Integrated molecular signature of disease: analysis of influenza virus-infected macaques through functional genomics and proteomics.

Recent outbreaks of avian influenza in humans have stressed the need for an improved nonhuman primate model of influenza pathogenesis. In order to further develop a macaque model, we expanded our previous in vivo genomics experiments with influenza virus-infected macaques by focusing on the innate immune response at day 2 postinoculation and on gene expression in affected lung tissue with viral genetic material present. Finally, we sought to identify signature genes for early infection in whole blood. For these purposes, we infected six pigtailed macaques (Macaca nemestrina) with reconstructed influenza A/Texas/36/91 virus and three control animals with a sham inoculate. We sacrificed one control and two experimental animals at days 2, 4, and 7 postinfection. Lung tissue was harvested for pathology, gene expression profiling, and proteomics. Blood was collected for genomics every other day from each animal until the experimental endpoint. Gross and microscopic pathology, immunohistochemistry, viral gene expression by arrays, and/or quantitative real-time reverse transcription-PCR confirmed successful yet mild infections in all experimental animals. Genomic experiments were performed using macaque-specific oligonucleotide arrays, and high-throughput proteomics revealed the host response to infection at the mRNA and protein levels. Our data showed dramatic differences in gene expression within regions in influenza virus-induced lesions based on the presence or absence of viral mRNA. We also identified genes tightly coregulated in peripheral white blood cells and in lung tissue at day 2 postinoculation. This latter finding opens the possibility of using gene expression arrays on whole blood to detect infection after exposure but prior to onset of symptoms or shedding.

Detection of beta-defensins secreted by human oral epithelial cells.

Human beta-defensins are antimicrobial peptides that may be critical in the innate immune response to infection. hBD1 and hBD2 are expressed in oral epithelial cells and are detected near the surface of oral tissue, consistent with a role in the epithelial protective barrier function. In this report, we examine secretion of beta-defensins in vitro and in biological fluid using ProteinChip(R) Array, surface enhanced laser desorption/ionization (SELDI) technology combined with time-of-flight mass spectrometry. We show that the 47-amino acid form of hBD1 and the 41-amino acid form of hBD2 are the major secreted forms. These forms are both expressed and secreted under conditions anticipated from previous analysis of beta-defensin mRNAs; specifically, hBD1 is detected in culture supernatant from both unstimulated and stimulated cells, and hBD2 is detected only in stimulated cells. Identity of hBD1 and hBD2 was confirmed by immunocapture on the ProteinChip surface. Both peptides are also present in gingival crevicular fluid that accumulates between the tissue and tooth surface, although hBD1 is also found in several smaller forms suggesting extracellular proteolysis. This methodology offers several technical advantages for detection of defensins in biological fluids, including ease and speed of screening, no need for HPLC preliminary processing, and small sample size.

Identification of prostate specific membrane antigen (PSMA) as the target of monoclonal antibody 107-1A4 by proteinchip; array, surface-enhanced laser desorption/ionization (SELDI) technology.

Recently we described the generation of the prostate tissue-specific monoclonal antibody (MAb) 107-1A4, its expression pattern and preliminary targeting of human prostate cancer xenografts. In this report we demonstrate that the target antigen for MAb 107-1A4 is prostate-specific membrane antigen (PSMA) using immunoaffinity absorption followed by SDS-PAGE and mass spectrometric analysis of peptides produced by in-gel tryptic digestion. The identity of the antigen has been confirmed by Western blots using MAbs of known specificity. MAb 107-1A4 is not reactive on Western blots. The conformational epitope for 107-1A4 is on the extracellular domain of PSMA. In competition studies, the binding of MAb 107-1A4 to LNCaP cells is inhibited by itself but not by any other of several other anti-PSMA MAbs, suggesting that the epitope may be unique. These results suggest that 107-1A4 is reactive to a conformational epitope in the external domain of PSMA that is unique among the panel of anti-PSMA MAbs in this study. Furthermore this work demonstrates the ability of mass spectroscopy to elucidate antibody-ligand interaction.

