nuclear antigen were increased significantly (P<0.05). CONCLUSION: The present study demonstrated that pancreaticobiliary maljunction can lead to the injuries of the liver, pancreas and gallbladder.

Ghrelin protects rats against traumatic brain injury and hemorrhagic shock through upregulation of UCP2.

OBJECTIVE: To determine the mechanism responsible for ghrelin's neuroprotective effects after traumatic brain injury (TBI) and hemorrhagic shock. BACKGROUND: Ghrelin, a gastrointestinal hormone, has been demonstrated to possess multiple functions, including upregulation of uncoupling protein 2 (UCP2) and stimulation of the vagus nerve. Recent evidence has indicated that ghrelin is neuroprotective. We, therefore, hypothesized that ghrelin protects rats against TBI and hemorrhagic shock through upregulation of UCP2, involving stimulation of the vagus nerve. METHODS: Brain injury was induced by dropping a 450 g of weight from 1.5 m onto a steel helmet attached to the skull of male adult rats. Immediately after TBI, a midline laparotomy was performed, and both lumbar veins were isolated and severed at the junction with the vena cava. The abdomen was kept open for 20 minutes. At 45 minutes after TBI and uncontrolled hemorrhage (UH), ghrelin (4, 8, or 16 nmol/rat) or 1 mL of normal saline (vehicle) was intravenously administered. The Neurological Severity Scale (NSS), morphological alterations and ß-amyloid precursor protein expression in the brain, systemic organ injury markers (ie, alanine aminotransferase, aspartate aminotransferase, and lactate), and UCP2 expression in the cortex were measured. To determine whether the protective effect of ghrelin is mediated through upregulation of UCP2, genipin, a specific UCP2 antagonist, was administered intravenously before the injection of ghrelin in animals with TBI and UH. The role of the vagus nerve was assessed by performing vagotomy immediately before ghrelin administration. RESULTS: Ghrelin attenuated brain injury and facilitated functional recovery after TBI and UH. Ghrelin increased UCP2 expression in the cortex, and administration of genipin abolished ghrelin's protection after TBI and UH. Furthermore, vagotomy prevented the beneficial effects of ghrelin and eliminated ghrelin-induced UCP2 upregulation after TBI and UH. CONCLUSIONS: The protective effects of ghrelin after TBI and UH seem to be related to upregulation of UCP2 expression in the brain and requiring the intact vagus nerve.

Activation of peroxisome proliferator-activated receptor ß/δ attenuates acute ischemic stroke on middle cerebral ischemia occlusion in rats.

BACKGROUND: Peroxisome proliferator-activated receptor (PPAR)-ß/δ is a transcription factor that belongs to the nuclear hormone receptor family. There is little information about the effects of the immediate administration of specific ligands of PPAR-ß/δ (GW0742) in animal models of acute ischemic stroke. Using a rat model of middle cerebral ischemia occlusion (MCAO) in vivo, we have investigated the effect of pretreatment with GW0742 before MCAO. METHODS: The neuroprotective effect of GW0742 against acute ischemic stroke was evaluated by the neurologic deficit score (NDS), dry-wet weight, and 2,3,5-triphenyltetrazolium chloride staining. The levels of interleukin (IL)-1ß, nuclear factor (NF)-κB, and tumor necrosis factor (TNF)-α were detected by an enzyme-linked immunosorbent assay. The expressions of inducible nitric oxide synthase (iNOS), Bax, and Bcl-2 were detected by Western blot. The apoptotic cells were counted by in situ terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assay. RESULTS: The pretreatment with GW0742 significantly increased the expression of Bcl-2, and significantly decreased in the volume of infarction, NDS, edema, expressions of IL-1ß, NF-κB, TNFα, and Bax, contents of iNOS and the apoptotic cells in infarct cerebral hemisphere compared with rats in the vehicle group at 24 hours after MCAO. CONCLUSIONS: The study suggests the neuroprotective effect of the PPAR-ß/δ ligand GW0742 in acute ischemic stroke by a mechanism that may involve its anti-inflammatory and antiapoptotic action.

