Translation regulatory factor RBM3 is a proto-oncogene that prevents mitotic catastrophe.

RNA-binding proteins play a key role in post-transcriptional regulation of mRNA stability and translation. We have identified that RBM3, a translation regulatory protein, is significantly upregulated in human tumors, including a stage-dependent increase in colorectal tumors. Forced RBM3 overexpression in NIH3T3 mouse fibroblasts and SW480 human colon epithelial cells increases cell proliferation and development of compact multicellular spheroids in soft agar suggesting the ability to induce anchorage-independent growth. In contrast, downregulating RBM3 in HCT116 colon cancer cells with specific siRNA decreases cell growth in culture, which was partially overcome when treated with prostaglandin E(2), a product of cyclooxygenase (COX)-2 enzyme activity. Knockdown also resulted in the growth arrest of tumor xenografts. We have also identified that RBM3 knockdown increases caspase-mediated apoptosis coupled with nuclear cyclin B1, and phosphorylated Cdc25c, Chk1 and Chk2 kinases, implying that under conditions of RBM3 downregulation, cells undergo mitotic catastrophe. RBM3 enhances COX-2, IL-8 and VEGF mRNA stability and translation. Conversely, RBM3 knockdown results in loss in the translation of these transcripts. These data demonstrate that the RNA stabilizing and translation regulatory protein RBM3 is a novel proto-oncogene that induces transformation when overexpressed and is essential for cells to progress through mitosis.

Comparison of dried plum supplementation and intermittent PTH in restoring bone in osteopenic orchidectomized rats.

UNLABELLED: Bone loss was confirmed after 90 days in 50 6-month-old male Sprague Dawley rats that were sham-operated or orchidectomized (ORX). In this study, we have shown that dried plum (DP) has potent effects on bone in terms of bone mass, microarchitecture, and strength in osteopenic male rats. Although these changes may be mediated through the suppression of bone resorption, the fact that the restoration in some of the bone structural and biomechanical parameter shares some similarities with parathyroid hormone (PTH) should not be overlooked. Further investigation is needed on a mechanistic level to clarify the influence of DP on bone metabolism. INTRODUCTION: This study was designed to investigate the extent to which DP reverses bone loss in osteopenic ORX rats and to compare its effects to PTH. MATERIALS AND METHODS: Fifty, 6-month-old male Sprague Dawley rats were sham-operated or ORX, and bone loss was confirmed after 90 days. The ORX groups were assigned to control (AIN-93M) diet, 25% DP diet, or PTH (80 microg/kg) for 90 days. RESULTS: DP induced an 11% increase in vertebral and femoral BMD compared to ORX-controls. BMD in the PTH-treated group was increased by 20.7% (vertebra) and 17.9% (femur). Vertebral trabecular bone volume (BV/TV) and number were increased by DP and trabecular separation was decreased compared to controls, which were similar to PTH. Alterations in trabecular bone of the femur were similar to those in the vertebra, but DP did not restore BV/TV to the same extent. Cortical thickness was improved by DP and further enhanced by PTH. DP tended to decrease urinary deoxypyridinoline and calcium, but did not alter alkaline phosphatase or osteocalcin. CONCLUSION: We conclude that though the degree of improvement was not equivalent to PTH with regard to all parameters, DP reverses bone loss due to ORX and the mechanisms should be further investigated.

Dried plum prevents bone loss in a male osteoporosis model via IGF-I and the RANK pathway.

