Tumor necrosis factor (cachectin) is an endogenous pyrogen and induces production of interleukin 1.

Recombinant human tumor necrosis factor (rTNF alpha) injected intravenously into rabbits produces a rapid-onset, monophasic fever indistinguishable from the fever produced by rIL-1. On a weight basis (1 microgram/kg) rTNF alpha and rIL-1 produce the same amount of fever and induce comparable levels of PGE2 in rabbit hypothalamic cells in vitro; like IL-1, TNF fever is blocked by drugs that inhibit cyclooxygenase. At higher doses (10 micrograms/kg) rTNF alpha produces biphasic fevers. The first fever reaches peak elevation 45-55 min after bolus injection and likely represents a direct action on the thermoregulatory center. During the second fever peak (3 h later), a circulating endogenous pyrogen can be shown present using passive transfer of plasma into fresh rabbits. This likely represents the in vivo induction of IL-1. In vitro, rTNF alpha induces the release of IL-1 activity from human mononuclear cells with maximal production observed at 50-100 ng/ml of rTNF alpha. In addition, rTNF alpha and rIFN-gamma have a synergistic effect on IL-1 production. The biological activity of rTNF alpha could be distinguished from IL-1 in three ways: the monophasic pyrogenic activity of rIL-1 was destroyed at 70 degrees C, whereas rTNF alpha remained active; anti-IL-1 neutralized IL-1 but did recognize rTNF alpha or natural cachectin nor neutralize its cytotoxic effect; and unlike IL-1, rTNF alpha was not active in the mitogen-stimulated T cell proliferation assay. The possibility that endotoxin was responsible for rTNF alpha fever and/or the induction of IL-1 was ruled-out in several studies: rTNF alpha produced fever in the endotoxin-resistant C3H/HeJ mice; the IL-1-inducing property of rTNF alpha was destroyed either by heat (70 degrees C) or trypsinization, and was unaffected by polymyxin B; pyrogenic tolerance to daily injections of rTNF alpha did not occur; levels of endotoxin, as determined in the Limulus amebocyte lysate, were below the minimum rabbit pyrogen dose; and these levels of endotoxin were confirmed by gas chromatography/mass spectrometry analysis for the presence of beta-hydroxymyristic acid. Although rTNF alpha is not active in T cell proliferation assays, it may mimic IL-1 in a T cell assay, since high concentrations of rTNF alpha induced IL-1 from epithelial or macrophagic cells in the thymocyte preparations. These studies show that TNF (cachectin) is another endogenous pyrogen which, like IL-1 and IFN-alpha, directly stimulate hypothalamic PGE2 synthesis. In addition, rTNF alpha is an endogenous inducer of IL-1.(ABSTRACT TRUNCATED AT 400 WORDS)

The diagnosis and treatment of non-cardiac thoracic trauma.

Penetrating and blunt force mechanisms frequently result in thoracic trauma. Thoracic injuries cover the spectrum from trivial to lethal, and more than half are associated with head, abdomen or extremity trauma. Fortunately over eighty percent of injuries can be managed non-operatively utilizing tube thoracostomy, appropriate analgesia and aggressive respiratory therapy. Patients requiring emergency thoracotomy are either in shock or have life threatening injuries and, as expected, have significant mortality and morbidity. Injury to the thorax directly accounts for approximately 25% of trauma related mortality and is a contributing factor in another 25%. Early mortality results from haemorrhage, catastrophic injury or associated head or abdominal trauma. Not unexpectedly, late deaths are related to sepsis and organ failure. Blunt injury to the thorax most commonly results from motor vehicle collisions, with motorcycle accidents, pedestrians struck and falls next in frequency. Stab wound and gunshot wounds comprise the vast majority of penetrating injuries. In general the mortality from penetrating injury is higher and related to vascular injury and shock. Mortality from blunt trauma often results from abdominal and, especially, head injury. Rapid assessment and interventions, such as tube thoracostomy and airway control, can be life saving. The patient's haemodynamic status drives early treatment, often necessitating emergency surgery. Detailed imaging studies are reserved for haemodynamically stable patients. The evaluation and treatment of specific thoracic injuries will be discussed, as well as some general principles in treating thoracic trauma.

Computational and experimental models of the human torso for non-penetrating ballistic impact.

