Systemic and pulmonary hypertension after resuscitation with cell-free hemoglobin.

Human hemoglobin (Hb) and Hb cross-linked between the alpha subunits with bis(3,5-dibromosalicyl)fumarate (alpha alpha Hb) were used to treat hemorrhagic shock in water-deprived swine. Water was withheld for 48 h to induce a 10% loss of body mass, and 25 ml/kg of blood were removed in 1 h to produce circulatory shock. Swine were resuscitated with 1) Hb, 2) alpha alpha Hb, 3) human serum albumin, or 4) Ringer lactate. Mild high-output renal failure was observed in the non-cross-linked Hb-treated animals but not in other groups of animals. Swine treated with Hb and alpha alpha Hb had increases in plasma creatine kinase and lactate dehydrogenase activity that were resolved within 7 days. Both Hb- and alpha alpha Hb-treated swine displayed marked elevations of mean blood pressure in the systemic (39 +/- 6 Torr) and pulmonary (20 +/- 6 Torr) circulations that continued for 3 h and were associated with reduced cardiac output and a doubling of the systemic and pulmonary vascular resistances. Oxygen delivery was equivalent, and the rate of correction of the lactic acidosis was equal in all groups.

Delayed partial liquid ventilation shows no efficacy in the treatment of smoke inhalation injury in swine.

In an earlier neonatal porcine model of smoke inhalation injury (SII), immediate postinjury application of partial liquid ventilation (PLV) had dramatic beneficial effects on lung compliance, oxygenation, and survival over a 24-h period. To explore the efficacy of PLV following SII, we treated animals at 2 and 6 h after SII and followed them for 72 h. Pigs weighing 8-12 kg were sedated and pharmacologically paralyzed, given a SII, and placed on volume-cycled, pressure-limited ventilation. Animals were randomized to three groups: group I (+SII, no PLV, n = 8), group II (+SII, PLV at 2 h, n = 6), and group III (+SII, PLV at 6 h, n = 7). Ventilatory parameters and arterial blood gasses were obtained at scheduled intervals. The PLV animals (groups II and III) followed a worse course than group I (no PLV); PLV groups had higher peak and mean airway pressures, oxygenation index, and rate-pressure product (a barotrauma index) and lower lung compliance and arterial partial pressure of oxygen-to-inspired oxygen fraction ratio (all P < 0.05). PLV conferred no survival advantage. The reported beneficial effects of PLV with other models of acute lung injury do not appear to extend to the treatment of SII when PLV is instituted in a delayed manner. This study was not able to validate the previously reported beneficial effects of PLV in SII and actually found deleterious effects, perhaps reflecting the predominance of airway over alveolar disease in SII.

Loss of chromosomal integrity drives rat mammary tumorigenesis.

Breast cancer incidence varies with diet and other environmental influences, including carcinogen exposure. However, the effects of carcinogens on cell growth control pathways are poorly understood. Here, we have examined processes that are activated in the mammary glands of rats treated with 1-methyl-1-nitrosourea (MNU). This synthetic carcinogen was used to study events occurring during mammary tumor initiation and development. In female Wistar-Furth rats, given 1 dose of MNU beginning at 50 days of age, 84% of the rats developed tumors by 46 weeks of age (latency 13-15 weeks). Changes in the gland occurred as early as 1-day post-MNU. Cells exhibited DNA damage, leading to chromosomal instability, supernumerary centrosomes and higher levels of Aurora A; these events correlated with the appearance of preneoplasia in the glands. In mammary tumors, elevated numbers of centrosomes coincided with genomic instability. Tumors were transplanted into syngeneic hosts and subsequent tumor generations displayed the same marker chromosomes in mostly aneuploid metaphases with hyperdiploid numbers of chromosomes, suggesting that clonality and aneuploidy were passed on from one generation to the next. Collectively, these data suggest that the carcinogen MNU induces changes resulting in genetic instability detectable before hyperplasia and tumors develop in the rat mammary gland.

Acute and subacute dermal toxicity of Break-Free CLP: a weapons cleaning and maintenance compound.