Previously, dietary supplementation with dried plums, a rich source of polyphenolic compounds with antioxidant and anti-inflammatory properties, has been shown to improve bone density, microstructure and biomechanics in female animal models of osteopenia. We designed this study to determine the extent to which dried plum prevents skeletal deterioration in gonadal hormone deficient male animals and to begin to understand its mechanism of action. Sixty 6-month-old male Sprague-Dawley rats were either sham-operated (Sham = 1 group) or orchidectomized (ORX = 4 groups) and randomly assigned to dietary treatments: standard semi-purified diet (Control) with either LD = 5%, MD = 15%, or HD = 25% (w/w) dried plum for 90 days. At the end of the treatment period, both the MD and HD dried plum completely prevented the ORX-induced decrease in whole body, femur, and lumbar vertebra bone mineral density (BMD). Biomechanical testing indicated that the MD and HD of dried plum prevented the ORX-induced decrease in ultimate load of the cortical bone as well as the compressive force and stiffness of trabecular bone within the vertebrae. Analyses of trabecular microarchitecture of the distal femur metaphysis and vertebral body revealed that HD dried plum protected against the decrease in trabecular bone volume (BV/TV) induced by ORX. In the distal femur, all doses of dried plum improved trabecular number (TbN) and separation (TbSp) compared to the ORX-control group, while MD and HD dried plum prevented the ORX-induced changes in vertebral TbN and TbSp. At the end of the 90-day treatment, no remarkable changes in serum osteocalcin or alkaline phosphatase in any of the treatment groups were observed, while serum insulin-like growth factor (IGF)-I was increased by dried plum. The ORX-induced increase in urinary deoxypyridinoline (DPD) excretion was completely prevented by all doses of dried plum coinciding with down-regulation of gene expression for receptor activator of NFkappa-B ligand (RANKL) and osteoprotegerin (OPG) in the bone. We conclude that dried plum prevents osteopenia in androgen deficient male rats, and these beneficial effects may be attributed in part to a decrease in osteoclastogenesis via down-regulation of RANKL and stimulation of bone formation mediated by IGF-I.

Systemic bone loss and induction of coronary vessel disease in a rat model of chronic inflammation.

Clinically, osteopenia or low bone mass has been observed in a variety of chronic inflammatory diseases, and elevated proinflammatory mediators have implicated this process. The purpose of this study was to develop an in vivo model of bone loss induced by chronic systemic inflammation. Time-release pellets designed to deliver one of three doses of LPS: Low (3.3 microg/day), High (33.3 microg/day), or Placebo over 90 days, were implanted subcutaneously in 3-month-old male Sprague-Dawley rats (n = 8/group). Neutrophil counts, indicative of ongoing inflammation, were elevated (P < 0.05) in both LPS groups at 30 days post-implant and remained significantly elevated in the High dose throughout the 90-day study period. At the end of the study, bone loss occurred in the femur as indicated by decreased bone mineral density (BMD) in both LPS-treated groups, but vertebral BMD was reduced in the High dose animals only. Microcomputed tomography revealed that trabecular bone volume (BV/TV) of the proximal tibial metaphysis tended to be reduced in the High dose LPS group. Deleterious effects on trabecular number (TbN) and trabecular separation (TbSp) were observed in both LPS-treated groups, but only the High dose group reached statistical significance. These alterations in trabecular microarchitecture resulted in compromised biomechanical properties. No changes in cortical thickness, porosity, or area of the tibia midshaft were evident at either dose of LPS. Up-regulation of the proinflammatory mediators, cyclooxygenase (COX)-2, interleukin (IL)-1, and tumor necrosis factor (TNF)-alpha was demonstrated in the metaphyseal region where the deleterious effects of LPS were observed. In addition to these alterations in bone, trichrome staining indicated changes in the coronary arterioles, consistent with vascular disease. Utilization of a LPS time-release pellet appears to provide an in vivo model of chronic inflammation-induced bone loss and a potentially novel system to study concurrent development of osteopenia and vascular disease.

Stress induced in heart and other tissues by rat dermal exposure to JP-8 fuel.

Limited information is available regarding the development of systemic organ stress by dermal exposure to JP-8 fuel. In this study, the systemic stress potential of this fuel is evaluated in a rat model subjected to dermal applications of JP-8 for 7 days at 300 microl per day. Tissue histology indicated that JP-8 induces morphological alterations that suggest that tissue stress in the heart is more substantial than stress in the kidney and liver. Immunoblot analysis of tissues revealed increased levels of the inducible heat shock protein 70 (HSP70) in the heart, kidney, and liver after this dermal JP-8 exposure. This exposure also leads to increased levels of heme oxygenase-1 (HO-1/HSP3) in the liver. Additionally during this exposure, a negative regulator of inflammation, IkappaBalpha (inhibitor of NF-kappaB), was increased in the liver, slightly increased in the kidney, and not increased in the heart. Two regions of the rat brain were also examined and HSP70 and IkappaBalpha were increased in the cerebellum but not significantly increased in the cortex. This study indicates dermal JP-8 exposure causes systemic alterations that are associated with cytoprotective activities (e.g., in the liver) as well as potentially toxic mechanisms (heart and kidney).

