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OBJECTIVE: To identify a safe and potentially effective recombinant tissue factor pathway inhibitor (rTFPI) dose for further clinical evaluation in patients with severe sepsis. DESIGN: Prospective, randomized, single-blind, placebo-controlled, dose escalation, multicenter, multinational phase II clinical trial. SETTING: Thirty-eight intensive care units in the United States and Europe. PATIENTS: Two hundred and ten subjects with severe sepsis who received standard supportive care and antimicrobial therapy. INTERVENTIONS: Subjects received a continuous intravenous infusion of placebo or rTFPI at 0.025 or 0.05 mg/kg/hr for 4 days (96 hrs). MEASUREMENTS AND MAIN RESULTS: There were no significant imbalances in demographics, severity of illness, or source of infection in patients randomized to placebo or either dose of rTFPI. A 20% relative reduction in 28-day all-cause mortality was observed when all rTFPI-treated patients were compared with all placebo patients. An improvement in pulmonary organ dysfunction score and in a composite intensive care unit score (pulmonary, cardiovascular, and coagulation) were also noted in the rTFPI-treated patients. Logistic regression modeling indicated a substantial treatment by baseline laboratory international normalized ratio (INR) interaction effect when only treatment and INR were in the model (p =.037) and when baseline Acute Physiology and Chronic Health Evaluation (APACHE II) and log10 interleukin 6 were adjusted for (p =.026). This interaction effect indicates that higher baseline INR is associated with a more pronounced beneficial rTFPI effect. There was no increase in mortality in subjects treated with either dose of rTFPI compared with placebo. Biological activity, as detected by a statistically significant reduction in thrombin-antithrombin complexes (TATc), was noted in the all rTFPI-treated patients compared with those receiving placebo. There were no major imbalances across all treatment groups with respect to safety. The frequency of adverse events (AEs) and severe adverse events (SAEs) was similar among the treatment groups, with a slight increase in SAEs and SAEs involving bleeding in the 0.05 mg/kg/hr rTFPI group. The overall incidence of AEs involving bleeding was 28% of patients in the all placebo group and 23% of patients in the all rTFPI-treated group; a slight but statistically insignificant increase in incidence of SAEs involving bleeding was observed in the all rTFPI group (9%) as compared with the all placebo group (6%; p =.39). CONCLUSIONS: Although the trial was not powered to show efficacy, a trend toward reduction in 28-day all-cause mortality was observed in the all rTFPI group compared with all placebo. This study demonstrates that rTFPI doses of 0.025 and 0.05 mg/kg/hr could be safely administered to severe sepsis patients. Additionally, rTFPI demonstrated bioactivity, as shown by reduction in TATc complexes and interleukin-6 levels. These findings warrant further evaluation of rTFPI in an adequately powered, placebo controlled, randomized trial for the treatment of severe sepsis.

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BACKGROUND: Despite the availability of potent antibiotics and intensive care, mortality rates from septic shock are 40-70%. We assessed the safety and efficacy of murine monoclonal antibody to human tumour necrosis factor alpha (TNF alpha MAb) in the treatment of septic shock. METHODS: In a randomised, multicentre, double-blind, placebo-controlled clinical trial in 105 hospitals in the USA and Canada, we randomly assigned 1879 patients a single infusion of 7.5 mg/kg TNF alpha MAb (n=949) or placebo (0.25% human serum albumin n=930). Our main outcome measurement was the rate of all-cause mortality at 28 days. FINDINGS: 382 (40.3%) of 948 patients who received TNF alpha MAb and 398 (42.8%) of 930 who received placebo had died at 28 days (95% CI -0.02 to 0.07, p=0.27). We found no association between therapy with TNF alpha MAb and increased rapidity in reversal of initial shock or prevention of subsequent shock. Similarly, baseline plasma interleukin-6 concentrations of more than 1000 pg/mL or detectable circulating TNF concentrations were not associated with improvement in survival after TNF alpha MAb therapy. Coagulopathy but not other organ or system failures, was significantly decreased in the TNF alpha MAb group compared with placebo (day 7, p<0.001; day 28, p=0.005). Serious adverse events were reported in 55.2% of patients given placebo and 54.1% in the TNF alpha MAb group. INTERPRETATION: We did not find an improvement in survival after septic shock with TNF alpha MAb. Therapy not solely dependent on TNF alpha blockade may be required to improve survival.

