Pretreatment with recombinant murine tumor necrosis factor alpha/cachectin and murine interleukin 1 alpha protects mice from lethal bacterial infection.

Tumor necrosis factor/cachectin (TNF/C) is the principal mediator of bacterial endotoxin-induced shock and death. We found that the C3H/HeJ mouse, which is less able to produce TNF/C in response to endotoxin, has a 1,000-fold greater susceptibility to lethal infection with Escherichia coli than the TNF-responsive congenic mouse, C3H/HeN. This surprising finding suggested that this lethal peptide may also be involved in host protection. To test this hypothesis we pretreated the C3H/HeJ mouse with a combination of recombinant murine TNF/C-alpha and IL-1 alpha. This combination protected these mice against an intraperitoneal bacterial challenge of greater than 20 LD50S (nearly 2 x 10(2) CFU) that grew to a level of greater than 10(7) CFU/ml of blood and per gram of liver in untreated mice. This suggests a significant role for these cytokines in host defenses against invasive infections that require bacterial replication within the host. These protective mechanisms may not be important for less virulent organisms. These findings may have important implications for the proposed use of anti-TNF/C agents in the treatment of septic shock.

Mobilization of sialidase from intracellular stores to the surface of human neutrophils and its role in stimulated adhesion responses of these cells.

Desialation of cell surfaces has been associated with the initiation or modification of diverse cellular functions. In these studies we have examined the subcellular distribution of sialidase (SE) in human neutrophils as well as the mobilization of this enzyme following neutrophil activation. Separation of subcellular fractions by density gradient centrifugation showed that SE is present not only in neutrophil primary and secondary granule populations, like lysozyme, but also in plasma membrane fractions. Neutrophil activation was associated with a redistribution of SE from secondary granule-enriched fractions to the plasma membrane. Furthermore, SE activity detected on the surface of intact neutrophils with a fluorescent SE substrate increased rapidly after activation with kinetics that matched both the loss of total cell-associated sialic acid and release of free sialic acid from the cells. These activation-dependent events were in each case blocked by incubation of neutrophils with the SE inhibitor, 2-deoxy-N-acetyl-neuraminic acid. Aggregation responses of neutrophils as well as adhesion responses to nylon and plastic surfaces were also inhibited by 2-deoxyNANA. Our findings indicate that the activation-dependent desialation of the neutrophil surface is associated with mobilization of an endogenous SE to the plasma membrane and has a role in stimulated adhesion responses of these cells.

The K1 capsule is the critical determinant in the development of Escherichia coli meningitis in the rat.

Although Escherichia coli strains possessing the K1 capsule are predominant among isolates from neonatal E. coli meningitis and most of these K1 isolates are associated with a limited number of 0 lipopolysaccharide (LPS) types, the basis of this association of K1 and certain 0 antigens with neonatal E. coli meningitis is not clear. The present study examined in experimental E. coli bacteremia and meningitis in newborn and adult rats whether or not the K1 capsule and/or O-LPS antigen are critical determinants in the development of meningitis. Rats received subcutaneously at K1 E. coli strain (018+K1+) or mutants lacking either the K1 capsule (018+K1-) or 0 side-chain (018-K1+). 12-24 h later, blood and cerebrospinal fluid (CSF) specimens were obtained for quantitative cultures. The isolation of E. coli from CSF was observed in both newborn and adult rats infected with K1+ strains regardless of LPS phenotype (018+ or 18-) who also developed a high degree of bacteremia (e.g., greater than 10(4) CFU/ml of blood). In contrast, none of the newborn and adult rats infected with 018+K1- and developing bacteremia of greater than 10(4) were found to have positive CSF cultures. These findings indicate that the presence of the K1 capsule and a high degree of bacteremia are key determinants in the development of E. coli meningitis, suggesting that there may be specific binding sites present in the brain which have an affinity for the K1 capsule and thus may be responsible for the entry of K1-encapsulated E. coli into the meninges.