Association between preoperative white blood cell counts and thirty-day surgical mortality after craniotomy in adult intracranial tumor patients.

Objective: White blood cell (WBC) counts has been identified as a prognostic biomarker which frequently predict adverse outcomes and mortality risk in various conditions. However, evidence for the association between WBC counts and short-term outcomes after intracranial tumor resection remains limited. This study aimed to explore associations between preoperative WBC counts and thirty-day surgical mortality after craniotomy in adult intracranial tumor patients. Methods: This retrospective cohort study performed secondary analysis of 18,049 intracranial tumor craniotomy patients from the ACS NSQIP database (2012-2015). The major exposure and outcome were preoperative WBC counts and thirty-day surgical mortality, respectively. Cox regression modeling assessed the linear association between them. Non-linear associations between them were evaluated by conducting smooth curve fitting using an additive Cox proportional hazard model in conjunction with segmented linear regression modeling. Subgroup analysis and interaction testing assessed effect modification. Sensitivity analysis evaluated result robustness. Results: The total thirty-day surgical mortality after craniotomy was 2.49% (450/18,049). The mean of preoperative WBC counts was 9.501 ± 4.402 × 10^9/L. Fully adjusted model shows that elevated preoperative WBC counts was independently associated with increased thirty-day surgical mortality (HR = 1.057, 95%CI: 1.040, 1.076). Further analysis revealed a non-linear association between them: below a WBC threshold of 13.6 × 10^9/L, higher WBC counts elevated thirty-day mortality (HR = 1.117; 95%CI: 1.077, 1.158), while risk plateaued and no significant mortality rise occurred above this level (HR = 1.015, 95%CI: 0.982, 1.050). Steroid usage status has a significant effect modification on the WBC-mortality association (P for interaction = 0.002). The non-linear WBC-mortality association was only present for non-steroid users (HR = 1.158, 95%CI: 1.108, 1.210) but not steroid users (HR = 1.009, 95%CI: 0.966, 1.055). The sensitivity analysis confirmed the result robustness. Conclusion: Elevated preoperative WBC counts were independently and non-linearly associated with an increased risk of thirty-day surgical mortality in adult non-steroid use patients undergoing craniotomy for intracranial tumors. As a convenient predictor, preoperative WBC data allows improved risk profiling and personalized management in adult intracranial tumor patients.

Ghrelin attenuates brain injury after traumatic brain injury and uncontrolled hemorrhagic shock in rats.

Traumatic brain injury (TBI) and hemorrhagic shock often occur concomitantly due to multiple injuries. Gastrointestinal dysfunction occurs frequently in patients with TBI. However, whether alterations in the gastrointestinal system are involved in modulating neuronal damage and recovery after TBI is largely neglected. Ghrelin is a "gut-brain" hormone with multiple functions including antiinflammation and antiapoptosis. The purpose of this study was to determine whether ghrelin attenuates brain injury in a rat model of TBI and uncontrolled hemorrhage (UH). To study this, brain injury was induced by dropping a 450-g weight from 1.5 m onto a steel helmet attached to the skull of male adult rats. Immediately after TBI, a midline laparotomy was performed and both lumbar veins were isolated and severed at the junction with the vena cava. At 45 min after TBI/UH, ghrelin (4, 8 or 16 nmol/rat) or 1 mL normal saline (vehicle) was intravenously administered. Brain levels of TNF-α and IL-6, and cleaved PARP-1 levels in the cortex were measured at 4 h after TBI/UH. Beam balance test, forelimb placing test and hindlimb placing test were used to assess sensorimotor and reflex function. In additional groups of animals, ghrelin (16 nmol/rat) or vehicle was subcutaneously (s.c.) administered daily for 10 d after TBI/UH. The animals were monitored for 28 d to record body weight changes, neurological severity scale and survival. Our results showed that ghrelin downregulated brain levels of TNF-α and IL-6, reduced cortical levels of cleaved PARP-1, improved sensorimotor and reflex functions, and decreased mortality after TBI/UH. Thus, ghrelin has a great potential to be further developed as an effective resuscitation approach for the trauma victims with brain injury and severe blood loss.