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The UDP-GlcNAc/MurNAc family of eukaryotic and prokaryotic enzymes use UDP-GlcNAc or UDP-MurNAc-pentapeptide as donors, dolichol-P or polyprenol-P as acceptors, and generate sugar-P-P-polyisoprenols. A series of six conserved sequences, designated A through F and ranging from 5 to 13 amino acid residues, has been identified in this family. To determine whether these conserved sequences are required for enzyme function, various mutations were examined in hamster UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT). Scramble mutations of sequences B-F, generated by scrambling the residues within each sequence, demonstrated that each is important in GPT. While E and F scrambles appeared to prevent stable expression of GPT, scrambling of B-D resulted in GPT mutants that could be stably expressed and bound tunicamycin, but lacked enzymatic activity. Further, the C and D scramble mutants had an unexpected sorting defect. Replacement of sequences B-F with prokaryotic counterparts from either the B.subtilis mraY or E.coli rfe genes also affected GPT by preventing expression of the mutant protein (B, F) or inhibiting its enzymatic activity (C-E). For the C-E replacements, no acquisition of acceptor activity for polyprenol-P, the fully unsaturated natural bacterial acceptor, was detected. These studies show that the conserved sequences of the UDP-GlcNAc/MurNAc family are important, and that the eukaryotic and prokaryotic counterparts are not freely interchangeable. Since several mutants were efficiently expressed and bound tunicamycin, yet lacked enzymatic activity, the data are consistent with these sequences having a direct role in product formation.

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STUDY OBJECTIVES: To determine the optimal treatment of empyema thoracis (within the fibrinopurulent phase of illness) comparing pleural drainage and fibrinolytic therapy vs video-assisted thoracoscopic surgery (VATS), with regard to efficacy and duration of hospitalization. DESIGN: Twenty patients with confirmed parapneumonic empyema thoracis were randomized to chest tube pleural drainage plus streptokinase (CT-SK) vs VATS. SETTING: University-based teaching hospital providing for Dallas County. PATIENTS AND METHODS: Equivalent groups of patients with parapneumonic empyema thoracis were randomized to receive either of two therapies: CT-SK (n=9) or VATS (n=11). Outcomes analysis with respect to treatment efficacy, hospital duration, chest tube duration, hospital costs, and need for subsequent procedures was performed. RESULTS: Each group suffered one mortality (p=not significant). When compared with the CT-SK group, the VATS group had a significantly higher primary treatment success [10/11, 91% vs 4/9, 44%; p<0.05 Fisher's Exact Test], lower chest tube duration (5.8+/-1.1 vs 9.8+/-1.3 days; p=0.03), and lower number of total hospital days (8.7+/-0.9 vs 12.8+/-1.1 days; p=0.009). Clinically relevant but not statistically significant differences in hospital costs ($16,642+/-2,841 vs $24,052+/-3,466, p=0.11) also favored the VATS group. Of note, all the CT-SK treatment failures could be salvaged with VATS, and none required thoracotomy. CONCLUSIONS: In patients with loculated, complex fibrinopurulent parapneumonic empyema thoracis, a primary treatment strategy of VATS is associated with a higher efficacy, shorter hospital duration, and less cost than a treatment strategy that utilizes catheter-directed fibrinolytic therapy.

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Nosocomial pneumonia is a large and growing problem in American hospitals. This article reviews the epidemiology, risk factors, microbiology, natural history, and presentation of nosocomial pneumonia. Pathogenesis is also reviewed, and practical measures for prevention are stressed.

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Spontaneous pneumothorax (SP) secondary to the acquired immunodeficiency syndrome (AIDS) emerged in the decade of the 1980s. It has become an increasingly difficult condition to treat successfully both for the pulmonary internist and the surgeon. AIDS-related SP is complicated by a virulent form of necrotizing subpleural necrosis that results in diffuse air leaks that are refractory to the standard, traditional forms of therapy which enjoy good success for SP related to classic subpleural bleb disease. AIDS-related SP carries a high mortality rate despite treatment, independent of the development of primary respiratory failure. In reviewing our experience of 46 patients from a single institution treated over the past 10 years, we found that due to the high primary and secondary treatment failure rates, an aggressive stepped-care management of large-bore intercostal tube drainage, chemical pleurodesis, and early video-assisted talc poudrage is recommended in an attempt to shorten the duration of hospital stay, hospital costs, and mortality.