Safety and immunogenicity of a Pseudomonas aeruginosa O-polysaccharide toxin A conjugate vaccine in humans.

Lipid A-free polysaccharide (PS) isolated from Pseudomonas aeruginosa immunotype 5 lipopolysaccharide (LPS) was covalently coupled to toxin A via reductive amination. The PS-toxin A conjugate was comprised of 29.8% PS and 70.2% toxin A, possessed a molecular weight of greater than 1 X 10(6), was nontoxic for animals and was nonpyrogenic for rabbits at a dose of 50 micrograms/kg body wt when administered intravenously. The conjugate evoked only mild, transient reactions upon subcutaneous administration to human volunteers. Vaccination engendered immunoglobulin G (IgG) antibody, which neutralized the cytotoxic effect of toxin A and promoted the uptake and killing of P. aeruginosa in the presence of human polymorphonuclear leukocytes. Passively transferred IgG isolated from the serum of immunized donors was far more effective at preventing fatal P. aeruginosa burn wound sepsis than paired preimmunization serum. These studies establish the potential usefulness of such a PS-toxin A conjugate as a vaccine against P. aeruginosa.

Efficacy of antilipopolysaccharide and anti-tumor necrosis factor monoclonal antibodies in a neutropenic rat model of Pseudomonas sepsis.

Monoclonal antibodies (MAb) directed against bacterial lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF) provide partial protection in experimental models of septic shock. To determine if additional benefit accrues from a combination of anti-TNF and anti-LPS MAb in the treatment of septic shock, a neutropenic rat model was developed to study active infection with Pseudomonas aeruginosa 12.4.4. Animals were treated intravenously with an irrelevant MAb (group 1); anti-TNF MAb (group 2); MAb directed against P. aeruginosa 12.4.4 LPS (group 3); or a combination of anti-TNF and anti-LPS MAb (group 4). None of the control animals in group 1 survived the 7-d period of neutropenia (0/16). In contrast, the survival rate was 44% in group 2 (P less than 0.02); 37% in group 3 (P less than 0.05); and 75% in group 4 (P less than 0.0002). The combination of monoclonal antibodies provided greater protection than either MAb given alone (P less than 0.05). Serum TNF levels during infection were significantly greater in groups 1 and 3 (20.1 +/- 3.3 U, mean +/- SE) than in groups 2 and 4 (0.9 +/- 0.8 U, P less than 0.0001). These results indicate that a combination of monoclonal antibodies to LPS and TNF have additive benefit in experimental Pseudomonas aeruginosa sepsis. This immunotherapeutic approach may be of potential utility in the management of serious, gram-negative bacterial infection in neutropenic patients.

Human neutrophils increase expression of C3bi as well as C3b receptors upon activation.

We used monoclonal antibodies and flow cytometry to study the expression of the receptors for the complement fragments C3bi (CR3) and C3b (CR1) on human polymorphonuclear neutrophil leukocytes (PMN). Expression of both receptors was minimal on cells stained in anticoagulated whole blood incubated at 0 degree or 37 degrees C. PMN isolated with Percoll density gradients and held at 0 degree C also had only minimal expression of both receptors. With the isolated cells, however, a spontaneous increase in expression of both receptors occurred upon warming to 37 degrees C. This did not represent complete expression of either receptor since additional increments in surface expression could be induced upon stimulation with N-formyl-methionyl-leucyl-phenylalanine or Raji cell supernatant. The increases in complement receptor (CR) expression appeared to be specific since there were no changes in expression of the Fc gamma receptor or beta-2-microglobulin under any of these conditions. The increased CR expression seems to involve translocation from an intracellular pool since it is complete within minutes and is not blocked by puromycin or cycloheximide. These results demonstrate that both CR3 and CR1 expression increase rapidly upon activation of PMN and that isolated cells can be used to study this phenomenon, which may be a critical part of neutrophil function in vivo.