Direct demonstration that homotetrameric chaperone SecB undergoes a dynamic dimer-tetramer equilibrium.

We have shown here that the cytosolic bacterial chaperone SecB is a structural dimer of dimers that undergoes a dynamic equilibrium between dimer and tetramer in the native state. We demonstrated this equilibrium by mixing two tetrameric species of SecB that can be distinguished by size. We showed that the homotetrameric species exchanged dimers, because when the mixture was analyzed both by size exclusion chromatography and native polyacrylamide gel electrophoresis a third hybrid tetrameric species was detected. Furthermore, treatment of SecB with 5,5'-dithiobis-(2-nitrobenzoic acid), which modifies the sulfhydryl group on cysteines, caused irreversible dissociation to a dimer indicating that cysteine must be involved in the stabilizing interactions at the dimer interface. It is clear that the two dimer-dimer interfaces of the SecB tetramer are differentially stable. Dissociation at one interface allows for a dynamic dimer-tetramer equilibrium. Because only dimers were exchanged it is clear that the other interface between dimers is significantly more stable, otherwise oligomers should have formed with a random distribution of monomers.

Complexes between protein export chaperone SecB and SecA. Evidence for separate sites on SecA providing binding energy and regulatory interactions.

During localization to the periplasmic space or to the outer membrane of Escherichia coli some proteins are dependent on binding to the cytosolic chaperone SecB, which in turn is targeted to the membrane by specific interaction with SecA, a peripheral component of the translocase. Five variant forms of SecB, previously demonstrated to be defective in mediating export in vivo (Gannon, P. M., and Kumamoto, C. A. (1993) J. Biol. Chem. 268, 1590-1595; Kimsey, H. K., Dagarag, M. D., and Kumamoto, C. A. (1995) J. Biol. Chem. 270, 22831-22835) were investigated with respect to their ability to bind SecA both in solution and at the membrane translocase. We present evidence that at least two regions of SecA are involved in the formation of active complexes with SecB. The variant forms of SecB were all capable of interacting with SecA in solution to form complexes with stability similar to that of complexes between SecA and wild-type SecB. However, the variant forms were defective in interaction with a separate region of SecA, which was shown to trigger a change that was correlated to activation of the complex. The region of SecA involved in activation of the complexes was defined as the extreme carboxyl-terminal 21 aminoacyl residues.

Is routine roentgenography needed after closed tube thoracostomy removal?

BACKGROUND: Efficacy of chest radiograph protocol after tube thoracostomy tube (CT) removal. METHODS: Retrospective review (July of 1995 to July of 1996) of 141 patients with CT followed throughout their hospitalization. Excluded patients died (23 patients) or had thoracotomy (13 patients) before CT removal. RESULTS: A total of 105 patients had 113 CT removed (mean age, 36.9 years; Injury Severity Score = 23.4; CT duration, 5.0 days). Protocol chest radiographs were performed on average at 7.9 and 22.1 hours. Recurrent pneumothorax (RHPTX = new interpleural air) occurring in 12 patients (11%) and persistent pneumothorax (PHPTX = same volume of interpleural air) occurring in 13 patients (12%) caused no clinical problems and were treated without tube replacement. Three patients had symptoms after removal; none had RHPTX. Two patients had clinical signs; one reaccumulated a hemothorax requiring CT replacement, the other improved without replacement. CONCLUSIONS: Clinically significant RHPTX/PHPTX after CT removal is infrequent. Signs not symptoms detect CT removal complications. At our institution, chest radiographs are obtained in a delayed manner from protocol and offer no benefit over clinical assessment.

The financial impact of teaching surgical residents in the operating room.

