Kavin G. Shah

A novel method to determine the engulfment of apoptotic cells by macrophages using pHrodo succinimidyl ester.

Apoptotic cell phagocytosis has recently raised considerable interest, particularly due to its intricate molecular mechanisms and negative immunologic impact of incompetent clearance of apoptotic cells. There is a need for simple and reliable methods to clearly determine the internalization of apoptotic cells. Labeling with pHrodo succinimidyl ester (SE), a pH-sensitive fluorescent dye, makes engulfed apoptotic cells detectable due to the increased post-phagocytic light emission. This is a valuable tool for phagocytosis studies via FACS. We designed an ex vivo assay, using apoptotic pHrodo-labeled lymphocytes as prey and anti-CD11b-labeled tissue macrophages. To demonstrate its validity of detecting internalized apoptotic lymphocytes, we used MFGE8(-/-) macrophages, known to have impaired phagocytic ability. Uptake of apoptotic lymphocytes was accelerated and enhanced in splenic macrophages after stimulation with recombinant MFGE8, while peritoneal macrophages were able to compensate for the delayed uptake. This novel assay is a quick and reliable method to evaluate the internalization of apoptotic cells.

Human Ghrelin Ameliorates Organ Injury and Improves Survival after Radiation Injury Combined with Severe Sepsis

In the terrorist radiation exposure scenario, radiation victims are likely to suffer from additional injuries such as sepsis. Our previous studies have shown that ghrelin is protective in sepsis. However, it remains unknown whether ghrelin ameliorates sepsis-induced organ injury and mortality after radiation exposure. The purpose of this study is to determine whether human ghrelin attenuates organ injury and improves survival in a rat model of radiation combined injury (RCI) and, if so, the potential mechanism responsible for the benefit. To study this, adult male rats were exposed to 5-Gy whole body irradiation followed by cecal ligation and puncture (CLP, a model of sepsis) 48 h thereafter. Human ghrelin (30 nmol/rat) or vehicle (saline) was infused intravenously via an osmotic minipump immediately after radiation exposure. Blood and tissue samples were collected at 20 h after RCI (68 h after irradiation or 20 h after CLP) for various measurements. To determine the longterm effect of human ghrelin after RCI, the gangrenous cecum was removed at 5 h after CLP and 10-d survival was recorded. In addition, vagotomy or sham vagotomy was performed in sham and RCI animals immediately prior to ghrelin administration, and various measurements were performed at 20 h after RCI. Our results showed that serum levels of ghrelin and its gene expression in the stomach were decreased markedly at 20 h after RCI. Administration of human ghrelin attenuated tissue injury markedly, reduced proinflammatory cytokine levels, decreased tissue myeloperoxidase activity, and improved survival after RCI. Furthermore, elevated plasma levels of norepinephrine (NE) after RCI were reduced significantly by ghrelin. However, vagotomy prevented ghrelin’s beneficial effects after RCI. In conclusion, human ghrelin is beneficial in a rat model of RCI. The protective effect of human ghrelin appears to be attributed to rebalancing the dysregulated sympathetic/parasympathetic nervous systems.

Ghrelin as a novel therapy for radiation combined injury.

The threat of nuclear terrorism has led to growing worldwide concern about exposure to radiation. Acute radiation syndrome, or radiation sickness, develops after whole-body or a partial-body irradiation with a high dose of radiation. In the terrorist radiation exposure scenario, however, radiation victims likely suffer from additional injuries such as trauma, burns, wounds or sepsis. Thus, high-dose radiation injuries and appropriate therapeutic interventions must be studied. Despite advances in our understanding of the pathophysiology of radiation injury, very little information is available on the therapeutic approaches to radiation combined injury. In this review, we describe briefly the pathological consequences of ionizing radiation and provide an overview of the animal models of radiation combined injury. We highlight the combined radiation and sepsis model we recently established and suggest the use of ghrelin, a novel gastrointestinal hormone, as a potential therapy for radiation combined injury.

Attenuation of renal ischemia and reperfusion injury by human adrenomedullin and its binding protein.