Progressive increase in antibiotic resistance of gram-negative bacterial isolates. Walter Reed Hospital, 1976 to 1980: specific analysis of gentamicin, tobramycin, and amikacin resistance.

During a five-year period at Walter Reed Army Hospital, Washington, DC, the frequency of aminoglycoside resistance among clinical bacterial isolates increased from less than 1% in 1976 to 13% of all isolates in later years. This resistance was seen among frequently isolated species of gram-negative bacilli isolated from patients throughout the hospital and from all anatomical sites, including blood. The incidence of gentamicin and amikacin resistance rose with increased administration of these antibiotics; however, the incidence of tobramycin resistance increased despite its minimal usage. From 1977 to 1978 and from 1979 to 1980, there was a decrease in the conjugal transmissibility of resistance to gentamicin and tobramycin. There was no detectable transmissible amikacin resistance. These findings suggest that high priority must be given to strategies that limit the emergence and dissemination of organisms resistant to these important antibiotics.

Infective endocarditis and access site infections in patients on hemodialysis.

Infective endocarditis has been a subtle and very often lethal complication of hemodialysis. Thirty-five episodes have been described to date. Antecedent infections, particularly those involving the access site, access manipulation, and dental work appear to predispose to IE. Once IE is acquired, factors associated with mortality are involvement of two or more valves, infection caused by enterococci, antecedent infection, steroid therapy, infection in the first year post-access insertion and patient age over 46. The incidence of access infection with the arteriovenous fistula is significantly less than that associated with the arteriovenous cannula. Staphylococci are the most common organisms in access infections and in IE. Gram-negative bacilli and particularly Pseudomonas aeruginosa are a frequent cause of access infection but an unusual cause of IE. Access removal may be madatory in the successful management of IE in patients on hemodialysis.

Kinetics and dose dependence of macrophage colony-stimulating factor-induced proliferation and activation of murine mononuclear phagocytes in situ: differences between lungs, liver, and spleen.

Alveolar macrophages (AM) play an important role in antimicrobial defense mechanisms of the lung. It therefore seems reasonable to use macrophage colony-stimulating factor (M-CSF) to enhance local resistance mechanisms. However, little is known about the in vivo activity of M-CSF on macrophages in various organs. We determined the effect of a single subcutaneous dose of M-CSF (10, 50, 100, and 500 ng, respectively) on the number and functional status of AM as well as of macrophages in liver and spleen of mice. Organs were investigated immunohistochemically on days 1 and 3 after injection using monoclonal antibodies specific for F4/80, Ia antigen, and MAC-1. We found a significant increase in the number of F4/80+ AM, Kupffer cells, and splenic macrophages reaching its maximum 24 h after injection of low doses (10 and 50 ng per mouse, respectively) of M-CSF and decreasing to a level seen in untreated mice at 72 h after M-CSF in liver and spleen, whereas at a dose of 50 ng per mouse the number of AM remained high. In contrast, the numbers of AM, Kupffer cells, and splenic macrophages did not increase significantly when high doses were used (500 ng). The expression of Ia antigen and MAC-1 was increased on macrophages in the spleen but not on AM or Kupffer cells. TNF-alpha was elevated in bronchoalveolar (BAL) fluid after 3 h and IL-6 at 6, 12, and 24 h after M-CSF injection in dose-dependent manner. Nitric oxide production was not increased after injection of M-CSF. Our results point to regional differences in the response of macrophages to M-CSF. These may caused by differences in the M-CSF-induced production of TNF-alpha and IL-6. These findings may be important for the therapeutic use of M-CSF in microbial infections.

IFN-gamma inhibits lipopolysaccharide-induced interleukin-1 beta in primary murine macrophages via a Stat1-dependent pathway.