Ghrelin maintains the cardiovascular stability in severe sepsis.

BACKGROUND: Cardiovascular dysfunction, characterized by reduced cardiac contractility and depressed endothelium-dependent vascular relaxation, is common in severe sepsis. Although it is known that ghrelin produces beneficial effects following various adverse circulatory conditions, it remains unknown whether ghrelin increases cardiac contractility and improves vascular responsiveness to vasoactive agents in severe sepsis. METHODS: Male adult rats were subjected to sepsis by cecal ligation and puncture (CLP). At 5 h after CLP, a bolus intravenous injection of 2 nmol ghrelin was followed by a continuous infusion of 12 nmol ghrelin via a primed mini-pump over 15 h. At 20 h after CLP (i.e., severe sepsis), the maximal rates of ventricular pressure increase (+dP/dt(max)) and decrease (-dP/dt(max)) were determined in vivo. In additional groups of animals, the thoracic aortae were isolated at 20 h after CLP. The aortae were cut into rings, and placed in organ chambers. Norepinephrine (NE) was used to induce vascular contraction. Dose responses for an endothelium-dependent vasodilator, acetylcholine (ACh), and an endothelium-independent vasodilator, nitroglycerine (NTG) were carried out. RESULTS: +dP/dt(max) and -dP/dt(max) decreased significantly at 20 h after CLP. Treatment with ghrelin significantly increased +dP/dt(max) and -dP/dt(max) by 36% (P < 0.05) and 35% (P < 0.05), respectively. Moreover, NE-induced vascular contraction and endothelium-dependent (ACh-induced) vascular relaxation decreased significantly at 20 h after CLP. Administration of ghrelin, however, increased NE-induced vascular contraction and ACh-induced vascular relaxation. In contrast, no significant reduction in NTG-induced vascular relaxation was seen in rats with severe sepsis irrespective of ghrelin treatment. CONCLUSIONS: Ghrelin may be further developed as a useful agent for maintaining cardiovascular stability in severe sepsis.

Polysaccharides Extracted from Angelica sinensis (Oliv.) Diels Relieve the Malignant Characteristics of Glioma Cells through Regulating the MiR-373-3p-Mediated TGF-ß/Smad4 Signaling Pathway.

Objectives: Angelica sinensis polysaccharide (ASP) is a traditional herbal medicine accompanied by antitumor potential. This study aims to explore the therapeutic potential of ASP on glioma, as well as the underlying mechanisms involving microRNA-373-3p (miR-373-3p) and the TGF-ß/Smad4 signaling pathway. Methods: U251 cells (a human glioma cell line) were treated with different concentrations of ASP. miR-373-3p was silenced in U251 cells by the transfection of the miR-373-3p inhibitor. Cell viability and apoptosis were measured by CCK-8 assay and flow cytometry, respectively. Cell migration and invasion were detected by wound healing and transwell assays, respectively. The miR-373-3p expression was measured by RT-qPCR. The protein expressions of TGF-ß and Smad4 were evaluated by both western blotting and immunofluorescence. Results: ASP inhibited the viability, migration, and invasion, and enhanced the apoptosis of U251 cells in a dose-dependent manner. ASP increased miR-373-3p expression and decreased TGF-ß and Smad4 expressions in U251 cells. Silencing of miR-373-3p weakened the effects of ASP on inhibiting cell viability, migration, and invasion, as well as promoting cell apoptosis. In addition, deleting miR-373-3p weakened the inhibiting effects of ASP on the TGF-ß/Smad4 pathway in U251 cells. Conclusions: ASP suppresses the malignant progression of glioma via regulating the miR-373-3p-mediated TGF-ß/Smad4 pathway.

Oncogene or Tumor Suppressor: The Coordinative Role of Lysine Methyltransferase SET7/9 in Cancer Development and the Related Mechanisms.

