Effect of anti-OprF-OprI immunoglobulin on APACHE II score in a porcine two-hit model of hemorrhagic shock/resuscitation and pseudomonas aeruginosa sepsis.

BACKGROUND: Up to now, randomized clinical trials of treatment of bacterial sepsis with immunoglobulins show conflicting results. This paper investigates the effect of prophylactic immunization with anti-OprF-OprI antiserum on the APACHE II score in a clinically relevant two-hit model of hemorrhagic shock/resuscitation followed by Pseudomonas aeruginosa sepsis in pigs. METHODS: Twenty-three German Landrace-Hybrid pigs underwent chronic implantation of vascular catheters (internal and external jugular vein, carotic and pulmonary artery), hemorrhagic shock (mean blood loss 40% of estimated blood volume) for 45 min, followed by resuscitation with crystalloid, colloid, and shed blood. Randomization was to a control group (no immunization, n=6), an F-I group (50 mg/kg i.p. anti-OprF-OprI immunoglobulin, n=6), an S group (50 mg/kg i.p. unspecific porcine immunoglobulins, n=6), and a PS group (50 mg/kg i.p. immunoglobulin against the antigens of heat-killed P. aeruginosa, n=5). After at least 18 h for recovery from anesthesia, the pigs underwent a continuous intravenous infusion of P. aeruginosa for 48 h. Thereafter, the animals were monitored for another 48 h and then dissected. RESULTS: The APACHE II score significantly increased from baseline value in all groups during bacterial challenge. However, there were no between-group differences in APACHE II score. In contrast, pigs of the F-I and PS groups showed significant lower lung concentrations of P. aeruginosa (p<0.05 vs. control group) at autopsy. CONCLUSION: These experimental data suggest that under comparable clinical conditions, a prophylactic immunization with anti-OprF-OprI immunoglobulin would not have an overall benefit to patients with P. aeruginosa sepsis.

Combination of enoxaparin and fibroblast growth factor-1 increases myocardial blood flow and capillary density after myocardial infarction in rabbits.

OBJECTIVE: The effect of enoxaparin and fibroblast growth factor-1 (FGF-1) on post-infarction capillary density and regional myocardial blood flow (RMBF) was examined. METHODS: New Zealand White rabbits received an intramyocardial injection of either physiological saline, FGF-1 + enoxaparin, FGF-1 or enoxaparin directly after ligation of the left anterior descending artery. RMBF and capillary density were investigated using fluorescent microspheres and histological examination. RESULTS: One week after infarction a significant difference in the number of capillaries could be demonstrated within the FGF-1 + enoxaparin group (p < 0.001 versus the control group), the FGF-1 group (p < 0.01) and the enoxaparin group (p < 0.05). Treatment with FGF-1 + enoxaparin resulted in a significantly increased number of capillaries compared to treatment with FGF-1 (p < 0.05) and enoxaparin (p < 0.05) alone. Additionally, all groups treated with FGF-1 and/or enoxaparin showed a significant increase of microvessel density in the treated ischemic border zone compared to the non-treated ischemic border zone (p < 0.001 for FGF-1 + enoxaparin, p < 0.01 for FGF-1, p < 0.05 for enoxaparin). RMBF was significantly increased within the FGF-1 + enoxaparin group compared to the control group (p < 0.05). Moreover, perfusion rates within the FGF-1 + enoxaparin-treated area did not significantly differ from the pre-infarction values. CONCLUSION: Treatment with either enoxaparin or FGF-1 or FGF-1 + enoxaparin resulted in increased microvessel growth. However, only the combination of enoxaparin with FGF-1 promotes capillary growth and RMBF. Thus, we conclude that enoxaparin enhances the angiogenic potential of intramyocardially injected FGF-1 in the acutely infarcted rabbit heart.

Impact of vector-priming on the immunogenicity of a live recombinant Salmonella enterica serovar typhi Ty21a vaccine expressing urease A and B from Helicobacter pylori in human volunteers.

Orally administered recombinant Salmonella vaccines represent an attractive option for mass vaccination programmes against various infectious diseases. Therefore, it is crucial to gather knowledge about the possible impact of preexisiting immunity to carrier antigens on the immunogenicity of recombinant vaccines. Thirteen volunteers were preimmunized with Salmonella typhi Ty21a in order to evaluate the effects of prior immunization with the carrier strain. Then, they received three doses of 1-2 x 10(10) viable organisms of either the vaccine strain S. typhi Ty21a (pDB1) expressing subunits A and B of recombinant Helicobacter pylori urease (n = 9), or placebo strain S. typhi Ty21a (n = 4). Four volunteers were preimmunized and boosted with the vaccine strain S. typhi Ty21a (pDB1). No serious adverse effects were observed in any of the volunteers. Whereas none of the volunteers primed and boosted with the vaccine strain responded to the recombinant antigen, five of the nine volunteers preimmunized with the carrier strain showed cellular immune responses to H. pylori urease (56%). This supports the results of a previous study in non-preimmunized volunteers where 56% (five of nine) of the volunteers showed a cellular immune response to urease after immunisation with S. typhi Ty21a (pDB1).