Vitamin E provides protection for bone in mature hindlimb unloaded male rats.

The deleterious effects of skeletal unloading on bone mass and strength may, in part, result from increased production of oxygen-derived free radicals and proinflammatory cytokines. This study was designed to evaluate the ability of vitamin E (alpha-tocopherol), a free-radical scavenger with antiinflammatory properties, to protect against bone loss caused by skeletal unloading in mature male Sprague-Dawley rats. A 2 x 3 factorial design was used with either hindlimb unloading (HU) or normal loading (ambulatory; AMB), and low-dose (LD; 15 IU/kg diet), adequate-dose (AD; 75 IU/kg diet), or high-dose (HD; 500 IU/kg diet) vitamin E (DL-alpha-tocopherol acetate). To optimize the effects of vitamin E on bone, dietary treatments were initiated 9 weeks prior to unloading and continued during the 4-week unloading period, at which time animals were euthanized and blood and tissue samples were collected. Serum vitamin E was dose-dependently increased, confirming the vitamin E status of animals. The HD treatment improved oxidation parameters, as indicated by elevated serum ferric-reducing ability and a trend toward reducing tissue lipid peroxidation. Histomorphometric analysis of the distal femur revealed significant reductions in trabecular thickness (TbTh), double-labeled surface (dLS/BS), and rate of bone formation to bone volume (BFR/BV) due by HU. AMB animals on the HD diet and HU animals on the LD diet had reduced bone surface normalized to tissue volume (BS/TV) and trabecular number (TbN); however, the HD vitamin E protected against these changes in the HU animals. Our findings suggest that vitamin E supplementation provides modest bone protective effects during skeletal unloading.

A rat model of syngeneic bone marrow transplantation during breast cancer therapy.

The purpose of this study was to develop a breast cancer model in rats, in which myeloablative chemotherapy and syngeneic bone marrow transplantation (SBMT) could be evaluated systematically for therapeutic effect. The Wistar-Furth (WF) DMBA-4 breast cancer cell line transplanted into naive WF rats produced rapidly growing tumors that were lethal within 2 months. SBMT was performed following preparation with a regimen (Bu-Cy), consisting of busulfan 16 mg/kg by gastric gavage on days -3 and -2 followed by 250 mg/kg of cyclophosphamide i.p. on day -1. Marrow was prepared from the femurs of donors and infused i.v. into the recipient on day 0. In all, 15 rats treated with Bu-Cy without marrow died, while 22 of 25 transplanted rats survived. In total, 16 rats with measurable tumors showed tumor responses following transplantation, but tumors recurred and survival was minimally prolonged. Of nine rats transplanted before clinical tumors were detected, five became long-term survivors that resisted further tumor challenge. It was concluded that the DMBA-4 breast cancer in WF rats could serve to evaluate SBMT following myeloablative doses of chemotherapy at various tumor loads. At large tumor loads therapy was not curative, but at low tumor burdens cures were possible and resistance to subsequent tumor challenge was demonstrated. The model may be useful for further studies of stem cell infusion in rodent tumor systems.

Down-regulation of renal endothelial nitric oxide synthase expression in experimental glomerular thrombotic microangiopathy.