SET7/9 is a member of the protein lysine methyltransferase family that methylates both histone 3 lysine 4 (H3-K4) and lysine(s) of other non-histone proteins. In recent years, dis-regulation of SET7/9 were frequently detected in various cancer types and SET7/9-mediated methylation has been recognized as an important mechanism that affects cancer initiation and development through regulation of a series of cellular processes. Here we review the currently identified histone and non-histone protein targets of SET7/9 that are closely correlated with human cancer and the function of SET7/9 in regulating the expression and stability of its protein targets. The review also discusses the putative role of SET7/9 as an oncogene or tumor suppressor in the development of various cancer types and the underlying mechanisms, which may help better evaluate the potential of SET7/9 as a novel candidate for cancer therapy.

Peripheral administration of human adrenomedullin and its binding protein attenuates stroke-induced apoptosis and brain injury in rats.

Stroke is a leading cause of death and the primary medical cause of acquired adult disability worldwide. The progressive brain injury after acute stroke is partly mediated by ischemia-elicited inflammatory responses. The vasoactive hormone adrenomedullin (AM), upregulated under various inflammatory conditions, counterbalances inflammatory responses. However, regulation of AM activity in ischemic stroke remains largely unknown. Recent studies have demonstrated the presence of a specific AM binding protein (that is, AMBP-1) in mammalian blood. AMBP-1 potentiates AM biological activities. Using a rat model of focal cerebral ischemia induced by permanent middle cerebral artery occlusion (MCAO), we found that plasma levels of AM increased significantly, whereas plasma levels of AMBP-1 decreased significantly after stroke. When given peripherally early after MCAO, exogenous human AM in combination with human AMBP-1 reduced brain infarct volume 24 and 72 h after MCAO, an effect not observed after the treatment by human AM or human AMBP-1 alone. Furthermore, treatment of human AM/AMBP-1 reduced neuron apoptosis and morphological damage, inhibited neutrophil infiltration in the brain and decreased serum levels of S100B and lactate. Thus, human AM/AMBP-1 has the ability to reduce stroke-induced brain injury in rats. AM/AMBP-1 can be developed as a novel therapeutic agent for patients with ischemic stroke.

Upregulation of Kupffer cell α2A-Adrenoceptors and downregulation of MKP-1 mediate hepatic injury in chronic alcohol exposure.

Alcohol-induced liver disease is associated with unacceptable morbidity and mortality. When activated, Kupffer cells (KCs), the resident macrophages in the liver, release proinflammatory cytokine TNF-α, a key mediator of hepatic damage. Although chronic alcohol causes increase in norepinephrine (NE) release leading to hepatic dysfunction, the mechanism of NE-induced hepatic injury in chronic alcohol exposure has not been elucidated. This study was conducted to determine whether chronic alcohol exposure increases NE and upregulates KC α(2A)-adrenoceptors (α(2A)-AR) to cause TNF-α release. We also examined the role of mitogen activated protein kinase (MAPK) phosphatase-1 (MKP-1) in this process. Male adult rats were fed the Lieber-DeCarli liquid diet containing alcohol as 36% of total calories. The animals were sacrificed after 6 weeks and blood and liver samples were harvested for further analysis. KCs from healthy male rats were cultured with alcohol for 7 days, and cells then harvested for RNA and protein analyses. Chronic alcohol exposure resulted in hepatic damage. Alcohol caused a 276% increase in circulating NE and 86% increase in TNF-α in the liver. There was a 75% and 62% decrease in MKP-1 mRNA and protein levels, respectively in the liver. In-vitro experiments revealed 121% and 98% increase in TNF-α and α(2A)-AR mRNA levels with alcohol exposure, respectively, and a 32% decrease in MKP-1 mRNA compared to controls. In summary, chronic alcohol exposure elevates NE and upregulates KC α(2A)-AR to release TNF-α. Alcohol induced downregulation of MKP-1 leads to further release of TNF-α and hepatic injury.