Interleukin-1 (IL-1) plays an important role in host defenses against microbial pathogens. Excessive production of this cytokine, however, may be responsible in part for the lethality observed during sepsis. Our studies show that interferon-gamma (IFN-gamma) downregulates lipopolysaccharide (LPS)-induced interleukin-1beta (IL-1beta) transcription in primary macrophages. This phenomenon does not occur in splenocytes or bone marrow-derived macrophages from signal transducer and activator of transcription (Stat1)-deficient mice, suggesting that Stat1, a transcription factor involved in IFN signaling, plays a critical role in this process. Moreover, nitric oxide (NO) was also involved in the downregulation of LPS-induced IL-1 by IFN, as addition of the inducible nitric oxide synthase (iNOS) inhibitor L-N(6)-(1-iminoethyl)lysine (NIL) negated the effect. Kinetic analysis of IL-1 and IFN levels in LPS-treated mice in vivo suggests that IFN-mediated inhibition of IL-1 might be an important negative feedback mechanism for limiting IL-1 generation in vivo.

Role of respiratory assistance devices in endemic nosocomial pneumonia.

The role of respiratory assistance devices and techniques in the acquisition of endemic hospital-associated pneumonia was prospectively studied in 13,086 patients over 11 months. Of these, 914 (7 percent) had a respiratory assistance device for at least 24 hours. Cultures of respirator effluent air and nebulizer fluid (taken after 24 hours), tracheostomy sites and irrigating solutions and respirometers were obtained in the 144 of 914 patients who had a respiratory assistance device for at least 72 hours. There were 108 episodes of hospital-associated pneumonia in 107 patients (0.82 percent incidence). Gram-negative organisms were associated with 70 percent of these episodes and Strep. pneumoniae with 5 percent. The risk of hospital-associated pneumonia was 0.3 percent in patients without a respiratory assistance device (35 percent of total hospital-associated pneumonia) versus (1.3 percent with endotracheal tubes and respirators (11 percent of hospital-associated pneumonia), 25 percent with tracheostomy (12 percent of hospital-associated pneumonia) and 66 percent in patients with tracheostomy and a respirator (9 percent of hospital-associated pneumonia). No case of hospital-associated pneumonia occurred in patients on respirators less than 24 hours, but the risk of hospital-associated pneumonia increased significantly after the fifth day of therapy. None of the 63 cultures of nebulizer fluid was positive. Although positive cultures of respiratory effluent, tracheal suction fluid or respirometer were not predictive of the acquisition of hospital-associated pneumonia, nine of 107 patients acquired this infection after a previously positive culture of a respiratory assistance device, and in five instances with the same organism. Since contaminated respiratory assistance devices are rarely a direct cause of hospital-associated pneumonia, routine in-use monitoring of respiratory assistance devices does not appear warranted.

Kinetics of cellular and cytokine responses in a chimeric mouse model for the study of staphylococcal enterotoxin B pathogenesis.

The mechanisms by which superantigens, such as staphylococcal enterotoxin B (SEB), contribute to microbial pathogenicity have been poorly defined. The study of such pathogenic processes has been hampered by the lack of an adequate animal model. We utilized a previously described murine chimeric model to determine the cytokines and cell populations that might be involved in SEB toxicity. In the absence of bone marrow transplantation (BMT), all total body irradiated (TBI) mice died, while all transplanted mice survived up to 6 months. Compared with non-TBI and non-BMT mice, chimeric mice had an increased percentage of CD11b (Mac-1)-positive splenocytes (17 vs. 59%, P < 0.05) and decreased CD45R-positive (B) cells (33 vs. 6%, P < 0.05) at 6 weeks after BMT. The relative numbers of splenocyte CD4 and CD8 cells were similar in chimeric and normal mice. Susceptibility of chimeric animals to 10 or 100 microg SEB was time-dependent: no mice challenged at 2 weeks post-BMT died, but 15% of mice challenged at 4 weeks and 50% of those challenged at 6-8 weeks died. Compared with TBI and non-BMT C3H/HeJ mice, SEB-challenged chimeric mice at 6-8 weeks had (1) increased splenocyte mRNA expression for: IFN-gamma (3.5 x optimally at 1 h), TNF-alpha (6.5 x at 2 h), IL-6 (4.8 x at 4 h), IL-1beta (8.4 x at 4 h), IL-2 (4.7 x at 4 h), and IL-10 (3 x at 16 h), and (2) increased and earlier peak serum levels of IFN-gamma, IL-6, IL-1beta and IL-2, but no increase in serum TNF-alpha or IL-4. These data support the hypothesis that the decreased percentage of B cells and increased macrophages in chimeric mice lead to enhanced T cell-macrophage interactions after SEB administration and a lethal burst of T cell and macrophage cytokine release. This model will provide insight into cell populations and mechanisms that mediate superantigen-induced toxicity.