BACKGROUND Acute renal failure secondary to ischemia and reperfusion (I/R) injury poses a significant burden on both surgeons and patients. It carries a high morbidity and mortality rate and no specific treatment currently exists. Major causes of renal I/R injury include trauma, sepsis, hypoperfusion, and various surgical procedures. We have demonstrated that adrenomedullin (AM), a novel vasoactive peptide, combined with AM binding protein-1 (AMBP-1), which augments the activity of AM, is beneficial in various disease conditions. However, it remains unknown whether human AM/AMBP-1 provides any beneficial effects in renal I/R injury. The objective of our study therefore was to determine whether administration of human AM/AMBP-1 can prevent and/or minimize damage in a rat model of renal I/R injury. METHODS Male adult rats were subjected to renal I/R injury by bilateral renal pedicle clamping with microvascular clips for 60 min followed by reperfusion. Human AM (12 microg/kg BW) and human AMBP-1 (40 microg/kg BW) or vehicle (52 microg/kg BW human albumin) were given intravenously over 30 min immediately following the clip removal (i.e., reperfusion). Rats were allowed to recover for 24 h post-treatment, and blood and renal tissue samples were collected. Plasma levels of AM were measured using a radioimmunoassay specific for rat AM. Plasma AMBP-1 was measured by Western analysis. Renal water content and serum levels of systemic markers of tissue injury were measured. Serum and renal TNF-alpha levels were also assessed. RESULTS At 24 h after renal I/R injury, plasma levels of AM were significantly increased while plasma AMBP-1 was markedly decreased. Renal water content and systemic markers of tissue injury (e.g., creatinine, BUN, AST, and ALT) were significantly increased following renal I/R injury. Serum and renal TNF-alpha levels were also increased post injury. Administration of human AM/AMBP-1 decreased renal water content, and plasma levels of creatinine, BUN, AST, and ALT. Serum and renal TNF-alpha levels were also significantly decreased after AM/AMBP-1 treatment. CONCLUSION Treatment with human AM/AMBP-1 in renal I/R injury significantly attenuated organ injury and the inflammatory response. Thus, human AM combined with human AMBP-1 may be developed as a novel treatment for patients with acute renal I/R injury.

Human ghrelin protects animals from renal ischemia-reperfusion injury through the vagus nerve

BACKGROUND Acute kidney injury secondary to renal ischemia and reperfusion injury is widely prevalent. Ghrelin, which is a stomach-derived peptide, has been shown to be anti-inflammatory. The purpose of this study was to examine whether human ghrelin has any beneficial effects after renal ischemia and reperfusion injury, and if so, whether ghrelins action in renal ischemia and reperfusion injury is mediated by the vagus nerve. METHODS Male adult rats were subjected to renal ischemia and reperfusion by bilateral renal pedicle clamping for 60 min, treated intravenously with human ghrelin (4 nmol/rat) or normal saline (vehicle) immediately after reperfusion. After 24 h, the animals were killed and samples were harvested. In separate groups, subdiaphragmatic vagotomy prior to renal ischemia and reperfusion was performed, treated with human ghrelin or vehicle, and at 24 h, blood and organs were harvested. RESULTS Renal ischemia and reperfusion injury caused significant increases in the serum levels of tissue injury markers compared with the sham operation. Human ghrelin treatment attenuated serum creatinine and blood urea nitrogen significantly by 55% and 53%, and liver enzymes (aminotransferase [AST] and alanine aminotransferase [ALT]) by 20% and 24%, respectively, compared with the vehicle-treated groups. Tissue water contents, plasma and kidney interleukin-6, and kidney myeloperoxidase activity were decreased. Bcl-2/Bax ratio was increased, and histology of the kidneys was improved. More importantly, prior vagotomy abolished ghrelins protective effect in tissue injury markers and tissue water contents in renal ischemia and reperfusion injured animals. CONCLUSION Human ghrelin treatment in renal ischemia and reperfusion injured rats attenuated systemic and kidney-specific inflammatory responses. The protection of human ghrelin in renal ischemia and reperfusion injury was mediated by the vagus nerve. These data suggest that ghrelin can be developed as a novel treatment for patients with acute kidney injury induced by renal ischemia and reperfusion injury.

Milk fat globule epidermal growth factor-factor 8 mitigates inflammation and tissue injury after hemorrhagic shock in experimental animals.

BACKGROUND: Insufficient clearance of apoptotic cells leads to increased inflammation and exaggerated organ injury. The opsonizing protein, milk fat globule epidermal growth factor-factor 8 (MFG-E8), upregulates apoptotic cell clearance. The purpose of this study was to determine the degree of apoptotic cell clearance, and whether inflammation, organ injury, and survival are improved after treatment with recombinant human MFG-E8 (rhMFG-E8) after hemorrhagic shock. METHODS: Male mice underwent a pressure-controlled (25 mm Hg ± 5 mm Hg) model of hemorrhagic shock for 90 minutes. They were resuscitated with normal saline with or without recombinant human MFG-E8 (rhMFG-E8) over 30 minutes. At 3.5-hour postresuscitation, blood and tissue were collected. MFG-E8 levels in the plasma, lungs, and spleen were measured. Apoptotic cell clearance was measured by cleaved caspase-3 levels and TUNEL staining. Neutrophil infiltration was assessed using myeloperoxidase activity in the lungs and spleen. Plasma and tissue levels of proinflammatory cytokines (IL-1&bgr;, IL-6, and TNF-&agr;) were measured by ELISA. Finally, a seven-day survival study was also conducted. RESULTS: MFG-E8 levels in the plasma, lungs, and spleen significantly decreased by 33%, 44%, and 55%, respectively, at 3.5 hour after hemorrhage and resuscitation with rhMFG-E8. Treatment with rhMFG-E8 significantly improved apoptosis, by reducing TUNEL+ cells after treatment and restoring cleaved caspase-3 expression back to baseline. Neutrophil infiltration was blunted by 29% and 41% in the lungs and spleen, respectively. Cytokine expression was also reduced significantly, by 64% to 73% in plasma, 24% to 58% in the lungs, and 49% to 76% in the spleen. Finally, animals demonstrated a superior survival rate over 7 days after treatment with rhMFG-E8. CONCLUSION: The administration of rhMFG-E8 is a potent treatment in animals after hemorrhagic shock.