Both computational finite element and experimental models of the human torso have been developed for ballistic impact testing. The human torso finite element model (HTFEM), including the thoracic skeletal structure and organs, was created in the finite element code LS-DYNA. The skeletal structure was assumed to be linear-elastic while all internal organs were modeled as viscoelastic. A physical human surrogate torso model (HSTM) was developed using biosimulant materials and the same anthropometry as the HTFEM. The HSTM response to impact was recorded with piezoresistive pressure sensors molded into the heart, liver and stomach and an accelerometer attached to the sternum. For experimentation, the HSTM was outfitted with National Institute of Justice (NIJ) Level I, IIa, II and IIIa soft armor vests. Twenty-six ballistic tests targeting the HSTM heart and liver were conducted with 22 caliber ammunition at a velocity of 329 m/s and 9 mm ammunition at velocities of 332, 358 and 430 m/s. The HSTM pressure response repeatability was found to vary by less than 10% for similar impact conditions. A comparison of the HSTM and HTFEM response showed similar pressure profiles and less than 35% peak pressure difference for organs near the ballistic impact point. Furthermore, the peak sternum accelerations of the HSTM and HTFEM varied by less than 10% for impacts over the sternum. These models provide comparative tools for determining the thoracic response to ballistic impact and could be used to evaluate soft body armor design and efficacy, determine thoracic injury mechanisms and assist with injury prevention.

The influence of carbohydrate structure on the clearance of recombinant tissue-type plasminogen activator.

Modification of the carbohydrate structures of recombinant tissue-type plasminogen activator (rt-PA) can increase or decrease its rate of clearance in rabbits. When rt-PA was treated with sodium periodate to oxidize carbohydrate residues, the rate of clearance was decreased from 9.6 +/- 1.9 ml min-1 kg-1 to 3.5 +/- 0.6 ml min-1 kg-1 (mean +/- SD, n = 5). A similar change in the clearance of rt-PA was introduced by the use of endo-beta-N-acetyl-glucosaminidase H (Endo-H), which selectively removes high mannose asparagine-linked oligosaccharides; the clearance of Endo-H-treated rt-PA was 5.0 +/- 0.5 ml min-1 kg-1. A mutant of rt-PA was produced with an amino acid substitution at position 117 (Asn replaced with Gln) to remove a potential glycosylation site that normally contains a high mannose structure. The clearance of this material was also decreased, similar to the periodate and Endo-H-treated rt-PA. Conversely, when rt-PA was produced in the CHO 15B cell line, which can produce only high mannose oligosaccharide structures on glycoproteins, the clearance was increased by a factor of 1.8. These results demonstrate that the removal of rt-PA from the blood depends significantly upon the nature of its oligosaccharide structures.

The protective effect of profound hypothermia on the canine central nervous system during one hour of circulatory arrest.

Circulatory arrest during profound hypothermia is a safe technique of cardiac surgery when used in selected instances. Despite its proven safety, the degree of cerebral protection offered by this technique is still poorly defined. Ten dogs anesthetized with Pentothal (thiopental sodium) were surface cooled to 32 degrees C. They were placed on cardiopulmonary bypass, cooled to 13 degrees C (cerebral temperature), and then underwent one hour of circulatory arrest. At the end of the arrest period, the dogs were rewarmed, resuscitated, and successfully weaned from bypass. A control group of 6 dogs were subjected to the same protocol but without the one-hour period of circulatory arrest. There were no group differences in animal weight, duration of surface cooling, cardiopulmonary bypass, or rewarming, mean flow, or mean arterial pressure. After a 7-day observation period, the dogs were killed with rapid tissue fixation using formalin. No neurological deficits were noted in any of the dogs during the observation period. The fixed brains were examined by a neuropathologist. No gross or microscopic evidence of cerebral hypoxia was seen in any of the animals. We conclude that one hour of circulatory arrest under profoundly hypothermic temperatures produces no detectable neurological changes or histological evidence of cerebral hypoxia.

Use of stapling instruments in surgery for aneurysms of the aorta.

Since their inception, surgical stapling devices have been used almost exclusively in pulmonary and gastrointestinal procedures. We present our experience with surgical staplers in operations for aneurysms of the aorta. Three illustrative case reports are presented that demonstrate the applicability of surgical stapling devices in excluding aortic aneurysms. Seven patients have undergone operation using this technique, all with excellent technical results. We believe that surgical stapling devices represent a safe, easy, and rapid means of excluding aneurysms of the aorta.

Thoracic splenosis.