BACKGROUND: There have been no published data regarding the cost of training surgical residents in the operating room. METHODS: At the University of Tennessee Medical Center-Knoxville, in addition to resident-performed teaching cases, some cases are performed without the assistance of residents by the same faculty. RESULTS: Sixty-two case categories involving 14,452 cases were compared for operative times alone. In 46 case categories (10,787 procedures), resident operative times were longer than faculty alone. In 16 case categories, resident operating times were shorter than faculty times. The net incremental operative time cost was 2,050 hours between July 1993 and March 1997. Assuming 4 years of operative training for 11 graduating chief residents, the cost per graduating resident was $47,970. CONCLUSION: Extrapolated to a national annual cost for the 1,014 general surgery residents who completed training in the 1997 academic year, the annual cost of training residents in the operating room is $53 million. This high monetary cost suggests the need for digital skills, selection criteria, the development of training curriculum and resource facilities, the pre-operating room need for suturing and stapling techniques, and perhaps the acquisition of virtual surgery training modules.

Kinetic partitioning. Poising SecB to favor association with a rapidly folding ligand.

Chaperones are a class of proteins that possess the remarkable ability to selectively bind polypeptides that are in a nonnative state. The selectivity of SecB, a molecular chaperone in Escherichia coli, for its ligands can be explained in part by a kinetic partitioning between folding of the polypeptide and association with SecB. It has clearly been established that kinetic partitioning can be poised to favor association with SecB by changing the rate constant for folding of the ligand. We now demonstrate that binding to SecB can be given a kinetic advantage over the pathway for folding by modulating the properties of the chaperone. By poising SecB to expose a hydrophobic patch, we were able to detect a complex between SecB and maltose-binding protein under conditions in which rapid folding of the polypeptide otherwise precludes formation of a kinetically stable complex. The data presented here are interpreted within the framework of a kinetic partitioning between binding to SecB and folding of the polypeptide. We propose that exposure of a hydrophobic patch on SecB increases the surface area for binding and thereby increases the rate constant for association. In this way association of SecB with the polypeptide ligand has a kinetic advantage over the pathway for folding.

Prospective study of blunt aortic injury: Multicenter Trial of the American Association for the Surgery of Trauma.

BACKGROUND: Blunt aortic injury is a major cause of death from blunt trauma. Evolution of diagnostic techniques and methods of operative repair have altered the management and posed new questions in recent years. METHODS: This study was a prospectively conducted multi-center trial involving 50 trauma centers in North America under the direction of the Multi-institutional Trial Committee of the American Association for the Surgery of Trauma. RESULTS: There were 274 blunt aortic injury cases studied over 2.5 years, of which 81% were caused by automobile crashes. Chest computed tomography and transesophageal echocardiography were applied in 88 and 30 cases, respectively, and were 75 and 80% diagnostic, respectively. Two hundred seven stable patients underwent planned thoracotomy and repair. Clamp and sew technique was used in 73 (35%) and bypass techniques in 134 (65%). Overall mortality was 31%, with 63% of deaths being attributable to aortic rupture; mortality was not affected by method of repair. Paraplegia occurred postoperatively in 8.7%. Logistic regression analysis demonstrated clamp and sew (p = 0.002) and aortic cross clamp time of > or = 30 minutes (p = 0.01) to be associated with development of postoperative paraplegia. CONCLUSIONS: Rupture after hospital admission remains a major problem. Although newer diagnostic techniques are being applied, at this time aortography remains the diagnostic standard. Aortic cross clamp time beyond 30 minutes was associated with paraplegia; bypass techniques, which provide distal aortic perfusion, produced significantly lower paraplegia rates than the clamp and sew approach.

Predictors of survival after inferior vena cava injuries.

