Asha Jacob

Recombinant human milk fat globule-EGF factor 8 produces dose-dependent benefits in sepsis.

PurposeAnimal milk fat globule-EGF factor 8 (MFG-E8) has been shown to be beneficial in attenuating the inflammatory response in sepsis. In this study, we examined the effect of recombinant human MFG-E8 (rhMFG-E8) in an animal model of sepsis in an effort to develop it as a potential therapy against sepsis in humans.MethodsRats were subjected to sepsis by cecal ligation and puncture (CLP), and at 5xa0h post-CLP, they were given different doses of rhMFG-E8 (20, 40, 80, 160xa0μg/kg BW) in normal saline. At 20xa0h post-CLP, samples were collected for further analysis. A 10-day survival study was also performed.ResultsAt 20xa0h after CLP, organ injury indicators, serum IL-6 and TNF-α, and plasma HMGB-1 levels were significantly increased as compared to sham-operated animals. Treatment with 20xa0μg/kg rhMFG-E8 significantly reduced these levels. With higher doses, further reductions in AST and ALT (59–62%), creatinine (65–68%), and lactate (46–57%), and serum IL-6 and TNF-α were obtained. The 160xa0μg/kg dose produced the greatest reduction in serum TNF-α. With treatment with 20xa0μg/kg rhMFG-E8, HMGB-1 levels decreased by 80%, returning back to sham values. In a 10-day survival study, vehicle-treated animals produced a 36% survival rate, while rhMFG-E8 significantly improved the survival rate to 68–72%. Treatment with increasing doses of rhMFG-E8 significantly reduced the number of apoptotic cells detected and markedly attenuated the tissue damages observed in the lungs.ConclusionsThese data suggest that recombinant human MFG-E8 is beneficial in ameliorating sepsis in an animal model of sepsis.

Milk fat globule--EGF factor VIII ameliorates liver injury after hepatic ischemia-reperfusion.

BACKGROUNDnHepatic ischemia-reperfusion (I/R) injury is a serious clinical complication that may compromise liver function because of extensive hepatocyte loss. Therefore, thexa0development of novel and effective therapies for hepatic I/R is critical for the improvement of patient outcome. It has been previously shown that administration of milk fat globule-EGF factor VIII (MFG-E8), a membrane-associated secretory glycoprotein, exerts significant beneficial effects under acute inflammatory conditions through multiple physiological processes associated with tissue remodeling.nnnMETHODSnTo determine whether administration of recombinant human (rh) MFG-E8 attenuates liver injury in an animal model of hepatic I/R, male adult rats were subjected to 70% hepatic ischemia for 90 min, followed by reperfusion. At the beginning of reperfusion, rats were treated intravenously with normal saline (vehicle) or rhMFG-E8 (160xa0μg/kg) over a period of 30 min. MFG-E8 levels and various measurements were assessed 4 h after reperfusion. In addition, survival study was conducted in MFG-E8(-/-) and rhMFG-E8-treated wild-type (WT) mice using a total hepatic ischemia model.nnnRESULTSnLiver and plasma MFG-E8 protein levels were significantly decreased after hepatic I/R. Administration of rhMFG-E8 significantly improved liver injury, suppressed apoptosis, attenuated inflammation and oxidative stress, and downregulated NF-κB pathway. We also noticed that rhMFG-E8 treatment restored the downregulated PPAR-γ expression after hepatic I/R. MFG-E8(-/-) mice showed deterioration on survival and, in contrast, rhMFG-E8-treated WT mice showed a significant improvement of survival compared with vehicle-treated WT mice.nnnCONCLUSIONSnMFG-E8-mediated multiple physiological events may represent an effective therapeutic option in tissue injury following an episode of hepatic I/R.

Cold-Inducible RNA-Binding Protein Is an Important Mediator of Alcohol-Induced Brain Inflammation

Binge drinking has been associated with cerebral dysfunction. Ethanol induced microglial activation initiates an inflammatory process that causes upregulation of proinflammatory cytokines which in turn creates neuronal inflammation and damage. However, the molecular mechanism is not fully understood. We postulate that cold-inducible RNA-binding protein (CIRP), a novel proinflammatory molecule, can contribute to alcohol-induced neuroinflammation. To test this theory male wild-type (WT) mice were exposed to alcohol at concentrations consistent to binge drinking and blood and brain tissues were collected. At 5 h after alcohol, a significant increase of 53% in the brain of CIRP mRNA was observed and its expression remained elevated at 10 h and 15 h. Brain CIRP protein levels were increased by 184% at 10 h and remained high at 15 h. We then exposed male WT and CIRP knockout (CIRP−/−) mice to alcohol, and blood and brain tissues were collected at 15 h post-alcohol infusion. Serum levels of tissue injury markers (AST, ALT and LDH) were significantly elevated in alcohol-exposed WT mice while they were less increased in the CIRP−/− mice. Brain TNF-α mRNA and protein expressions along with IL-1β protein levels were significantly increased in WT mice, which was not seen in the CIRP−/− mice. In cultured BV2 cells (mouse microglia), ethanol at 100 mM showed an increase of CIRP mRNA by 274% and 408% at 24 h and 48 h respectively. Corresponding increases in TNF-α and IL-1β were also observed. CIRP protein levels were markedly increased in the medium, suggesting that CIRP was secreted by the BV2 cells. From this we conclude that alcohol exposure activates microglia to produce and secrete CIRP and possibly induce pro-inflammatory response and thereby causing neuroinflammation. CIRP could be a novel mediator of alcohol-induced brain inflammation.