Infection with certain strains of Escherichia coli and endotoxemia results in renal glomerular thrombotic microangiopathy (TMA) characterized by endothelial swelling and prominent glomerular microthrombus formation. Nitric oxide (NO) is an endogenous biologic modulator with diverse physiologic functions including vasodilation and inhibition of platelet adhesion and aggregation. NO is synthesized from conversion of L-arginine to L-citrulline by a family of NO synthases (NOS), which include constitutive and inducible isoforms. Indirect evidence supports the hypothesis that TMA is associated with depressed intrarenal NO production. However, the effect of TMA on renal tissue NOS expression has not been fully elucidated. We studied rats with TMA induced by iv bolus injection of high dose (20 mg/kg) E. coli endotoxin. Subgroups of six animals each were sacrificed before or at 30, 90, 180, 360, and 720 minutes after the administration of endotoxin. Renal histology and tissue expression of endothelial and inducible nitric oxide synthases (eNOS and iNOS) were examined. Additionally, we examined the effect of endotoxin on glomerular NO production, and eNOS and iNOS protein expression in vitro. Glomerular capillary thrombosis developed by 180 minutes after endotoxin administration in approximately half of the animals. The glomeruli without thrombotic lesions apparent by light microscopy disclosed early signs of TMA characterized by endothelial swelling, platelet accumulation/adhesion, and patchy fibrinogen deposition. These morphologic changes were associated with a marked reduction of renal tissue eNOS expression beyond 180 minutes after the endotoxin administration. The fall in eNOS expression was coupled with a significant rise in iNOS protein abundance, which was expressed largely by glomerular circulating neutrophils and endothelial cells, peritubular vascular endothelium, and collecting ducts of cortex and medulla. In vitro incubation of isolated glomeruli with endotoxin also resulted in a marked reduction in eNOS expression and a significant rise in iNOS content. Administration of E. coli endotoxin leads to a sustained fall in renal eNOS expression both in vivo and in vitro. The associated decline in intrarenal endothelial NO production/availability may result in renal vasoconstriction and a hypercoagulative state, which may contribute to the pathogenesis of endotoxin-induced TMA.

Expression of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) and p53 tumor suppressor in dysplastic progression and adenocarcinoma in Barrett esophagus.

BACKGROUND: Barrett esophagus predisposes individuals to esophageal carcinoma, which develops from intermediate stages of tissue dysplasia primarily in the vicinity of the gastroesophageal junction. Understanding the cellular and molecular events in the progression of Barrett esophagus to adenocarcinoma may contribute to its early diagnosis and treatment. Mutation and overexpression of the tumor suppressor p53 have previously been observed in Barrett high grade dysplasia and adenocarcinoma. The expression of the cyclin-dependent kinase (CdK) inhibitor p21 can be up-regulated by p53, resulting in the down-regulation of cell division at the G(1)/S-phase transition. The current study examined the correlation between the expression of p21 and p53 by quantifying their levels during the progression of dysplasia and adenocarcinoma in Barrett esophageal tissues. METHODS: Barrett esophageal tissue samples that were negative or indefinite for dysplasia, contained dysplasia, and contained adenocarcinoma were examined by immunohistochemistry. Paraffin embedded sections of lining and glandular epithelia were adsorbed with primary murine antibodies against human p21 or p53 followed by horseradish peroxidase secondary antibody. An immunoreactivity score for each primary antibody and section was obtained by multiplying a staining intensity factor by the percent of positively stained cells. RESULTS: Nuclear p21 expression was detectable immunohistochemically in Barrett esophagus that was negative for dysplasia, but it was significantly elevated (P

Shock after blast wave injury is caused by a vagally mediated reflex.

OBJECTIVE: Bomb blast survivors occasionally suffer from profound shock and hypoxemia without signs of external injury. We hypothesize that a vagally mediated reflex such as the pulmonary defensive reflex is the cause of shock from blast wave injury. This study was a prospectively randomized, controlled animal study. METHODS: By using a previously described model of blast wave injury, we randomized rats to one of four groups: control, blast-only, bilateral cervical vagotomy plus atropine 200 microg/kg i.p. only, and bilateral cervical vagotomy plus atropine 200 microg/kg i.p. before blast injury. Cardiopulmonary parameters were recorded for 90 minutes after the blast or until death. RESULTS: Bradycardia, hypotension, and absence of compensatory peripheral vasoconstriction, typically seen in animals subjected to a blast pressure injury, were prevented by bilateral cervical vagotomy and intraperitoneal injection of atropine methyl-bromide. Hypoxia and lung injury were not statistically significant between the blasted groups, suggesting equivalent injury. CONCLUSION: Our data implicate a vagally mediated reflex such as the pulmonary defensive reflex as the cause of shock seen immediately after a blast pressure wave injury.

Prevention of rat mammary carcinoma utilizing leuprolide as an equivalent to oophorectomy.