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Cell-surface carbohydrates mediate the adherence of many pathogenic bacteria to epithelial cells. Because gram-negative bacteria adhere especially well to respiratory epithelial cells obtained from severely ill patients, we compared respiratory epithelial cell-surface carbohydrate levels of normal subjects with those of critically ill patients. Lectins were used to quantitate the amount of mannose, galactose, fucose, and sialic acid on buccal and tracheal cells. Fifteen critically ill patients, 20 normal subjects, and 10 minimally ill hospitalized patients were studied. The severely ill patients' buccal cells had decreased amounts of sialic acid and galactose. No differences were found between the normal and critically ill patients' tracheal-cell carbohydrates. The results obtained with a sialic acid-specific lectin were confirmed by direct measurement of buccal-cell sialic acid. We conclude that severely ill patients have decreased amounts of galactose and sialic acid on their upper-airway epithelial cells, and that loss of these two monosaccharides may explain the high prevalence of gram-negative bacterial colonization and pneumonia in the critically ill.

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Exoglycosidases remove peripheral monosaccharides from oligosaccharides and hence are capable of altering respiratory epithelial cell surface carbohydrates. We obtained saliva and tracheal secretions from 34 critically ill patients and saliva from 23 healthy subjects. Compared with the normal subjects, the ill patients had large amounts of mannosidase, fucosidase, hexosaminidase, and sialidase activity. Sialidase increased adherence of several gram-negative bacteria to epithelial cell monolayers and pure glycoproteins. Pretreatment of glycoproteins with some of the patients' saliva samples also increased bacterial adherence to the glycoproteins. We conclude that respiratory tract exoglycosidase activity increases during critical illness. By altering normal cell surface carbohydrates, exoglycosidases may facilitate bacterial adherence and respiratory tract colonization.

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One mechanism by which severe illness or stress might facilitate adherence and colonization of GNB to respiratory epithelium is by altering epithelial cell surface carbohydrates. To investigate this possibility we used radiolabeled lectins to quantitate carbohydrate levels on intact buccal and tracheal epithelium. A rat model of GNB colonization, in which renal infarction was performed to produce colonization, was used. Buccal and tracheal epithelial surface carbohydrate levels from normal rats and rats 48 hours after renal infarction were compared. Buccal and tracheal epithelium from the renal infarction animals had decreased amounts of sialic acid and fucose, and decreased levels of these sugars occurred at the same time that heavy oropharyngeal GNB colonization developed. Tracheas obtained from the infarcted animals bound three times more Type 1 piliated GNB than normal tracheas. Sialic acid and fucose levels are decreased early after stress, and we speculate that altered epithelial carbohydrates may predispose to GNB colonization by exposing binding sites for GNB.

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We used Chinese hamster ovary (CHO) cell lines to define the structures of glycoproteins responsible for Type 1 piliated bacterial adherence. CSH 50 Escherichia coli, a Type 1 piliated bacteria, adhered significantly better than an isogenic nonpiliated E. coli to all CHO lines tested. CSH 50 E. coli adhered least well to CHO cells expressing intact complex type oligosaccharides on cell surface glycoproteins. CSH 50 adherence increased when shorter oligosaccharides were present and was maximal when mannose groups were present in terminal, nonreducing positions. Five high mannose type glycoproteins, with molecular weights of 79, 75, 55, 50, and 37 kD, were identified as high affinity ligands for Type 1 piliated bacteria. Our results suggest that alterations in cell surface carbohydrates may increase adherence of Type 1 piliated gram-negative bacteria to cells.