Immature dendritic cell-derived exosomes rescue septic animals via milk fat globule epidermal growth factor-factor VIII [corrected].

Sepsis, a highly lethal systemic inflammatory syndrome, is associated with increases of proinflammatory cytokines (e.g., TNF-alpha, HMGB1) and the accumulation of apoptotic cells that have the potential to be detrimental. Depending on the timing and tissue, prevention of apoptosis in sepsis is beneficial; however, thwarting the development of secondary necrosis through the active removal of apoptotic cells by phagocytosis may offer a novel anti-sepsis therapy. Immature dendritic cells (IDCs) release exosomes that contain milk fat globule EGF factor VIII (MFGE8), a protein required to opsonize apoptotic cells for phagocytosis. In an experimental sepsis model using cecal ligation and puncture, we found that MFGE8 levels decreased in the spleen and blood, which was associated with impaired apoptotic cell clearance. Administration of IDC-derived exosomes promoted phagocytosis of apoptotic cells and significantly reduced mortality. Treatment with recombinant MFGE8 was equally protective, whereas MFGE8-deficient mice suffered from increased mortality. IDC exosomes also attenuated the release of proinflammatory cytokines in septic rats. Liberation of HMGB1, a nuclear protein that contributes to inflammation upon release from unengulfed apoptotic cells, was prevented by MFGE8-mediated phagocytosis in vitro. We conclude that IDC-derived exosomes attenuate the acute systemic inflammatory response in sepsis by enhancing apoptotic cell clearance via MFGE8.

Milk fat globule epidermal growth factor 8 attenuates acute lung injury in mice after intestinal ischemia and reperfusion.

RATIONALE: Milk fat globule epidermal growth factor 8 (MFG-E8) is a potent opsonin for the clearance of apoptotic cells and is produced by mononuclear cells of immune competent organs including the spleen and lungs. It attenuates chronic and acute inflammation such as autoimmune glomerulonephritis and bacterial sepsis by enhancing apoptotic cell clearance. Ischemia-reperfusion (I/R) injury of the gut results in severe inflammation, apoptosis, and remote organ damage, including acute lung injury (ALI). OBJECTIVES: To determine whether MFG-E8 attenuates intestinal and pulmonary inflammation after gut I/R. METHODS: Wild-type (WT) and MFG-E8(-/-) mice underwent superior mesenteric artery occlusion for 90 minutes, followed by reperfusion for 4 hours. A group of WT mice was treated with 0.4 microg/20 g recombinant murine MFG-E8 (rmMFG-E8) at the beginning of reperfusion. Four hours after reperfusion, MFG-E8, cytokines, myeloperoxidase activity, apoptosis, and histopathology were assessed. A 24-hour survival study was conducted in rmMFG-E8- and vehicle-treated WT mice. MEASUREMENTS AND MAIN RESULTS: Mesenteric I/R caused severe widespread injury and inflammation of the small intestines and remote organs, including the lungs. MFG-E8 levels decreased in the spleen and lungs by 50 to 60%, suggesting impaired apoptotic cell clearance. Treatment with rmMFG-E8 significantly suppressed inflammation (TNF-alpha, IL-6, IL-1beta, and myeloperoxidase) and injury of the lungs, liver, and kidneys. MFG-E8-deficient mice suffered from greatly increased inflammation and potentiated ALI, whereas treatment with rmMFG-E8 significantly improved the survival in WT mice. CONCLUSIONS: MFG-E8 attenuates inflammation and ALI after gut I/R and may represent a novel therapeutic agent.

Discovery of a new class of integrin antibodies for fibrosis.