A clinical trial is currently under way to examine the effectiveness of leuprolide as a breast cancer chemopreventive agent and contraceptive. This trial, as well as similar proposed studies, is based on the assumption that leuprolide is as effective as surgical castration in preventing the onset of mammary tumors; however, this has not been well documented in the DMBA animal model. We directly compared leuprolide and oophorectomy in this model and examined a combined therapy of leuprolide/bromocriptine. Twenty-seven day old female Sprague-Dawley rats were randomly allocated into one of eight groups. All rats received a 20-mg dose of DMBA at the age of 55 days. Group 1 (n = 10), no treatment; Group 2 (n = 9), leuprolide (100 microg/kg/day) for eight weeks beginning four weeks prior to DMBA; Group 3 (n = 10), oophorectomy four weeks prior to DMBA with replacement estrogen beginning four weeks following DMBA. Estrogen replacement was achieved with a 0.05-mg estradiol tablet releasing 0.833 microg/day over a 60-day period. Group 4 (n = 10), leuprolide (100 microg/kg/day) initiated two weeks prior to DMBA and continuing for two weeks following DMBA; Group 5 (n = 9), oophorectomy two weeks prior to DMBA with 0.05 mg of estradiol in depot form, releasing 0.833 microg/day, beginning four weeks following DMBA and continuing until week 16 of the study; Group 6 (n = 10), leuprolide (100 microg/kg/day) beginning two weeks prior to DMBA and continuing for the duration of the experiment; Group 7 (n = 10), leuprolide (100 microg/kg/day) for eight weeks beginning two weeks prior to DMBA; Group 8 (n = 9), leuprolide (100 microg/kg/day) and bromocriptine (83 microg/day) for eight weeks beginning two weeks prior to DMBA. At nineteen weeks (15 weeks post DMBA), animals were sacrificed and autopsies performed. One hundred percent of untreated animals developed tumors. No animals undergoing oophorectomy four weeks prior to DMBA or receiving leuprolide four weeks prior to and simultaneously with DMBA developed tumors. In animals pretreated two weeks prior to DMBA with leuprolide or oophorectomy, each group had one animal with tumor development. No tumors developed in the animals receiving ongoing injections of leuprolide. However, one tumor developed in those receiving leuprolide for the first eight weeks beginning two weeks prior to DMBA administration. One animal receiving both leuprolide and bromocriptine developed one tumor. We conclude that chemical oophorectomy (with leuprolide) is as effective as surgical oophorectomy in inhibiting DMBA induced carcinogenesis.

Global primary blast injury: a rat model.

Blast wave injury from bombs cause a unique but poorly understood spectrum of injuries. Previous blast wave models involved high energy explosives detonated in an open field without the sophisticated monitoring of laboratory equipment. We characterized a rodent model that produces a global blast injury in a safe laboratory environment. Male rats, prospectively randomized to four groups of ten, were anesthetized and subjected to a blast at 2.0 cm, 2.5 cm, or 3.5 cm from the blast nozzle. The control group received no blast. Intensity of the blast (80-120 psi peak pressure, 1-2 msec duration) was controlled by varying the distance of the blast wave generator to the rat. The rats were monitored for three hours following the blast and then euthanized. Bradycardia was an immediate but transient response to blast injury. Mean arterial pressure was bimodal with severe hypotension occurring immediately after the blast and, again, two to three hours later. The characteristic injuries from a blast wave, such as pulmonary hemorrhage with increased lung weight, intestinal serosal hemorrhage, and hemoperitoneum, were found in the rats subjected to the blast pressure wave. In conclusion, our rodent model accurately reproduces the clinical spectrum of injuries seen in blast victims and will provide a powerful tool for studying the pathophysiology and potential treatments of bomb blast victims.

Prevention of DMBA-induced rat mammary carcinomas comparing leuprolide, oophorectomy, and tamoxifen.