In patients with inferior vena cava (IVC) injuries, predictors of survival are investigated. From 1987 to 1995, 27 IVC injuries were identified among 514 patients with vascular trauma. The ability of clinical determinants to predict survival were retrospectively assessed. IVC injuries occurred in 7 females and 20 males (mean age, 27.7 +/- 2.5 years) from both blunt (n = 14) and penetrating (n = 13) trauma. The mean revised trauma score was 10.2 +/- 0.6. Injuries were treated by primary repair (n = 22), ligation (n = 4), or prosthetic grafting (n = 1). Thirteen patients died (48%), 10 within 12 hours of admission. Suprahepatic (n = 2), retrohepatic (n = 12), suprarenal (n = 1), and infrarenal (n = 12) injuries were associated with 100, 67, 100, and 20 per cent mortality, respectively. Blood transfusions (16 +/- 4 vs 23 +/- 4 units), coagulation factor replacement (7 +/- 2 vs 7 +/- 2 units), and electrolyte solution use (8.6 +/- 1.4 vs 9.6 +/- 1.4 L) were similar among survivors and nonsurvivors. Four complications [venous hypertension (n = 2), IVC thrombosis (n = 1), and pulmonary embolus (n = 1)] occurred in the 14 survivors (28.6%). Blunt injury, revised trauma score, free perforation, injury location, intraoperative hypotension, and blood loss were predictive of mortality. IVC injuries remain extremely lethal, and improved survival is associated with infrarenal penetrating injuries and a contained hematoma.

Concomitant orthopedic and vascular injuries as predictors for limb loss in blunt lower extremity trauma.

Lower extremity trauma with concomitant orthopedic and vascular injury is associated with a high degree of limb loss. Despite successful arterial repair, many patients will ultimately require amputations. The effect of associated orthopedic injuries on limb loss in patients with lower extremity arterial injuries is investigated. From 1989 to 1994, 52 infrainguinal arterial injuries were identified among 365 vascular trauma patients. Clinical determinants were retrospectively assessed for the ability to predict postoperative amputations. Femoral artery injuries were present in 23 patients, with 53 per cent the result of blunt trauma. The incidence of lower extremity fractures was 53 per cent (60% open). Seventy-nine per cent of femoral artery injuries were repaired with saphenous vein grafts. Popliteal artery injuries were present in 13 patients, with 77 per cent the result of blunt trauma. The incidence of lower extremity fractures and posterior knee dislocations was 85 per cent (73% open) and 38 per cent, respectively. Ninety-two per cent of injuries were repaired with saphenous vein grafts. Tibial artery injuries were present in 16 patients, with 44 per cent the result of blunt trauma. The incidence of lower extremity fractures was 81 per cent (46% open). Twenty-five per cent of tibial artery injuries were treated with arterial repair, and 31 per cent with arterial ligation. Fourteen above-knee (AKA) and two below-knee amputations were performed. Amputation rates were 26.1 per cent (three AKAs) for femoral, 53.8 per cent (seven AKAs) for popliteal, and 38 per cent (four AKAs, two below-knee amputations) for tibial artery injuries. At the popliteal and femoral locations, greater than two long-bone fractures was predictive of amputation. For tibial arteries, one-vessel (n = 10), two-vessel (n = 3), and three-vessel (n = 3) injuries were associated with 20, 33, and 100 per cent amputations rates, respectively. Blunt injury, pulseless extremity, need for arterial repair (rather than ligation or no therapy), increasing number of injured tibial vessels, and multiple long-bone fractures were predictors of amputation (P < 0.05). Distal vascular injuries combined with complex orthopedic fractures are more likely to result in limb loss. Two or more long-bone fractures is predictive of amputation at all three locations.

Regulation of GLUT1-mediated sugar transport by an antiport/uniport switch mechanism.