Thoracic splenosis is a rare pathologic entity resulting from seeding of splenic tissue in the pleural cavity after thoracoabdominal trauma. A 45-year-old man with a history of splenectomy secondary to abdominal trauma presented with a left lung mass and an inconclusive tissue diagnosis after needle biopsy. Thoracic splenosis was not suspected preoperatively, considered on an intraoperative frozen section, and established on permanent pathologic biopsy specimens obtained during thoracotomy. A history of thoracoabdominal trauma, combined with radiologic and radionuclide imaging studies, may establish the diagnosis without thoracotomy.

Cricothyroidotomy for prolonged ventilatory support after cardiac operations.

Forty-nine patients required prolonged ventilatory support after cardiac operations. Cricothyroidotomy was used routinely in these patients after approximately 7 days of endotracheal intubation. There were no infections of the median sternotomy wounds despite frequent colonization of the stoma. The only immediate complication was mild stomal bleeding in a patient taking anticoagulants. Nineteen patients (39%) died of underlying disease. The average duration of cricothyroidotomy was 59 days (range, 3 to 270 days). Cannulas were successfully removed in all survivors after an average of 38 days (range, 6 to 187 days). All of the patients were followed by personal interview, telephone contact, or contact with the referring physician. The average length of follow-up was 17 months (range, 2 to 50 months). All symptomatic patients were evaluated by laryngoscopy and bronchoscopy. One patient required endoscopic removal of granulation tissue from the stomal site; 2 others required tracheal resection for stenosis at the balloon site. There were no instances of subglottic stenosis. There were 4 late deaths, none of which was related to the cricothyroidotomy. Based on these findings, we suggest that cricothyroidotomy, with its low complication rate, is the procedure of choice for patients requiring prolonged mechanical ventilation after cardiac operations.

The structure of the exopolysaccharide of Pseudomonas fluorescens strain H13.

An acidic exopolysaccharide was isolated from P. fluorescens strain H13. The structure of the polysaccharide repeating unit was determined using chemical methods and 1D and 2D NMR techniques. The repeating unit was characterized as a trisaccharide composed of D-glucose, 2-acetamido-2-deoxy-D-glucose and 4-O-acetyl-2-acetamido-2-deoxy-D-mannuronic acid.

Preparation, isolation, and characterization of cutin monomers and oligomers from tomato peels.

Cutin in tomato peels was depolymerized in methanolic base to yield cutin monomers or a mixture of cutin oligomers. These products were isolated by typical solvent extraction methods or by precipitation, and the isolates were characterized by chromatographic and spectroscopic analyses. It was determined that the compositions of the isolates from both isolation procedures were similar, although solvent extraction gave higher yields. However, the precipitation method, which is easy to carry out and avoids the use of undesirable organic solvents, may be preferable in commercial processes for recovering these compounds.

Chromatography of recombinant proteins.

Variants of intact polypeptides/proteins ranging in mass from 6,500 to 70,000 Da were easily separated using reversed-phaseHPLC (rpHPLC) or affinity chromatography. A variant of rhlGF-I, where the racemization of a serine residue was detected in the intact molecule, was resolved from rhlGF-I within 25 minutes by rpHPLC. Other variants of rhlGF-I separated by this method include methionine sulphoxide at position 59, des Gly1, des Gly1Pro2, Glu for Asp substitution at position 20 and incorrectly folded IGF-I. For rhDNase (approximately 40 kDa), affinity chromatography was able to clearly resolve three different amino acids (Asn, Asp and iso-Asp) at position 74 of the intact glycoprotein. The presence or absence of O-linked sugars on Thr -37 of recombinant human thrombopoietin was rapidly demonstrated by rpHPLC. While the separation of these types of variants is essential, the demonstration of biological activity is critical for designing specifications that allow the administration of these proteins into humans. Once a correlation exists between the variant and its biological activity, control of the manufacturing process can be better achieved with analytical methodology.

A pacemaker monitoring system.

The use of cardiac pacemaker technology has greatly decreased the morbidity and mortality caused by certain cardiac arrhythmias. Its use, however, has not been without complications, including broken or dislodged pacemaker leads, failure of the pacemaker to sense the QRS complex, and cardiac arrhythmias. This paper presents a straightforward and inexpensive monitoring system which detects four specific pacemaker failure states.

A report of the treatment of coronary artery aneurysm without cardiopulmonary bypass.