Break-Free CLP((R)) is a commercial cleaning, lubricating and preserving compound used in both the military and civilian sectors for maintenance of small- and large-caliber weapons. Like many commercial mixtures, there is very little information available on the toxicity of Break-Free CLP. Studies were conducted to characterize the biological effects of single or repeat dermal application of Break-Free CLP to the clipped backs of CD-1 mice. Break-Free CLP was applied neat, 50 microl three times of week for up to 2 weeks. Foci of epithelial ulceration were observed in skin sections from 22% of Break-Free CLP-treated animals in conjunction with markedly thickened epithelium suggesting that robust epithelial regeneration was occurring in these animals. Skin histopathology of Break-Free CLP-treated animals closely matched the histopathology from mice treated repeatedly with 2% croton oil in acetone (dermal irritation positive control). Serum alkaline phosphatase activity was significantly (P < 0.05) lower for mice treated with Break-Free CLP, 2% croton oil or 7,12-dimethylbenz[a]anthracene (DMBA) compared with negative and vehicle control mice. Skin nitric oxide (NO) levels were not significantly elevated for mice treated with Break-Free CLP but were significantly elevated for mice treated with dermal irritation positive control compound DMBA. The cumulative skin changes in Break-Free CLP-treated animals support conducting a subchronic dermal application study. The observed decreases in serum alkaline phosphatase activity suggest that future studies should include the liver and bone as possible target organs. Additionally, dermal penetration studies could provide key health risk assessment information for characterizing the potential health risks associated with chronic dermal exposure to Break-Free CLP.

Microtubules: a brief historical perspective.

Microtubules are cylindrical organelles of varying length, an overall diameter of 25 nm, and a central hollow core of approximately 5 nm thick. They constitute one of three major structural components of the cytoskeleton and are found in almost all eukaryotic cells, where they perform a variety of essential functions, including chromosome movement, vesicular traffic, beating of cilia and flagella, and maintenance of cell form and morphogenesis. Although microtubules were discovered by transmission electron microscopy in the late 1950s, biophysical evidence for their dynamic structure and function in mitosis was obtained almost a decade earlier through polarizing microscopy. Research on the morphology and molecular structure of microtubules spans 3 decades and is now entering an exciting new "biophysical" era, as described in this issue.

Systemic chemotherapy of transitional cell carcinoma of the urothelium.

Although the incidence of bladder cancer has increased in recent years, survival has also improved. Chemotherapy has made a substantial impact on this disease and now is used in patients with advanced or metastatic disease as well as in select patients with high-risk muscle invasive disease. While cisplatin remains the most active single antineoplastic agent, several other agents including methotrexate, vinblastine, and Adriamycin (doxorubicin) have important activity. More recently, paclitaxel and gemcitabine have shown promising activity in bladder cancer. Multidrug combination therapy has provided more frequent and durable responses than single agent therapy. Regimens containing cisplatin and methotrexate have been shown to be most effective in the treatment of advanced disease. Adjuvant chemotherapy regimes typically have included cisplatin or cisplatin and methotrexate combinations. However, studies have been limited and further prospective trials are required to determine the role of adjuvant chemotherapy. Multiple studies have investigated neoadjuvant chemotherapy with cisplatin and methotrexate combinations or anthracycline-based regimens, but study results are mixed. Further trials will be required to define the role of neoadjuvant chemotherapy in bladder cancer.

Intraoperative use of the absorbable fibrin adhesive bandage: long term effects.

PURPOSE: The absorbable fibrin adhesive bandage (AFAB) reduces acute blood loss in experimental trauma models, but the effects on wound healing and subsequent function have heretofore not been investigated. Retropubic prostatectomy was selected to evaluate short and long term effects of using the AFAB intraoperatively. MATERIALS AND METHODS: Dogs undergoing prostatectomy were randomly assigned to one of four treatments: CONTROL- sponges and manual pressure were applied after transecting the prostatic pedicles. Sponges were removed when the prostate was delivered. Vessels were isolated and ligated if bleeding continued after removal. AFAB- hemostatically active bandages were applied to the prostatic bed prior to sponges and pressure. Additional bandages were applied at the urethrovesical junction after completing the anastomosis. PLACEBO- visually identical (hemostatically inert) bandages were applied in an identical fashion. LIQUID SEALANT- concentrated thrombin and fibrinogen solution was applied to the vessels prior to sponges and pressure. Additional sealant solution was applied around the anastomosis. RESULTS: Blood loss and time to achieve hemostasis were significantly less in the AFAB group compared with the other treatments. There were no differences in days to anastomotic integrity, continence, or intra-abdominal adhesions at necropsy six weeks later. CONCLUSIONS: The AFAB can reduce surgery time and blood loss, with no decrement in wound healing or subsequent function.

Staphylococcus aureus enterotoxin B challenge of monkeys: correlation of plasma levels of arachidonic acid cascade products with occurrence of illness.