Avian erythrocyte sugar transport is stimulated during anoxia and during exposure to inhibitors of oxidative phosphorylation. This stimulation results from catalytic desuppression of the cell surface glucose transporter GLUT1 [Diamond, D., & Carruthers, A. (1993) J. Biol. Chem. 268, 6437-6444]. The present study was undertaken to investigate the mechanisms of GLUT1 suppression/desuppression. Sugar uniport (sugar uptake or exit in the absence of sugar at the opposite side of the membrane) is absent in normoxic avian erythrocytes, but sugar antiport (sugar uptake coupled to sugar exit) is present. Exposure to cyanide and/or to FCCP (mitochondrial inhibitors) stimulates erythrocyte sugar uniport but not sugar antiport. K(m)(app) for 3-O-methylglucose uniport and antiport are unaffected by metabolic poisoning. Ki(app) for inhibitions of 3-O-methylglucose uniport by cytochalasin B and forskolin (sugar export site ligands) are unaffected by progressive stimulation of sugar uniport. Cyanide and FCCP stimulation of 3-O-methylglucose uniport are associated with increased AMP-activated protein kinase activity. Purified human GLUT1 is not phosphorylated by exposure to cytosol extracted from poisoned avian erythrocytes. FCCP does not stimulate GLUT1-mediated 3-O-methylglucose uptake in K562 cells but does increase K562 AMP-activated protein kinase activity. FCCP stimulation of 3-O-methylglucose uniport in resealed erythrocyte ghosts requires cytosolic ATP and/or glutathione. The nonmetabolizable ATP analog AMP-PNP cannot be substituted for ATP in this action. These results are contrasted with allosteric regulation of human erythrocyte sugar transport and suggest that avian erythrocyte sugar transport suppression results from inhibition of carrier uniport function. Uniport suppression is not mediated by interaction with cytosolic molecular species that bind to the sugar export site. The antiport to uniport switch mechanism requires ATP hydrolysis, is associated with elevated AMP-activated kinase function, and, if triggered by this kinase, is mediated by factors absent in K562 cells and downstream from the kinase.

Early tracheostomy in trauma patients.

A retrospective analysis of 118 trauma patients who underwent tracheostomy for airway and pulmonary management was undertaken. Timing of the procedure was defined as early (0-3 days), intermediate (4-7 days), and late (> 7 days). Head injury patients received tracheostomy early (p < 0.00003). Aspiration evaluated by modified bedside aspiration test was a frequent occurrence in all three groups with no difference in incidence (p < 0.34). Pneumonia was less frequent in the early group compared with the intermediate and late groups (p < 0.0034). The incidence of pneumonia in the early group was not different from that observed in early extubated patients (n = 282; p < 0.23). Our study suggests that early tracheostomy may decrease pulmonary septic complications in trauma patients. Although no change in length of stay can be attributed to the early performance of tracheostomy, preventing pneumonia in the intensive care unit setting with its resulting high expense is beneficial.

Blunt carotid injury: reassessing the role of anticoagulation.

Twenty patients with blunt mechanism injuries to the internal carotid artery were treated over a 10-year period. The purpose of this review is to assess the necessity and timing of anticoagulation as a primary therapeutic modality in patients with this injury. Sixteen patients (80%) survived, the majority with normal neurologic function. Twelve patients with either unilateral or bilateral carotid mural injury were anticoagulated. Ten survived with normal neurologic function. Five patients in the heparin anticoagulant group had a significant delay in the initiation of anticoagulation. All recovered without deficits. Two patients were treated with aspirin alone and recovered without deficits. Two patients received no treatment; one survived without deficits, and the nonsurvivor succumbed to a severe head injury. Therefore, a total of nine patients were either not treated with heparin or had a significant delay in the initiation of heparin. Eight of nine (88%) in this group recovered without deficit. A delay in the initiation of heparin therapy, no therapy, or the institution of antiplatelet therapy may all be appropriate in the initial management of mural injuries of the internal carotid artery.

Craniofacial trauma and cerebrospinal fluid leakage: a retrospective clinical study.

PURPOSE: The study examines the efficacy of antibiotic treatment in patients with traumatic cerebrospinal fluid (CSF) leakage and identifies a consistent clinical approach for better management of these patients. PATIENTS AND METHODS: A retrospective study of traumatic CSF leakage in 88 patients with craniofacial fractures was performed. The mechanism of injury, etiology and source of leakage, decision to treat conservatively (without surgery) versus aggressively (with surgery), decision to use early versus late repair, and decision to provide prophylactic antibiotic coverage were recorded. Both timing of repair and the decision to use antibiotics were compared with the incidence of meningitis resulting from CSF leakage. RESULTS: Of 48 patients treated with antibiotics, 5(10.4%) developed meningitis. In the remaining 40 who were not treated with antibiotics, only one patient acquired meningitis. Five of 53 (9.4%) patients with conservative treatment developed meningitis versus 1 of 35 (2.9%) after aggressive treatment. CONCLUSION: There was no statistically significant difference in the rate of occurrence of meningitis between the conservative and the aggressive treatment group. However, the data suggest that there may be no benefit in treating these patients with antibiotics to prevent meningitis, whereas aggressive treatment and early repair of facial fractures may, in fact, be helpful.