Coronary artery aneurysm (CAA) is a fairly rare pathologic entity whose exact incidence is unknown but has been reported from 1.4% in autopsy series [Daoud, 1963] to 4.9% in the Coronary Artery Surgery Registry [Swaye, 1984]. While atherosclerosis is the most common cause of true coronary artery aneurysms, pseudoaneurysms most often occur as complications of percutaneous transluminal coronary angioplasty (PTCA) or directional artherectomy [Dralle, 1995]. We report the successful treatment of a coronary artery aneurysm with comcomitant coronary revascularization without the use of cardiopulmonary bypass in a patient with impaired pulmonary and myocardial function. The use of an intracoronary shunt, previously described by one of the authors (EW), facilitated the surgical procedure [Franzone 1977].

alpha and beta-glycopyranosyl phosphates and 1.2-phosphates. Assignments of conformations in solution by 13C and 1H NMR.

The 1H and 13C NMR parameters of the anomeric pairs of aldopyranosyl phosphates and their rigid 1,2-phosphate derivatives are reported.The derivatives of D-glucose, D-galactose, and D-mannose exist in the 4C1 conformation while the L-fuco derivatives are in the C4 conformation. As judged by 31P--1H and 31P--13C coupling constants, all of the alpha anomers of the aldopyranosyl phosphates have the phosphate moiety predominantly trans to C(2) while in the beta anomers other rotamers make significant contributions. This relationship remains the same for the biologically important nucleoside diphosphate sugars (UDPGlc, UDPGal, GDPMan, and GDPFuc). From the pH dependence of 13C chemical shifts, observed in 0.5 M solutions, the pK'a2 of the alpha anomers is 6.1 while the pK'a2 of the beta anomers is 0.6--0.8 pH unit lower. In the 1.2-phosphates, the chair conformation of the parent aldose is retained while an envelope conformation is formed by the cyclic phosphate. In the alpha anomers, the plane is formed between C(2), C(1), O(1), and P while O(2) is above the plane. In the beta anomers, O(1) is out of the plane formed by the other atoms. The beta anomers have phosphorus coupled to C(3) with coupling constants of 10.8--11.7 Hz, approximately 2 Hz greater than the maximum reported for trans coupling (Lapper, R. D., & Smith, I. C. P. (1973) J. Am. Chem. Soc. 95, 2880).

Control of recombinant DNA produced pharmaceuticals by a combination of process validation and final product specifications.

Traditional methods of product release for human pharmaceutical use are designed and selected to ensure control of purity, potency, safety and identity. The selection of these tests depends upon the nature of the product. In addition to the control achieved by these methods, control of the efficiency of the manufacturing process in removing substances that cannot be tested for in the final product is achieved by "process validation". Process validation allows the use of sophisticated experimental techniques that do not lend themselves to final product testing or the use of others which, when applied to the final product, are insufficiently sensitive to be used for product release. The removal of such components as DNA and process materials can be demonstrated by these studies. Demonstrations that the manufacturing process reproducibly removes these components, eliminate the need for these tests on every batch. The essential elements of a process validation study are development of a sensitive assay technique for the component of interest and quantitation of the efficiency of individual process steps in removing that component. Even when that component is undetectable in normal process samples, the efficiency of any process step can still be determined by the addition of that component during the validation studies. The values of process validation in demonstrating the removal of DNA and in the determination of E. coli protein contamination levels during the manufacture of methionyl human growth hormone is illustrated.

A convenient microscale colorimetric method for terminal galactose on immunoglobulins.

A new approach for quantitative determination of terminal galactose (Gal) residues of immunoglobulins was developed by combining exoglycosidase digestion with the classical colorimetric estimation of reducing sugars. The ferricyanide colorimetric method was modified to increase the stability of the chromophore (Prussian blue) and adapted to determine the amount of terminal Gal residues present in immunoglobulins. The method involves the release of covalently bound Gal from immunoglobulins by Diplococcus pneumoniae beta-D-galactosidase (specific for beta(1,4) linked galactose), removal of the glycoprotein and enzyme from the reaction mixture by heat denaturation or ethanol precipitation, followed by colorimetric measurement of the released sugar using the ferricyanide assay. The ferricyanide method was modified to enhance the solubility and stability of the chromophore by increasing the concentration of aqueous sulfuric acid and sodium dodecyl sulfate (SDS). The linear range of the modified method was from approximately 11 to 111 microM Gal. Typical variation in assay results was on the order of 5%. Using the modified method, the terminal Gal content of a recombinant chimeric monoclonal antibody (anti-CD20, rIgG) expressed in Chinese hamster ovary (CHO) cells was determined and evaluated for batch-to-batch consistency. The method was used to optimize pH, time, temperature, and enzyme concentration for beta-galactosidase digestion for maximal release of terminal Gal residues from rIgG.