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Numerous investigators have reported an increased incidence of pneumonia caused by Gram-negative bacilli and other secondary pathogens in transplant recipients infected by cytomegalovirus (CMV). To determine if CMV infections are related to colonization of the upper respiratory tract by Gram-negative bacilli, we examined prospectively 22 renal transplant recipients with sequential bacteriological, virological and biochemical examinations performed just prior to and at various times after transplantation. Only 11% of subjects had Gram-negative bacilli isolated from gargle specimens prior to transplantation, as compared to 54% after transplantation. More importantly, after transplantation, subjects with active CMV infections were more likely to have prolonged oropharyngeal carriage of Gram-negative bacilli than subjects without CMV infections (36% v. 25%). During active CMV infections, the rate at which Gram-negative bacilli were isolated from gargle specimens rose from 28 to 47%. During culture-positive CMV infections, the isolation rate reached 57% and was significantly different from that of CMV-negative samples (P less than 0.01). The increased rate of Gram-negative bacillary isolation from gargle specimens during CMV infections was not a function of type of immunosuppressive agents used, rejection episodes, antibiotic administration, concomitant hepatitis B, Epstein-Barr (EBV) virus, or herpes simplex virus infections, or alterations in salivary fibronectin concentrations.

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PURPOSE: To evaluate the effect of different levels of glycemic control on the pulmonary function of subjects with type I insulin-dependent diabetes mellitus. PATIENTS AND METHODS: Eighteen subjects with type I insulin-dependent diabetes mellitus with no history or physical findings of respiratory disease. Patients were given insulin therapy with a standard twice-daily insulin injection regimen (standard treatment group) or a subcutaneous insulin infusion device (insulin pump) (intensive treatment group). Glycosylated hemoglobin (HbA1c) levels were determined at quarterly intervals in both groups of patients (standard treatment group, n = 10; intensive treatment group, n = 8). Pulmonary function and diffusing capacity for carbon monoxide (DLCO) were measured after 6 years of continuous follow-up. RESULTS: The average HbA1c in the standard treatment group was significantly higher than that of the intensive treatment group throughout the 6 years of follow-up (p less than 0.001). The forced vital capacity of the standard treatment group was 85 +/- 3% of predicted as compared with 106 +/- 4% of predicted in the intensive treatment group (p less than 0.001). The DLCO was also significantly diminished in the standard treatment group as compared with that in the intensive treatment group (65 +/- 2% versus 87 +/- 4% of predicted) (p less than 0.001). CONCLUSION: These data confirm previous reports of abnormal respiratory function in subjects with insulin-dependent diabetes mellitus and suggest that long-term near-normoglycemia may be beneficial in preventing the deterioration of pulmonary function associated with diabetes mellitus.

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Adherence of gram-negative bacteria (GNB) to epithelial surfaces is important for GNB colonization to occur. Pili, rodlike structures projecting from the outer membrane of GNB, and GNB surface hydrophobicity have been shown to enhance GNB adherence. We investigated the types of pili and the hydrophobicity of aerobic GNB colonizing the stomach, oropharynx, and trachea of critically ill patients. Piliation and hydrophobicity of oral, tracheal, and gastric GNB were compared with that of commensal GNB isolated from patients' rectums. Significantly more oropharyngeal than rectal GNB were piliated, and the most common type of pili present was type 1, or mannose-sensitive pili. Mannose-resistant and P pili were present less often, and no colonizing GNB had S pili. Colonizing GNB were hydrophilic rather than hydrophobic, and no differences in hydrophobicity were noted between colonizing GNB and rectal isolates. Our results suggest that pili may be important for oropharyngeal GNB colonization.

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Septic shock (SS) is the most common type of shock encountered by internists, and its prevalence appears to be increasing. SS complicates all types of infections. The hemodynamic characteristics of SS include a low systemic vascular resistance and an elevated, but relatively inadequate, cardiac output. A cardiomyopathy frequently occurs. The major endogenous mediator of SS is tumor necrosis factor, and interleukins-1 and -2 may also contribute. Important secondary phenomena include release of platelet activating factor, vasodilator prostaglandins, and upregulation of adhesion molecules on polymorphonuclear leukocytes and endothelial cells. Current therapy is often ineffectual, and potentially promising new therapeutic approaches are reviewed.