Leuprolide, a gonadotropin releasing hormone agonist, is currently being evaluated in a pilot study of premenopausal women for the prevention of breast cancer. However, little data is available regarding the efficacy of leuprolide in experimental animal models of carcinoma when administered prior to the carcinogen. In the present study the capacity of leuprolide to prevent tumor development was evaluated by comparing its pretreatment effects in the DMBA-induced rat mammary carcinoma model to pretreatment with tamoxifen and oophorectomy. Fifty-five day old, female Sprague-Dawley rats were randomly allocated to one of four groups: 1) no treatment; 2) oophorectomy two weeks prior to DMBA; 3) leuprolide, 40 microg/kg/day; and 4) tamoxifen, 10 mg/kg/week. All animals received four 5 mg doses of DMBA for a total dose of 20 mg. Leuprolide and tamoxifen treatments began two weeks prior to DMBA and ended one week after DMBA administration. Animals were assessed weekly to determine palpable tumor onset, number, size, and volume. At the conclusion of the study (16 weeks), autopsies were performed and tumor tissue was collected for confirmation of malignancy. Seventy-eight percent of the untreated rats developed tumors. No tumors developed in the oophorectomy group, while the number of rats with tumors was significantly reduced (p<0.05) with both leuprolide (30%) and tamoxifen (21.9%) compared to controls (77.8%). There were no significant differences in the tumor number for each tumor-bearing rat or in tumor volume between treated and control groups. Using our dosage regimen, 'chemical oophorectomy' with leuprolide was not as effective as surgical oophorectomy in the prevention of chemical carcinogenesis by DMBA but was comparable to the results obtained with tamoxifen.

Expression of peroxisome proliferator activated receptor mRNA in normal and tumorigenic rodent mammary glands.

The peroxisome proliferator activated receptors (PPARs) alpha, beta/delta, and gamma are novel nuclear hormone receptors activated by long chain fatty acids and synthetic ligands and which regulate lipid metabolism. Recent studies have detected PPARgamma mRNA in human mammary tumor cell lines. The current study examined the expression profile of PPAR mRNAs in normal and malignant rodent mammary tissues. Virgin murine mammary glands contained PPAR alpha, beta/delta, and gamma mRNAs based on northern blot analysis. The PPARgamma isoform was predominantly gamma2 based on quantitative PCR analysis. During pregnancy and lactation, the PPARalpha and gamma mRNAs decreased while the PPAR beta/delta mRNA remained relatively unchanged. NMuMG cells, an epithelial line derived from normal murine mammary gland, expressed PPAR alpha, beta/delta, and gamma mRNAs, independent of the presence or absence of compounds modifying PPAR activity. In rats, the physiologic expression pattern of PPARgamma mRNA paralleled the murine model; levels were detected in virgin but not lactating mammary glands. In addition, the PPARgamma mRNA was not detected in several histologically distinct 7,12-dimethylbenz(a)anthracene induced mammary tumors. These findings suggest that PPARs may regulate mammary epithelial and stromal cell function in response to physiologic or pathologic stimuli that profoundly alter lipid metabolism.

Cardiopulmonary physiology of primary blast injury.

OBJECTIVE: Bomb blast survivors are occasionally found in profound shock and hypoxic without external signs of injury. We investigated the cardiovascular and pulmonary responses of rats subjected to a blast pressure wave. DESIGN: Prospectively randomized, controlled animal study. MATERIALS AND METHODS: Rats were instrumented and subjected to a blast pressure wave of different intensities from a blast wave generator. Cardiopulmonary parameters were recorded for 3 hours or until death. MEASUREMENTS AND MAIN RESULTS: The cardiovascular response to a blast pressure wave was immediate bradycardia, hypotension, and low cardiac index. Three hours later, the rats developed hypotension, low cardiac index, and low stroke volume. Interestingly, systemic vascular resistance remained unchanged. The pulmonary response was a decreased PaO2 and stable PacO2, suggesting a ventilation-perfusion mismatch from massive pulmonary hemorrhage. CONCLUSIONS: Blast-induced circulatory shock resulted from immediate myocardial depression without a compensatory vasoconstriction. Hypoxia presumably resulted from a ventilation-perfusion mismatch caused by pulmonary hemorrhage.

A synthetic lipopolysaccharide-binding peptide based on the neutrophil-derived protein CAP37 prevents endotoxin-induced responses in conscious rats.