Arachidonic acid cascade products have been shown to be increased in vitro in Staphylococcus aureus enterotoxin B (SEB)-treated epithelial cell cultures in our laboratory. In order to confirm that these products were clinically related to SEB intoxication, monkeys were administered SEB by nasogastric intubation. It caused emesis in five of six monkeys (less than 4 h), and the sixth monkey showed signs of mild illness. The monkeys which vomited continued to display signs of gastrointestinal illness beyond 8 h but were without any apparent signs of illness by 24 h. Blood samples were collected prior to SEB administration, upon first indication of illness, and at twice that time interval. One week prior to SEB treatment, the same monkeys were administered saline by nasogastric intubation and in every way handled similarly in order to serve as their own controls. Blood samples were taken from the control animals at 0, 4, and 8 h. The plasma concentrations of prostaglandin E2 (PGE2), leukotriene B4 (LTB4), and 5-hydroxyeicosatetraenoic acid (5-HETE) did not vary significantly throughout the 8-h experiment for saline-treated controls, nor did they differ from the concentrations found in the plasma of monkeys just before administration of SEB. When the SEB-treated monkeys showed the first indication of illness (less than 4 h), the mean of the concentration in plasma of PGE2 increased 1.44-fold, that of LTB4 increased 2.23-fold, and that of 5-HETE was essentially unchanged. At twice the time interval of the first display of illness (less than 8 h), PGE2 was still elevated (1.48-fold), LTB4 had decreased slightly to 1.66-fold, and 5-HETE had soared (3,45-fold), suggesting a divergence in the enzymatic utilization of the parent compound of the latter two metabolites, 5-hydroperoxyeicosatetraenoic acid. These studies suggest that arachidonic acid cascade metabolites were a consequence of SEB intoxication and may provide a logical site for metabolic interference in SEB-induced toxicity.

The C terminus of mitosin is essential for its nuclear localization, centromere/kinetochore targeting, and dimerization.

Mitosin is a novel 350-kDa nuclear phosphoprotein that dramatically relocates from the evenly nuclear distribution in S phase to the centromere/kinetochore and mitotic apparatus in M phase. The dynamic relocalization of mitosin is accompanied by the phosphorylation of itself, suggesting that mitosin plays a role in mitotic progression. The molecular basis of nuclear localization and targeting of mitosin to the centromere/kinetochore were characterized using a set of epitope-tagged deletion mutants. The data indicate that the extreme C terminus (amino acids 2,487-3,113) of mitosin has both an independent centromere/kinetochore targeting domain and an unusually spaced bipartite nuclear localization signal. Moreover, the same centromere/kinetochore targeting domain was shown to be essential for the ability of mitosin to bind to itself or other putative mitosin-associated proteins through use of the yeast two-hybrid system. These results suggest that the C terminus of the mitosin is essential for its role in influencing cell cycle progression.

The subcellular localization of acetyl-CoA carboxylase 2.

Animals, including humans, express two isoforms of acetyl-CoA carboxylase (EC ), ACC1 (M(r) = 265 kDa) and ACC2 (M(r) = 280 kDa). The predicted amino acid sequence of ACC2 contains an additional 136 aa relative to ACC1, 114 of which constitute the unique N-terminal sequence of ACC2. The hydropathic profiles of the two ACC isoforms generally are comparable, except for the unique N-terminal sequence in ACC2. The sequence of amino acid residues 1-20 of ACC2 is highly hydrophobic, suggesting that it is a leader sequence that targets ACC2 for insertion into membranes. The subcellular localization of ACC2 in mammalian cells was determined by performing immunofluorescence microscopic analysis using affinity-purified anti-ACC2-specific antibodies and transient expression of the green fluorescent protein fused to the C terminus of the N-terminal sequences of ACC1 and ACC2. These analyses demonstrated that ACC1 is a cytosolic protein and that ACC2 was associated with the mitochondria, a finding that was confirmed further by the immunocolocalization of a known human mitochondria-specific protein and the carnitine palmitoyltransferase 1. Based on analyses of the fusion proteins of ACC-green fluorescent protein, we concluded that the N-terminal sequences of ACC2 are responsible for mitochondrial targeting of ACC2. The association of ACC2 with the mitochondria is consistent with the hypothesis that ACC2 is involved in the regulation of mitochondrial fatty acid oxidation through the inhibition of carnitine palmitoyltransferase 1 by its product malonyl-CoA.

Rapid purification of staphylococcal enterotoxin B by high-pressure liquid chromatography.