Resuscitation of uncontrolled traumatic hemorrhage induced by severe liver injury: the use of human adrenomedullin and adrenomedullin binding protein-1.

BACKGROUND The liver is a major organ that is susceptible to injury after blunt or penetrating trauma to the abdomen. No specific nonoperative treatment exists for traumatic hepatic injury (THI). Adrenomedullin (AM), a vasoactive peptide, combined with its binding protein, AM protein (AMBP-1), is beneficial in various disease conditions. In this study, we propose to analyze whether human AM combined with human AMBP-1 provides benefit in a model of THI in the rat. METHODS Male adult rats were subjected to trauma hemorrhage by resection of ∼50% of total liver tissues and allowed bleeding for 15 minutes. Immediately thereafter, human AM (48 μg/kg birth weight) plus human AMBP-1 (160 μg/kg birth weight) were given intravenously over 30 minutes in 1-mL normal saline. After 4 hours, the rats were killed, blood was collected, and tissue injury indicators were assessed. A 10-day survival study was also conducted. RESULTS At 4 hours after THI, plasma AMBP-1 levels were markedly decreased. Plasma levels of liver injury indicators (i.e., aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase) were significantly increased after THI. Similarly, lactate, creatinine, and tumor necrosis factor-α levels were significantly increased after THI. Administration of human AM/AMBP-1 after THI produced significant decreases of 64%, 23%, and 19% of plasma aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase levels, respectively. Similarly, plasma levels of lactate, creatinine, and tumor necrosis factor-α were also decreased by 42%, 28%, and 46% after human AM/AMBP-1 treatment, respectively. In a 10-day survival study, although vehicle treatment produced 41% survival, human AM/AMBP-1 treatment improved the survival rate to 81%. CONCLUSIONS Administration of human AM/AMBP-1 significantly attenuated tissue injury and inflammation and improved survival after THI. Thus, human AM/AMBP-1 can be developed as a novel treatment for victims with uncontrolled traumatic hemorrhage.

Partial closure of skin wounds after kidney transplantation decreases the incidence of postoperative wound infections.

Wound infections are a major cause of morbidity after kidney transplantation. The purpose of our study was to evaluate an improved technique of wound closure. Data corresponding to 104 consecutive live donor kidney recipients were prospectively collected and analyzed. Our routine standard technique involved closure of the abdominal wall muscle and fascia in one layer with interrupted nonabsorbable full thickness sutures. No drains were used. The skin was closed with interrupted 2-0 nylon sutures 4 to 5 cm apart, leaving the skin and subcutaneous tissue in between partially open. Patients were allowed to shower starting on the first postoperative day. Examination of the wounds was continued for at least 1 month postoperatively, and then routinely as needed. All patients were thoroughly informed preoperatively of our technique. There were no immediate postoperative wound infections. There were no instances of dehiscence, evisceration, or need for revision. All patients were able to continue with their routine daily activities. Cosmetic results were satisfactory in all cases. We did not experience any patient complaints with respect to our technique. Patient satisfaction scores conducted by Press Ganey and Associates ranked in the 99 percentile with respect to peers undergoing kidney transplantation. Three patients returned six months postoperatively with suture granulomas which were treated nonoperatively. Partial closure of the skin wound with no associated drains is an effective and cosmetically desirable way to decrease the incidence of postoperative infections in kidney transplantation.

Treatment of Late Class II Antibody-Mediated Rejection Status Postkidney Transplantation: Two Case Reports

We are describing the successful treatment of two cases of late Class II antibody mediated rejection status postkidney transplantation. The first patient was treated with a combination of plasmapheresis, intravenous immunoglobulin (IVIG), and stenting of the transplanted renal artery. The second was treated with IVIG and pulse steroids.