Association between insertion/deletion polymorphism in angiotensin-converting enzyme gene and acute lung injury/acute respiratory distress syndrome: a meta-analysis

BackgroundA previous meta-analysis reported a positive association between an insertion/deletion (I/D) polymorphism in the angiotensin-converting enzyme gene (ACE) and the risk of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Here, we updated this meta-analysis and additionally assessed the association of this polymorphism with ALI/ARDS mortality.MethodsWe searched electronic databases through October 2011 for the terms “angiotensin-converting enzyme gene”, “acute lung injury”, and “acute respiratory distress syndrome,” and reviewed all studies that reported the relationship of the I/D polymorphism in ACE with ALI/ARDS in humans. Seven studies met the inclusion criteria, comprising 532 ALI/ARDS patients, 3032 healthy controls, and 1432 patients without ALI/ARDS. We used three genetic models: the allele, dominant, and recessive models.ResultsThe ACE I/D polymorphism was not associated with susceptibility to ALI/ARDS for any genetic model. However, the ACE I/D polymorphism was associated with the mortality risk of ALI/ARDS in Asian subjects ( Palleleu2009<u20090.0001, Pdominantu2009=u20090.001, Precessiveu2009=u20090.002). This finding remained significant after correction for multiple comparisons.ConclusionsThere is a possible association between the ACE I/D polymorphism genotype and the mortality risk of ALI/ARDS in Asians.

A deficiency in cold-inducible RNA-binding protein accelerates the inflammation phase and improves wound healing.

Chronic or non-healing wounds are a major concern in clinical practice and these wounds are mostly associated with diabetes, and venous and pressure ulcers. Wound healing is a complex process involving overlapping phases and the primary phase in this complex cascade is the inflammatory state. While inflammation is necessary for wound healing, a prolonged inflammatory phase leads to impaired healing. Cold-inducible RNA-binding protein (CIRP) belongs to a family of cold-shock proteins that are expressed in high levels under stress conditions. Recently, we demonstrated that a deficiency in CIRP led to decreased inflammation and mortality in an experimental model of hemorrhagic shock. Thus, we hypothesized that a deficiency in CIRP would accelerate the inflammatory phase and lead to an improvement in cutaneous wound healing. In this study, to examine this hypothesis, a full-thickness wound was created on the dorsum of wild-type (WT) and CIRP-/- mice. The wound size was measured every other day for 14 days. The wound area was significantly decreased in the CIRP-/- mice by day 9 and continued to decrease until day 14 compared to the WT mice. In a separate cohort, mice were sacrificed on days 3 and 7 after wounding and the skin tissues were harvested for histological analysis and RNA measurements. On day 3, the mRNA expression of tumor necrossis factor (TNF)-α in the skin tissues was increased by 16-fold in the WT mice, whereas these levels were increased by 65-fold in the CIRP-/- mice. Of note on day 7, while the levels of TNF-α remained high in the WT mice, these levels were significantly decreased in the CIRP-/- mice. The histological analysis of the wounded skin tissue indicated an improvement as early as day 3 in the CIRP-/- mice, whereas in the WT mice, infiltrated immune cells were still present on day 7. On day 7 in the CIRP-/- mice, Gr-1 expression was low and CD31 expression was high, whereas in the WT mice, Gr-1 expression was high and CD31 expression was low, indicating that the CIRP-/- mice have already moved into the angiogenesis and tissue formation phase, whereas the WT mice were still in the inflammatory state. These data collectively suggest that a deficiency in CIRP accelerates the wound healing process.