The Staphylococcus aureus enterotoxins represent a group of proteins that cause emesis and diarrhea in humans and other primates. We have developed a rapid two-step high-pressure liquid chromatography (HPLC) procedure for purification of staphylococcal enterotoxin B (SEB). Sterile filtrates (2.5 liters) of strain 10-275 were adsorbed directly onto a reversed-phase column (50 mm by 30 cm Delta Pak; 300 A [30 nm], 15 microns, C18). SEB was obtained by using a unique sequential gradient system. First, an aqueous ammonium acetate to acetonitrile gradient followed by an aqueous trifluoroacetic acid (TFA) wash was used to remove contaminants. A subsequent TFA to acetonitrile-TFA gradient eluted the bound SEB. Further purification was obtained by rechromatography on a cation-exchange column. From 35 to 45% of the SEB in starting filtrates was recovered. Analysis by immunoblotting of samples separated on sodium dodecyl sulfate-polyacrylamide gels indicated that HPLC-purified SEB exhibited immunological and biochemical properties similar to those of the SEB standard. Induction of an emetic response in rhesus monkeys showed that the HPLC-purified toxin also retained biological activity.

Effect of JP-8 jet fuel on molecular and histological parameters related to acute skin irritation.

Organic chemicals such as jet fuels and solvents can cause skin irritation after dermal exposure. The molecular responses to these chemicals resulting in acute irritation are not understood well enough to establish safe exposure limits. Male F-344 rats were dermally exposed to JP-8 jet fuel for 1 h using Hill Top Chambers. Whole skin samples were collected at 0, 1, 2, 4, and 6 h after the beginning of the exposures, homogenized, and analyzed for interleukin (IL)-1alpha and inducible nitric oxide synthase (iNOS) protein and nitrite levels. IL-1alpha levels (determined by ELISA) ranged from approximately 11 to 34% above the 0-h samples over the observed time period. At 1 and 2 h, significantly higher (p < 0.05) levels of IL-1alpha were detected when compared to the 0-h samples. Western blot analysis revealed significantly higher (p < 0.05) levels of iNOS at 4 and 6 h compared to 0-h samples. Increases in IL-1alpha and iNOS expression were also observed in the skin immunohistochemically. Nitrite concentrations in skin samples were measured to estimate nitric oxide production. Although nitrite concentrations in the skin increased approximately 6-27% above the 0-h samples over the observed time period, no significant changes in nitrite levels were detected. Pathological changes in the skin following JP-8 exposure were evaluated histologically. Increased numbers of granulocytes were observed infiltrating the skin at 2 h and were more prominent by 6 h. These data show that a 1-h exposure to JP-8 results in a local inflammatory response, which can be detected by changes in molecular and histological parameters.

Effects of long-term hemofiltration on circulating mediators and superoxide production during continuous endotoxin administration.

BACKGROUND: The purpose of this study was to test whether continuous hemofiltration eliminates cytokines and eicosanoids, or stimulates granulocyte function. METHODS: Nineteen pigs were divided into a control group (n = 7), a hemofiltration group (n = 7), and an extracorporeal circuit only group (n = 5). All animals received the same amount of intravenous endotoxin and resuscitation fluid. Zero-balanced hemofiltration was started 30 minutes after initiation of endotoxemia and continued throughout the experiment. Plasma endotoxin, tumor necrosis factor-alpha, eicosanoids, superoxide production, and other physiologic parameters were measured before challenge and at scheduled intervals thereafter. RESULTS: Eicosanoids were filtered but plasma concentrations were not reduced. Tumor necrosis factor-alpha was not filtered or adsorbed. There were no significant differences between groups in any measured parameters. CONCLUSION: Continuous hemofiltration could not efficiently remove tumor necrosis factor-alpha or eicosanoids. Also, continuous hemofiltration did not stimulate production of the proinflammatory mediators measured, nor improve respiratory distress.

Effects of burns on inhalation injury.

BACKGROUND: There are few studies of smoke injury combined with thermal burn. METHODS: Seven sheep (G1) received smoke injury alone; eight (G2) received a 40% full-thickness scald burn immediately after smoke injury. All animals were resuscitated with lactated Ringer's solution and killed 48 hours after injury. Cardiopulmonary variables and blood gases were measured serially. Ventilation perfusion distribution was analyzed using the multiple inert gas elimination technique. Lung wet to dry weight ratio and malondialdehyde levels were determined. RESULTS: G2 resulted in early significant hemodynamic changes. Serum total protein concentration was significantly lower and malondialdehyde significantly higher in G2. However, PaO2, lung wet to dry weight ratio, and ventilation perfusion mismatching in G2 did not differ from those in G1. CONCLUSIONS: Although the addition of burn injury exaggerated the lung lipid peroxidation and hypoproteinemia in the presence of more pronounced hemodynamic changes, the pulmonary dysfunction was not accentuated.