Soluble serum E-cadherin as a marker of tumour progression in colorectal cancer patients.

A pilot study was conducted to determine the concentrations of soluble serum E-cadherin in 36 patients with colorectal cancer or a high-grade dysplasia by the use of an ELISA technique. The results were compared with staging characteristics and concentrations of routine serum carcinoembryonic antigen (CEA). Sixteen patients with benign diseases and nine healthy volunteers served as internal or negative controls. Tumour specimens from seven patients were analysed by immunohistochemistry to compare concentrations of soluble serum E-cadherin with patterns of cell-bound E-cadherin or beta-catenin. Serum E-cadherin concentrations were increased in colorectal cancer patients (P = 0.009), but also in benign disease controls (P = 0.005), correlating with the T- (P < 0.05), but not N- or M-stage, and with serum CEA (P = 0.002) in case of existing liver metastases. Compared with other staining patterns, concentrations of soluble serum E-cadherin were higher in case of an exclusive membrane-bound localization of cellular beta-catenin (P = 0.071). The results suggest marker characteristics of soluble serum E-cadherin in colorectal cancer patients, but lacking specificity argues against a routine clinical use.

Recombinant OprF-OprI as a vaccine against Pseudomonas aeruginosa infections.

A vaccine against Pseudomonas aeruginosa based on recombinant outer membranes has been developed. After intramuscularly injecting into patients with severe burns, antibodies against P. aeruginosa were induced. Vaccination was well tolerated. Intranasal application of the vaccine into volunteers, induced specific s-IgA antibodies. We conclude that the newly developed vaccine may be suitable for protection of the main risk groups of P. aeruginosa infections. In particular, for the protection of burn patients and patients with cystic fibrosis.

Safety and immunogenicity of live recombinant Salmonella enterica serovar Typhi Ty21a expressing urease A and B from Helicobacter pylori in human volunteers.

Helicobacter pylori urease was expressed in the common live typhoid vaccine Ty21a yielding Ty21a(pDB1). Nine volunteers received Ty21a(pDB1) and three control volunteers received Ty21a. No serious adverse effects were observed in any of the volunteers. Ten out of 12 volunteers developed humoral immune responses to the Salmonella carrier as detected by antigen-specific antibody-secreting cells but only two volunteers seroconverted. A total of five volunteers showed responses in one or two out of three assays for cellular responses to the carrier (proliferation, IFN-gamma-secretion, IFN-gamma-ELISPOT). Three of the volunteers that had received Ty21a(pDB1) showed a weak but significant T-cell response to Helicobacter urease, while no volunteer had detectable humoral responses to urease. Ty21a(pDB1) is a suitable prototype to optimize Salmonella-based vaccination for efficient cellular responses that could mediate protective immunity against Helicobacter.

Secondary peritonitis: severity of disease and activation of peritoneal cells.

OBJECTIVE: To compare the degree of the inflammatory response of human peritoneum with the severity of peritonitis. DESIGN: Clinical laboratory study. SETTING: University hospital, Germany. SUBJECTS: 15 patients with diffuse secondary peritonitis and 5 having conventional cholecystectomy (controls) had peritoneal specimens taken from the site of incision. MAIN OUTCOME MEASURES: Correlation between presence of indicators of the inflammatory response: interleukin 1 (IL-1), interleukin 6 (IL-6), intercellular adhesion molecule-1 (ICAM-1), antibacterial protein (defensin 3 reflecting the activation of granulocytes), the antibody clone HAM 56 (for detection of local macrophages), and antibodies against macrophage migration inhibiting factor (MIF)-related proteins 8 and 14 (MRP 8 and 14), and clinical state evaluated by the Mannheim Peritonitis Index (MPI), the Peritonitis Index Altona II (PIA II) and the Acute Physiology Score (APS). C-reactive protein (CRP) concentrations were measured preoperatively in the serum. RESULTS: Expression of MRP 8 and 14, HAM 56, and defensin 3 was significantly higher in patients with peritonitis than in controls (p < 0.05). Expression of IL-1 and IL-6 was almost undetectable. ICAM-1 expression correlated significantly with phagocytic activation. There was no correlation between clinical scores, CRP, and immunohistochemically detectable variables. CONCLUSION: The pattern of peritoneal inflammatory reactions is relatively uniform and does not correlate with the clinical grading of severity.