Hypothermic blood substitution enables resuscitation after hemorrhagic shock and 2 hours of cardiac arrest.

Uncontrollable hemorrhage accounts for a large proportion of total mortality in both civilian (31%) and military (47%) trauma victims. Hypothermia is a relatively safe method that could provide total body protection during hypovolemic shock and facilitate surgical intervention as a potentially life-saving procedure. This study tested the hypothesis that profound hypothermia and complete blood replacement in an established canine model, would facilitate resuscitative therapy from exsanguinating hypovolemic shock. Adult dogs were prepared for extracorporeal bypass using closed-chest peripheral cannulation under general anesthesia. Controlled hypotensive, hemorrhagic shock (mean arterial blood pressure < 50 mmHg) was induced for 30 min at normal temperature followed by temporary resuscitation using crystalloid infusion for approximately 10 min. Using our established procedure, the dogs were then cooled externally to 27 degrees C before initiating blood substitution with Hypothermosol (Cryomedical Sciences, Inc. Rockville, MD) via the extracorporeal pump. The heart was arrested during further cooling to below 10 degrees C and Hypothermosol was recirculated for 2 hr, with (3 dogs) or without (5 dogs) 1 hr of circulatory arrest. During rewarming the animals were autotransfused, weaned from the pump, and allowed to recover. All dogs (n = 8) survived, all but one with complete neurologic recovery: blood chemistry samples examined immediately after the procedure showed significant differences (p < 0.05) in only a few parameters, including creatine kinase (CK-BB and CK-MB), compared with the previous group of control dogs. The consistent survival of dogs showing apparently normal neurologic, physiologic, and biochemical recovery supports the concept that profound hypothermia using a protective hypothermic blood substitute could provide time for therapeutic resuscitation of currently intractable trauma cases.

Interaction of SecB with intermediates along the folding pathway of maltose-binding protein.

SecB, a molecular chaperone involved in protein export in Escherichia coli, displays the remarkable ability to selectively bind many different polypeptide ligands whose only common feature is that of being nonnative. The selectivity is explained in part by a kinetic partitioning between the folding of a polypeptide and its association with SecB. SecB has no affinity for native, stably folded polypeptides but interacts tightly with polypeptides that are nonnative. In order to better understand the nature of the binding, we have examined the interaction of SecB with intermediates along the folding pathway of maltose-binding protein. Taking advantage of forms of maltose-binding protein that are altered in their folding properties, we show that the first intermediate in folding, represented by the collapsed state, binds to SecB, and that the polypeptide remains active as a ligand until it crosses the final energy barrier to attain the native state.

The role of a substance abuse consultation team in a trauma center.

OBJECTIVE: Trauma is a leading cause of morbidity and mortality in the U.S., and substance abuse makes an enormous contribution to this problem as well as to the increased cost of health care. A substance abuse consultation (SAC) team was developed to evaluate and intervene with trauma victims who are suspected of having problems with alcohol and/or other drugs. This study was undertaken to determine the effectiveness of this service. METHOD: This study is a retrospective review of 100 consecutive trauma admissions who were felt to be at high risk for substance abuse and were referred to the SAC team for evaluation and intervention. This study was undertaken in a tertiary care teaching hospital in a cooperative effort between the trauma center and the addictions program. RESULTS: All 100 patients were diagnosed with psychoactive substance use disorders and 78 were referred for alcohol and other drug rehabilitation. Of these patients, 62% accepted a referral for drug and alcohol treatment. CONCLUSIONS: From these preliminary data, it appears that a SAC team may be effective in intervening with hospitalized trauma patients who have alcohol and other drug problems.