Effect of Sulfo Lewis C on smoke inhalation injury in an ovine model.

OBJECTIVE: To evaluate the effect of Sulfo Lewis C (SO3-3âGal1-3GlcNAc-O(CH2)8-COOMe), a putative ligand of selectins, on smoke inhalation injury. DESIGN: Prospective animal study with concurrent controls. SETTING: An animal laboratory. SUBJECTS: Twelve 1-yr-old female sheep, weighing 24 to 33 kg. INTERVENTIONS: Twelve sheep received nine exposure units of smoke generated by thermolysis of pine woodchips (80 g). Group 1 (n = 6) was untreated. Group 2 (n = 6) was treated with an intravenous infusion of Sulfo Lewis C after smoke exposure. Animals were killed 48 hrs after injury. MEASUREMENTS AND MAIN RESULTS: Cardiopulmonary variables and blood gases were measured serially. Granulocyte free-radical production was measured before smoke exposure and at 4 and 48 hrs after injury. Ventilation/perfusion distribution (VA/Q) was analyzed using the multiple inert gas elimination technique. Granulocyte free-radical production was increased after smoke exposure in both groups. Oxygenation was significantly improved by the administration of Sulfo Lewis C. VA/Q analysis demonstrated significantly less blood flow to low VA/Q lung segments in treated animals. CONCLUSIONS: Selectin blockade attenuated lung injury after smoke exposure. These data support the hypothesis that neutrophils play a pivotal role in smoke inhalation injury.

Selectin blockade worsened lipopolysaccharide-induced lung injury in a swine model.

BACKGROUND: Polymorphonuclear leukocytes have been reported to play an important role in various acute lung injuries. Neutrophil recruitment into tissues is a multistep process involving sequential engagement of adhesion molecules. The objective of this study was to determine the effect of selectin inactivation with Sulfo Lewis C (SO3-3betaGal1-3betaGlcNAc-O(CH2)8-COOMe) on the pulmonary response to lipopolysaccharide (LPS) infusion. METHODS: All animals (n = 11) were pretreated with an intramuscular injection of a priming dose of Escherichia coli LPS (10 microg/kg). Eighteen hours later, animals received an intravenous infusion of LPS (20 microg/kg) over 20 minutes. All animals were resuscitated with a lactated Ringer's solution. Group I (G1; n = 5) received no additional treatment. Group II (G2; n = 6) received a bolus injection of Sulfo Lewis C (10 mg/kg) 10 minutes before LPS insult followed by a continuous infusion (1 mg/kg per hour) for the rest of the study. Animals were observed for 5 hours from initiation of the LPS infusion and killed. Cardiopulmonary variables and blood gases were measured serially. The multiple inert gas elimination technique (MIGET) was used to evaluate the matching of air flow and blood flow in the lung 5 hours after LPS infusion. Histologic evaluation of the parenchymal injury was performed by using light microscopy. The number of polymorphonuclear leukocytes and red blood cells in the alveolar spaces per field at 400x magnification were counted in 10 randomly selected fields. RESULTS: Hypoxemia, indexed as Pao2/FIO2, was exacerbated by the administration of Sulfo Lewis C (G1:437+/-33 vs. G2: 241+/-63 mm Hg at 5 hours, p<0.03). This finding is supported by the multiple inert gas elimination technique analysis, which demonstrated significantly greater blood flow to true shunt in G2 (G1:4.42+/-1.75 vs. G2:23.2+/-5.69, p<0.02). There was no difference between the two groups in red blood cell counts in the alveolar spaces. However, polymorphonuclear leukocyte counts were significantly greater in G2 (G1:1.8+/-0.58 vs. G2:9.9+/-2.34, p<0.01). CONCLUSION: Selectin blockade significantly worsened lung injury induced by LPS infusion, and greater numbers of neutrophils were observed in alveolar spaces in the group treated with Sulfo Lewis C. These findings are supported by the multiple inert gas elimination technique analysis, which demonstrated significantly greater blood flow to the true shunt compartment in treated animals. Further studies are required to determine the role of selectins in sepsis-induced lung injury.