The Caulobacter crescentus outer membrane protein Omp58 (RsaF) is not required for paracrystalline S-layer secretion.

To identify the outer membrane protein component of the Caulobacter crescentus CB2 surface-layer export machinery we used the Serratia marcescens LipD protein to find homologs in the CB2 genome. From two homologous sequences found, one encodes a putative OMP with a predicted molecular mass of 57.5 kDa, termed Omp58 (formerly RsaF). Comparison of membrane protein profiles revealed a protein with an appropriate molecular mass present in wild-type, but not CB2 omp58::kanamycin, a mutant strain with an inactivated omp58 gene. Disruption of omp58 did not affect surface-layer production, suggesting that Omp58 is not involved in surface-layer protein secretion and, thus, may not be the outer membrane protein component of the C. crescentus surface-layer export system.

Safety and immunogenicity of an intranasal Pseudomonas aeruginosa hybrid outer membrane protein F-I vaccine in human volunteers.

A hybrid protein [Met-Ala-(His)(6) OprF(190-342)-OprI(21-83)] consisting of the mature outer membrane protein I (OprI) and amino acids 190-342 of OprF of Pseudomonas aeruginosa was expressed in Escherichia coli and purified by Ni(2+) chelate-affinity chromatography. After several studies in healthy volunteers, as well as in patients, had proven the tolerability and immunogenicity of the the OprF-OprI vaccine, after intra-muscular application, we developed an emulgel for intranasal immunization. For this purpose we combined a highly concentrated OprF-I with sodium dodecylsulfate as vehicle and the gel matrix natriumlauryl sulfate. After safety and pyrogenicity evaluations in animals, eight healthy adult human volunteers received the OprF-I gel intranasally three times at 2-week intervals. The vaccination was well tolerated and no side effects were observed. An antibody induction (IgG and IgA) could be detected in the sera. These data support continued clinical investigation of the protection against infections in cystic fibrosis patients and patients prone to P. aeruginosa infections.

Inflammatory consequences of the translocation of bacteria and endotoxin to mesenteric lymph nodes.

BACKGROUND: Translocation of intestinal bacteria to mesenteric lymph nodes (MLNs) has been documented in humans under a variety of circumstances, yet its clinical significance remains to be established. The aim of this study was to correlate detectable translocation to MLNs of bacteria and endotoxin with local and systemic signs of inflammation. METHODS: From each of 10 patients with carcinoma of the cecal region two MLNs were harvested prior to resection. The presence of bacteria and endotoxin in the lymphatic tissue and blood was determined by culture methods and DNA preparation (PCR) and by a Limulus assay, respectively. Inflammatory mediators were determined in plasma and in MLN homogenates. RESULTS: Viable bacteria were detected in MLNs of 7 patients and in 9 of 20 lymph nodes. PCR revealed traces of bacteria in 4 patients and in 6 of their MLNs. Combining both modalities, the translocation rate was 80% and 55% for patients and MLNs, respectively. There was no detectable bacteremia. Endotoxin was found in the plasma of 7 patients and in 9 MLNs from 5 patients. There was no correlation between culture findings and endotoxin concentrations. Moreover, bacteriological data did not correspond to local or systemic inflammation. The group of MLN with detectable endotoxin differed significantly from LPS-negative nodes with respect to interleukin-6, interleukin-10, and sCD14. Systemic concentrations of endotoxin and inflammatory parameters did not correspond to levels within MLNs. CONCLUSION: Translocation to MLNs occurs in patients with cecal carcinoma. This, however, seems not to be of major clinical significance if no additional physiologic insults are encountered. Irrespective of the presence of bacteria, there are variations in inflammatory reactions between lymph nodes from one and the same patient, probably reflecting fluctuating response mechanisms to low-grade translocation.

Immunogenic efficacy of differently produced recombinant vaccines candidates against Pseudomonas aeruginosa infections.

Three different variants of the recombinant hybrid outer membrane protein OprF (aa 190-342)-OprI (aa 21-83) could be obtained in high yield after expression in Escherichia coli. The hybrid protein was modified N terminally, either with a minimal histidine tag or with a homologous sequence of OprF. Both recombinant proteins were purified by nickel chelate affinity chromatography under native and denaturing conditions, and this produced three suitable candidates for a vaccination trial, protein His-F-I, which was purified in its native as well as in its refolded form; and the native purified N terminally extended protein, ex-F-I. In mice, significantly higher antibody titers and survival rates after challenge with Pseudomonas aeruginosa were observed following immunization with protein His-F-I, purified under native conditions.