The lipid A component of lipopolysaccharide (LPS) derived from Escherichia coli has been implicated as a significant mediator in the development of circulatory and metabolic dysfunction and lethality associated with sepsis. A synthetic peptide corresponding to amino acid residues 20 through 44 of the neutrophil-derived 37-kDa cationic antimicrobial protein (CAP37 P(20-44)) possesses lipid A binding characteristics which may be useful in attenuating in vivo responses induced during circumstances of endotoxemia, including sepsis. The E. coli LPS to be used in the in vivo study was shown to be attenuated by CAP37 P(20-44) in a dose-dependent manner in the in vitro reaction with Limulus amoebocyte lysate. Intravenous infusion of CAP37 P(20-44) (1.5 or 3.0 mg/kg of body weight) with E. coli LPS (250 microg/kg over 30 min) into conscious, unrestrained rats prevented LPS-induced hyperdynamic and hypodynamic circulatory shock, hyperlactacidemia, and leukopenia in a dose-related fashion. CAP37 P(20-44) (0.2, 1.0, and 5.0 mg/kg) administered intravenously to conscious, actinomycin D-sensitized rats following a lethal dose of LPS neutralized LPS toxicity, resulting in dose-dependent 7-day survival rates of 30, 50, and 80%, respectively. CAP37 P(20-44) (5.0 mg/kg) significantly inhibited the endotoxin-induced increase in circulating tumor necrosis factor alpha in sensitized rats. These data demonstrate that CAP37 P(20-44) has the capacity to abolish in vivo biological responses to LPS that are relevant to human sepsis and to significantly neutralize the toxicity of circulating E. coli LPS.

Dose-response effect of in vivo administration of endotoxin on polymorphonuclear leukocytes oxidative burst.

In vitro, endotoxin primes polymorphonuclear leukocytes (PMNs) to respond with a greater oxidative burst. The purpose of the present study was to investigate the in vivo effect of a wide range of single endotoxin bolus doses using a rat model. PMNs were subsequently challenged in vitro with phorbol ester to produce reactive oxygen intermediates (ROI). Flow cytometric determination of ROI production by large doses induced a decrease in ROI production by the few PMNs that remained in the circulation. By 6 h after injection, ROI production had returned to basal levels after a high dose, and was still increasing after a low dose. Neutropenia occurred immediately after endotoxin injection. After 6 h, PMN counts returned to almost normal levels with a high dose, but rebound neutrophilia occurred with a small dose. In contrast to in vitro studies, in vivo injection showed a response pattern that varied widely with dose and time of observation.

Development of circulatory and metabolic shock following transient portal triad occlusion.

Liver ischemia is purposefully induced by portal triad occlusion (PTO) in several clinical situations including liver surgery for trauma, tumor, and transplantation. Despite significant morbidity from PTO, the hemodynamic and metabolic effects of PTO have not been evaluated relative to duration of ischemia. We investigated this using a total hepatic ischemia model. Rats received isoflurane anesthesia, carotid artery and jugular vein cannulation, and serial measurements of cardiac output (CO), mean arterial pressure (MAP), heart rate (HR), central venous pressure (CVP), stroke volume (SV), systemic vascular resistance (SVR), superior mesenteric artery blood flow (SMAF), intestinal vascular resistance (IVR), pH, pCO2, pO2, lactate, glucose, hematocrit (HCT), white blood cell count (WBC), and total neutrophils. Each group received 0, 15, 30, 45, or 60 min of PTO followed by 2 hr of reperfusion. All sham ischemia animals remained hemodynamically stable throughout the study. However, in the ischemic groups, there were significant time-dependent decreases in MAP, HR, CO, CVP, SV, SMAF, and pH, and increases in SVR, IVR, HCT, and lactate, while pCO2, pO2, glucose, and WBC remained stable. All of the ischemic animals survived except those that received 60 min of PTO. In this group, all of the animals survived the ischemic period; however, only one animal survived beyond 60 min of reperfusion. These data demonstrate a time-dependent circulatory and metabolic shock following PTO heralded by intestinal venous pooling and loss of intravascular fluid, and culminating in death. Careful hemodynamic monitoring and restoration of blood volume in the trauma patient may reduce morbidity and mortality.