Combination of Adrenomedullin with Its Binding Protein Accelerates Cutaneous Wound Healing

Cutaneous wound continues to cause significant morbidity and mortality in the setting of diseases such as diabetes and cardiovascular diseases. Despite advances in wound care management, there is still an unmet medical need exists for efficient therapy for cutaneous wound. Combined treatment of adrenomedullin (AM) and its binding protein-1 (AMBP-1) is protective in various disease conditions. To examine the effect of the combination treatment of AM and AMBP-1 on cutaneous wound healing, full-thickness 2.0-cm diameter circular excision wounds were surgically created on the dorsum of rats, saline (vehicle) or AM/AMBP-1 (96/320 μg kg BW) was topically applied to the wound daily and wound size measured. At days 3, 7, and 14, skin samples were collected from the wound sites. AM/AMBP-1 treated group had significantly smaller wound surface area than the vehicle group over the 14-day time course. At day 3, AM/AMBP-1 promoted neutrophil infiltration (MPO), increased cytokine levels (IL-6 and TNF-α), angiogenesis (CD31, VEGF and TGFβ-1) and cell proliferation (Ki67). By day 7 and 14, AM/AMBP-1 treatment decreased MPO, followed by a rapid resolution of inflammation characterized by a decrease in cytokines. At the matured stage, AM/AMBP-1 treatment increased the alpha smooth muscle actin expression (mature blood vessels) and Masson-Trichrome staining (collagen deposition) along the granulation area, and increased MMP-9 and decreased MMP-2 mRNA expressions. TGFβ-1 mRNA levels in AM/AMBP-1 group were 5.3 times lower than those in the vehicle group. AM/AMBP-1 accelerated wound healing by promoting angiogenesis, collagen deposition and remodeling. Treatment also shortened the days to reach plateau for wound closure. Thus, AM/AMBP-1 may be further developed as a therapeutic for cutaneous wound healing.

Milk Fat Globule-EGF Factor VIII Attenuates CNS Injury by Promoting Neural Stem Cell Proliferation and Migration after Cerebral Ischemia

The mediators in activating neural stem cells during the regenerative process of neurogenesis following stroke have not been fully identified. Milk fat globule-EGF Factor VIII (MFG-E8), a secreted glycoprotein serves several cellular functions by binding to its receptor, αv β3-integrin. However, its role in regulating neural stem cells after stroke has not been determined yet. We therefore, aim to reveal whether MFG-E8 promotes neural stem cell proliferation and migration during stroke. Stroke was induced in wild-type (Wt) and MFG-E8-deficinet (Mfge8-/-) mice by transient middle cerebral artery occlusion (tMCAO). Commercially available recombinant mouse MFG-E8 (rmMFG-E8) was used for mechanistic assays in neural stem cell line, while the in house prepared recombinant human MFG-E8 (rhMFG-E8) was used for in vivo administration into rats with tMCAO. The in vitro effects of recombinant rmMFG-E8 for the neural stem cell proliferation and migration were determined by BrdU and transwell migration assay, respectively. The expression of cyclin D2, p53 and netrin-1, was analyzed by qPCR. We report that the treatment of rhMFG-E8 significantly improved the neurological deficit score, body weight lost and neural stem cell proliferation in a rat model of tMCAO. Conversely, decreased neural stem cell proliferation was observed in Mfge8-/- mice in comparison with the Wt counterparts underwent tMCAO. rmMFG-E8 stimulated the proliferation of mouse embryonic neural stem cells via upregulation of cyclin D2 and downregulation of p53, which is mediated by αv β3-integrin. rmMFG-E8 also promoted mouse embryonic neural stem cell migration via αv β3-integrin dependent manner in upregulating netrin-1. Our findings suggest MFG-E8 to promote neural stem cell proliferation and migration, which therefore establishes a promising therapeutic strategy for cerebral ischemia.

Deficiency in milk fat globule-epidermal growth factor-factor 8 exacerbates organ injury and mortality in neonatal sepsis

INTRODUCTIONnNeonatal sepsis is a systemic inflammation occurring in neonates because of a proven infection within the first 28days of birth. It is the third leading cause of morbidity and mortality in the newborns. The mechanism(s) underlying the systemic inflammation in neonatal sepsis has not been completely understood. We hypothesize that the deficiency of milk fat globule-epidermal growth factor-factor 8 (MFG-E8), a protein commonly found in human milk, could be responsible for the increased inflammatory response leading to morbidity and mortality in neonatal sepsis.nnnMETHODSnMale and female newborn mice aged 5-7days were injected intraperitoneally with 0.9mg/g body weight cecal slurry (CS). At 10h after CS injection, they were euthanized, and blood, lungs and gut tissues were obtained for further analyses. Control newborn mice underwent similar procedures with the exception of the CS injection. In duplicate newborn mice after CS injection, they were returned to their respective cages with their mothers and were closely monitored for 7days and survival rate recorded.nnnRESULTSnAt 10h after CS injection, serum LDH in the MFG-E8 knockout (KO) newborn mice was significantly increased by 58% and serum IL-6, IL-1β and TNF-α in the MFG-E8KO newborn mice were also significantly increased by 56%, 65%, and 105%, respectively, from wild type (WT) newborn mice. There were no significant difference between WT control and MFG-E8 control newborn mice. The lung architecture was severely damaged and a significant 162% increase in injury score was observed in the CS MFG-E8KO newborn mice. The MPO, TUNEL staining, and cytokine levels in the lungs and the intestine in CS MFG-E8KO newborn mice were significantly increased from CS WT newborn mice. Similarly, intestinal integrity was also compromised in the CS MFG-E8KO newborn mice. In a survival study, while the mortality rate within 7days was only 29% in the CS WT newborn mice, 80% of the CS MFG-E8KO newborn mice died during the same time period with the majority of mortality occurring within 48h.nnnCONCLUSIONnThe deficiency in MFG-E8 caused increases in inflammation, tissue injury, neutrophil infiltration and apoptosis, which led to morbidity and mortality in murine neonatal sepsis. These studies suggest that MFG-E8 has a protective role in fighting against neonatal sepsis.