A hospital-wide outbreak of septicemia due to a few strains of Staphylococcus aureus.

During a 6-month period at Walter Reed Army Hospital the monthly attack rate of Staphylococcus aureus bacteremia increased to 3.8 +/- 0.5 (mean +/- SEM) from 2.5 +/- 0.2 cases per 1,000 dispositions for the previous 48 months (P less than 0.05). A predominant phage pattern, designated S, was found in 12 (39%) of 31 bacteremic isolates typed and another strain, delta, was associated with four catheter-related infections. Two other strains also accounted for infections. Patients with isolates of the S phage pattern had a higher mortality (59%) than patients with non-S isolates (37%). Thirty-eight per cent of S. aureus carriers among hospital personnel harbored S or delta strains. Limitation of intravascular devices, strict handwashing, and the use of gloves were associated with a significant decrease in the incidence of S. aureus bacteremia to 1.9 +/- 0.5/1,000 dispositions over the next 6 months (P less than 0.05). S and delta strains were reduced to 20% of these isolates despite their persistence in 32% of staphylococcal carriers upon reculture of personnel. We conclude that S. aureus persists as an important pathogen in the hospitals, and that phage typing S. aureus isolates remains an important tool in hospital epidemiology. The presence of multiple S. aureus strains causing this outbreak and the extent of their dissemination among patients and personnel reported here emphasizes the need to reevaluate strategies of nosocomial staphylococcal control.

Exposure to febrile temperature modifies endothelial cell response to tumor necrosis factor-alpha.

Fever is an important regulator of inflammation that modifies expression and bioactivity of cytokines, including tumor necrosis factor (TNF)-alpha. Pulmonary vascular endothelium is an important target of TNF-alpha during the systemic inflammatory response. In this study, we analyzed the effect of a febrile range temperature (39.5 degrees C) on TNF-alpha-stimulated changes in endothelial barrier function, capacity for neutrophil binding and transendothelial migration (TEM), and cytokine secretion in human pulmonary artery endothelial cells (EC). Permeability for [(14)C]BSA tracer was increased by treatment with TNF-alpha, and this effect was augmented by incubating EC at 39.5 degrees C. Treating EC with 2. 5 U/ml TNF-alpha stimulated an increase in subsequent neutrophil adherence and TEM. Incubating EC at 39.5 degrees C caused a 30% increase in TEM but did not modify the enhancement of neutrophil adherence or TEM by TNF-alpha treatment. Analysis of cytokine expression in EC cultures exposed to TNF-alpha at either 37 degrees or 39.5 degrees C revealed three patterns of temperature and TNF-alpha responsiveness. Granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin (IL)-8 were not detectable in untreated EC but were increased after TNF-alpha exposure, and this increase was enhanced at 39.5 degrees C. IL-6 expression was also increased with TNF-alpha exposure, but IL-6 expression was lower in 39.5 degrees C EC cultures. Transforming growth factor-beta(1) was constitutively expressed, and its expression was not influenced either by TNF-alpha or exposure to 39.5 degrees C. These data demonstrate that clinically relevant shifts in body temperature might cause important changes in the effects of proinflammatory cytokines on the endothelium.

Clinical trials for severe sepsis. Past failures, and future hopes.