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Cytolytic lymphocytes play an important role in defense against viral and neoplastic disease. Integral to the function of these cells is the content of lysosomal granules. Recent attention has focused on a family of proteases present in the granules of natural killer (NK) cells, interleukin-2 (IL-2)-activated NK cells (LAK cells), and cytotoxic T lymphocytes (CTL). In the current investigation, lymphocytes were obtained from human lung parenchyma and peripheral blood. Following activation with IL-2, both groups of lymphocytes exhibited comparable cytolytic activity against K562 targets. Lysosomal granules obtained from these cells contained two serine proteases with molecular weights of 30 and 28 kD. These proteases were capable of hydrolyzing benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT-ester), a substrate of cytolytic lymphocyte proteases. When compared to blood, unactivated lung lymphocytes contained significantly higher levels of protease content. Although IL-2 produced a significant increase in blood lymphocyte protease content, no change in lung lymphocyte granule protease activity was observed. We conclude that cytolytic lung lymphocytes contain high levels of lysosomal granule protease but differ from blood lymphocytes in the ability to increase protease content following activation with IL-2. The high level of protease content in cytolytic lung lymphocytes suggests that these cells could produce local tissue injury during the release of lysosomal granules.

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Fibronectin (FN) has been postulated to prevent gram-negative bacillary (GNB) colonization of the oropharynx by covering epithelial cell GNB receptors. We investigated the distribution of FN along the luminal surface of oropharyngeal epithelium in animals and humans. Examination of buccal epithelial biopsies obtained from normal rats revealed no luminal surface FN by either immunofluorescent or immunoperoxidase staining. Extraction of epithelial surface proteins and quantitation of FN by rocket immunoelectrophoresis and electrophoretic transfer to nitrocellulose followed by immunologic detection also detected no FN from normal animals' oropharyngeal biopsies. Buccal epithelial biopsies from three normal humans were examined for FN using electrophoretic transfer to nitrocellulose followed by immunologic detection, and no FN was demonstrable. Our results suggest that FN is not present on the oral epithelial surface of healthy rodents or humans, and that FN may not be involved in the pathogenesis of bacillary colonization.

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Little is known about the functions of alveolar macrophages during the later resolving phases of pulmonary inflammation. We have used an animal model of resolving pulmonary inflammation to obtain inflammatory macrophages (IMs) and have compared several IM functions with those of resident macrophages (RMs). IMs were frequently peroxidase positive and contained large amounts of myeloperoxidase activity. IMs also contained significant amounts of a serine protease type of elastase. The procoagulant activity of IMs was less than that of RMs, and IMs exhibited increased plasminogen activator activity when incubated on fibrin matrices. IMs also degraded fibrin directly, without plasminogen, and this activity was due to two different enzymes of molecular weights 39 and 63 kD that were present in IM granules and plasma membranes. These results suggest that, in vivo, IMs take up PMN enzymes and alter their procoagulant and fibrinolytic activity to maximize fibrin removal. These IM functions may be important for successful resolution of inflammatory injury.

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We have investigated the role of pili in mediating gram-negative bacterial adherence to an intact tracheal epithelium. Type 1 pili, but not P or Pseudomonas pili, markedly increased bacterial adherence. The adherence-promoting effect of Type 1 pili was due to the mannose-binding Type 1 pili adhesin, as both alpha-methyl mannoside and concanavalin A blocked adherence of Type 1 piliated bacteria. The Type 1 pili-binding site on tracheal epithelium appears to be a mannose-containing glycoprotein. Clearance of Type 1 piliated bacteria from the lung parenchyma was assessed by depositing the bacteria into a lobe; no difference in clearance rates between Type 1 and nonpiliated bacteria was present. Type 1 pili may enhance the ability of gram-negative bacteria to adhere to and colonize the lower respiratory tract.

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The Adult Respiratory Distress Syndrome (ARDS) is a fulminant form of respiratory failure affecting many seriously ill patients. The early manifestations of ARDS are caused by increased permeability of the alveolo-capillary barrier leading to pulmonary edema, stiff lungs, and a large right-to-left intrapulmonary shunt. Polymorphonuclear leukocytes (PMNS) are involved in the pathogenesis of most ARDS, and multiple PMN mechanisms can effect pulmonary injury; interactions between PMN adherence, proteolytic enzyme release, and oxygen radical production are emphasized. ARDS therapy remains largely supportive and has had little impact on mortality. The complications of infection and multiorgan failure play important roles in determining ARDS outcome.

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