Inhibition of lipogenesis reduces inflammation and organ injury in sepsis

BACKGROUNDnSepsis is a life-threatening acute inflammatory condition associated with metabolic complications. Accumulation of free fatty acids (FFAs) induces inflammation and causes lipotoxic effects in the liver. Because fatty acid metabolism plays a role in the inflammatory response, we hypothesized that the administration of C75, a fatty acid synthase inhibitor, could alleviate the injury caused by sepsis.nnnMETHODSnMale mice were subjected to sepsis by cecal ligation and puncture (CLP). At 4xa0h after CLP, different doses of C75 (1- or 5-mg/kg body weight) or vehicle (20% dimethyl sulfoxide in saline) were injected intraperitoneally. Blood and liver tissues were collected at 24xa0h after CLP.nnnRESULTSnC75 treatment with 1- and 5-mg/kg body weight significantly lowered FFA levels in the liver after CLP by 28% and 53%, respectively. Administration of C75 dose dependently reduced serum indexes of organ injury (aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase) and serum levels of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). In the liver, C75 treatment reduced inflammation (TNF-α and IL-6) and oxidative stress (inducible nitric oxide synthase and cyclooxygenase 2) in a dose-dependent manner. The 5-mg dose improved the 10-d survival rate to 85% from that of 55% in the vehicle. In the presence of C75, TNF-α release in RAW 246.7 cells with 4-h lipopolysaccharide stimulation was also significantly reduced.nnnCONCLUSIONSnC75 effectively lowered FFA accumulation in the liver, which was associated with inhibition of inflammation and organ injury as well as improvement in survival rate after CLP. Thus, inhibition of FFA by C75 could ameliorate the hepatic dysfunction seen in sepsis.

Treatment with milk fat globule epidermal growth factor-factor 8 (MFG-E8) reduces inflammation and lung injury in neonatal sepsis

Background. Sepsis remains one of the leading causes of infant death worldwide. It is characterized by uncontrolled inflammatory responses due to proven bacterial infection. Despite improvement in supportive care and the availability of effective antibiotics, no specific therapy targeting the dysregulated inflammatory response is available for neonatal sepsis. Milk fat globule epidermal growth factor‐factor 8 (MFG‐E8) is a secretory glycoprotein abundantly present in human milk. MFG‐E8 suppresses the systemic inflammatory responses in adult murine injury models by improving the clearance of dying cells. We hypothesized that exogenous administration of recombinant mouse MFG‐E8 could inhibit the exaggerated inflammatory response and lung injury in a murine model of neonatal sepsis. Methods. Neonatal sepsis was induced in 5‐ to 7‐day‐old male and female C57BL6 mice using an intraperitoneal injection of cecal slurry. At 1 hour after sepsis induction, a single dose of 40 &mgr;g/kg recombinant mouse MFG‐E8 or vehicle was administered via retro‐orbital injection. All neonates were returned to their mothers as a group. At 10 hours after cecal slurry injection, pups were killed and blood and lung tissues were collected. Control mice underwent a similar procedure with the exception of cecal slurry intraperitoneal injection. Results. Serum lactate dehydrogenase, IL‐1&bgr;, and IL‐6 were significantly increased 10 hours after cecal slurry injection. Treatment with recombinant mouse MFG‐E8 decreased these levels by 30%, 56%, and 37%, respectively. Lung morphology was significantly compromised in the vehicle group after cecal slurry injection, whereas the recombinant mouse MFG‐E8–treated groups demonstrated a 48% improvement in the lung injury score. Lung IL‐6 and MIP‐2 protein levels were significantly reduced with recombinant mouse MFG‐E8 treatment. Lung neutrophil infiltration as observed by Gr‐1 staining and, TUNEL‐positive cells were also significantly reduced with recombinant mouse MFG‐E8 treatment. Conclusion. Treatment with recombinant mouse MFG‐E8 attenuated inflammation and lung injury in murine neonatal sepsis. Thus, MFG‐E8 could be developed as a possible therapy for neonatal sepsis.