Recent clinical trials with experimental immunotherapeutic agents for severe sepsis and septic shock have been largely unsuccessful despite seemingly convincing preclinical evidence of significant benefit of these antisepsis therapies. This article reviews basic therapeutic rationale, preclinical evaluation, and clinical trial design of past clinical trials of innovative sepsis treatments. Lessons learned from past failures should provide insights into the design and implementation of successful clinical trials for new anti-sepsis agents in the future.

Vaccines and antibodies in the prevention and treatment of sepsis.

Antibodies to various core glycolipid antigens have been shown to correlate with survival from Gram-negative sepsis. Recent preclinical data also support efficacy of the anti-core glycolipid antibodies in the treatment of sepsis. Failure of some of the previous clinical trials with anti-core glycolipid antibody was probably due to inadequate levels of antibody in those preparations. Future clinical trials must ensure that sufficient amounts of anti-core glycolipid antibodies are present in the circulation of patients with sepsis.

Escherichia coli and Klebsiella vaccines and immunotherapy.

A polyvalent vaccine has been prepared from the capsular polysaccharide of 24 different serotypes of Klebsiella spp. Nearly 200 volunteers have received this vaccine. It is very well tolerated and elicits both binding (ELISA) and functional antibody to 21 of 24 antigens. Antibodies were also detected against 10 serotypes not included in the vaccine. An immunoglobulin for intravenous use (IVIG) was more protective in mouse lethality assays and enhanced opsonophagocytic killing of bacteria more than standard, nonhyperimmune globulin. A monovalent E. coli conjugate vaccine against O18ac antigen was safe and highly immunogenic in humans. A 12-valent conjugate vaccine elicits good levels of antibody in rabbits, and will soon undergo phase I testing in humans. These vaccines might best be used for inducing antibody in donor plasma that could be made into IVIG for passive administration.

Evaluation of a competitive ELISA method for the determination of Klebsiella O antigens.

Strains of Klebsiella spp. are often inagglutinable by O-specific antisera because of the copious capsule produced by most isolates. A competitive ELISA method based on the observation that bacterial supernates containing homologous O antigen specifically inhibited the reaction of type-specific antisera with purified LPS coated on ELISA plates was used to examine the O antigen of 82 isolates of different Klebsiella species and subspecies. The O antigens O1/2ab (19 isolates), O2ab (13 isolates), O2ac (11 isolates) and O3 (16 isolates) were found to account for > 70% of the O antigenic types. Overall, 65 (79%) of the strains could be assigned to a specific O serogroup. The method is suitable for examining the role of individual O antigens in systemic klebsiella infections such as nosocomial septicaemia and pneumonia.

Circulating levels of tumour necrosis factor, interleukin 6 and proteolytic activity in a murine model of burn and infection.

Cytokines and proteinases have both been implicated as mediators in the inflammatory response associated with trauma and sepsis. Using a burned-infected mouse model, it was previously found that mortality is proportional to the amount of proteolytic activity (PA) in the circulation. However, little is known about circulating cytokine levels in hosts that are both burned and infected. With this mouse model, both tumour necrosis factor (TNF) and interleukin 6 (IL-6) were upregulated by a burn and by an infection. Burn plus infection produced an additive effect on each cytokine, but IL-6 levels correlated better with mortality. Treating mice with the proteinase inhibitor aprotinin immediately preburn and infectious challenge significantly decreased IL-6, PA and mortality. This may be a clinically relevant model for studying mediators in burned and/or septic hosts.

Intravenous immunoglobins (IVIGs) to prevent and treat infectious diseases.

The availability of IVIGs for the prevention and treatment of bacterial diseases has presented may challenges. While a role for prophylaxis against infection has been suggested for some conditions characterized by hypogammaglobulinemia (or physiologic hypogammaglobulinemia), it has been difficult to demonstrate a convincing effect when IVIG is used as treatment for infectious diseases. The development of IVIGs enriched in antibody against specific pathogens may yet show therapeutic efficacy, but cost effective strategies for generating such reagents must be defined.