Lung fibrosis, or the scarring of the lung, is a devastating disease with huge unmet medical need. There are limited treatment options and its prognosis is worse than most types of cancer. We previously discovered that MK-0429 is an equipotent pan-inhibitor of αv integrins that reduces proteinuria and kidney fibrosis in a preclinical model. In the present study, we further demonstrated that MK-0429 significantly inhibits fibrosis progression in a bleomycin-induced lung injury model. In search of newer integrin inhibitors for fibrosis, we characterized monoclonal antibodies discovered using Adimab's yeast display platform. We identified several potent neutralizing integrin antibodies with unique human and mouse cross-reactivity. Among these, Ab-31 blocked the binding of multiple αv integrins to their ligands with IC50s comparable to those of MK-0429. Furthermore, both MK-0429 and Ab-31 suppressed integrin-mediated cell adhesion and latent TGFß activation. In IPF patient lung fibroblasts, TGFß treatment induced profound αSMA expression in phenotypic imaging assays and Ab-31 demonstrated potent in vitro activity at inhibiting αSMA expression, suggesting that the integrin antibody is able to modulate TGFß action though mechanisms beyond the inhibition of latent TGFß activation. Together, our results highlight the potential to develop newer integrin therapeutics for the treatment of fibrotic lung diseases.

Milk fat globule EGF factor 8 attenuates sepsis-induced apoptosis and organ injury in alcohol-intoxicated rats.

BACKGROUND: Despite advances in our understanding of excessive alcohol-intake-related tissue injury and modernization of the management of septic patients, high morbidity and mortality caused by infectious diseases in alcohol abusers remain a prominent challenge. Our previous studies have shown that milk fat globule epidermal growth factor-factor VIII (MFG-E8), a protein required to opsonize apoptotic cells for phagocytosis, is protective in inflammation. However, it remains unknown whether MFG-E8 ameliorates sepsis-induced apoptosis and organ injury in alcohol-intoxicated rats. The purpose of this study was to determine whether recombinant murine MFG-E8 (rmMFG-E8) attenuates organ injury after acute alcohol exposure and subsequent sepsis. METHODS: Acute alcohol intoxication was induced in male adult rats by a bolus injection of intravenous alcohol at 1.75 g/kg BW, followed by an intravenous infusion of 300 mg/kg BW/h of alcohol for 10 hours. Sepsis was induced at the end of 10-hour alcohol infusion by cecal ligation and puncture (CLP). rmMFG-E8 or vehicle (normal saline) was administered intravenously 3 times (i.e., at the beginning of alcohol injection, the beginning of CLP, and 10 hours post-CLP) at a dose of 20 microg/kg BW each. Blood and tissue samples were collected 20 hours after CLP in alcoholic animals for various measurements. RESULTS: Acute alcohol exposure per se did not affect the production of MFG-E8; however, it primed the animal and enhanced sepsis-induced MFG-E8 downregulation in the spleen. Administration of rmMFG-E8 reduces alcohol/sepsis-induced apoptosis in the spleen, lungs, and liver. In addition, administration of rmMFG-E8 after alcohol exposure and subsequent sepsis decreases circulating levels of TNF-alpha and interleukin-6 and attenuates organ injury. CONCLUSIONS: rmMFG-E8 attenuates sepsis-induced apoptosis and organ injury in alcohol-intoxicated rats.

Targeting N-myristoylation for therapy of B-cell lymphomas.

Myristoylation, the N-terminal modification of proteins with the fatty acid myristate, is critical for membrane targeting and cell signaling. Because cancer cells often have increased N-myristoyltransferase (NMT) expression, NMTs were proposed as anti-cancer targets. To systematically investigate this, we performed robotic cancer cell line screens and discovered a marked sensitivity of hematological cancer cell lines, including B-cell lymphomas, to the potent pan-NMT inhibitor PCLX-001. PCLX-001 treatment impacts the global myristoylation of lymphoma cell proteins and inhibits early B-cell receptor (BCR) signaling events critical for survival. In addition to abrogating myristoylation of Src family kinases, PCLX-001 also promotes their degradation and, unexpectedly, that of numerous non-myristoylated BCR effectors including c-Myc, NFκB and P-ERK, leading to cancer cell death in vitro and in xenograft models. Because some treated lymphoma patients experience relapse and die, targeting B-cell lymphomas with a NMT inhibitor potentially provides an additional much needed treatment option for lymphoma.