In vitro peritonitis: basic inflammatory reactions in a two-chamber coculture model of human peritoneum.

We developed an in vitro model of the peritoneum by coculturing human umbilical vein endothelial cells (HUVEC) and human peritoneal mesothelial cells (HPMC) to gather information on peritoneal physiology and to closer reflect the in vivo situation in humans. HUVEC and HPMC were seeded on collagen-coated polytetraflourethylene-insert membranes of pore size 3 microm. HUVEC were grown on the bottom of the membrane and HMPC on the top. The confluent cells were monitored by measuring transepithelial resistance and by confocal microscopy. The transmigration of PMNs as an important mechanism during secondary peritonitis was studied in this two-chamber model. PMNs were isolated by density separation. After stimulation of HMPC with the complement factor 5 split product C5a (1 ng/ml) or tumor necrosis factor-alpha (TNF-alpha; 10 or 50 microg/ml) for 1 h, 1 x 10(6) PMN were given to the lower compartment. Controls were cocultured cells without stimulation. After 1, 2, and 6 h nonadherent PMNs in the upper compartment were harvested and counted, interleukin-8 was measured in each compartment, and cells on the membrane were paraffin-embedded for immunohistochemistry. Each experiment was performed four times. Cells grew to confluence within 2-5 days and were detected on their respective seeding side by CD34 and cytokeratin 18 counterstaining. Transmigration of PMNs after C5a or TNF-alpha stimulation showed a significant time-dependent increase between 1 h and 6 h (P<0.05). PMNs were found in significantly higher numbers after stimulation with either C5a or TNF-alpha at 1, 2, and 6 h than without stimulation. After stimulation of HPMC, interleukin-8 secretion to the apical compartment increased in a time-dependent fashion, resulting in a gradient between the two chambers. Linear regression analysis revealed significant correlation between transmigrated PMN and interleukin-8 in stimulated cocultures; no correlation was found in controls. This new in vitro peritoneum consisting of cocultured mesothelial and endothelial cells may allow more detailed assessment of peritoneal pathophysiology. Generation of an interleukin-8 gradient affecting the migration of PNMs across the cocultured membrane represents a parameter which may be addressed in further studies.

Molecular analysis of hemolysin-mediated secretion of a human interleukin-6 fusion protein in Salmonella typhimurium.

Previously, we reported a plasmid-bearing Salmonella typhimurium strain capable of secreting human interleukin-6 (hIL-6) when genetically fused to the Escherichia coli hemolysin transport signal (HlyA(S)). Stationary phase culture supernatants of this strain revealed three major forms of hIL-6-HlyA(S) fusion protein (apparent molecular masses 32.4, 30.3, 27.0 kDa), at which the largest protein presumably represented full-length hIL-6-HlyA(S). The biological activity of the hIL-6-HlyA(S) protein mixture was similar to that of mature hIL-6. Accumulation of hIL-6-HlyA(S) in the culture supernatant occurred only during the initial growth phase, whereas in stationary phase and under in vitro conditions successive cleavage into the two truncated forms was observed. On the other hand, in whole cell lysates only full-length hIL-6-HlyA(S) could be detected, accounting for more than 50% of the totally synthesized protein. Upon cell fractionation, cellular hIL-6-HlyA(S) was exclusively found in the membrane fraction. These results suggest, that in S. typhimurium production and secretion of hIL-6-HlyA(S) is restricted to growing cells. A specific processing by a Salmonella-derived protease did not affect the biological activity of the fusion protein.

Local inflammatory peritoneal response to operative trauma: studies on cell activity, cytokine expression, and adhesion molecules.

OBJECTIVE: To test the hypothesis that different surgical procedures may lead to different degrees of activation of the human peritoneal response. DESIGN: Clinical laboratory study. SETTING: University Hospital, Germany. MATERIAL: Peritoneal specimens taken from the incision or parietal resection margins at the beginning and end of laparoscopic or open cholecystectomy, or other conventional open operations (n = 5 in each group). MAIN OUTCOME MEASURES: Detection of indicators of the inflammatory response: interleukin 1 (IL-1), interleukin 6 (IL-6), intercellular adhesion molecule- (ICAM-1), antibacterial protein (defensin 3 that reflects the activation of granulocytes), the antibody clone HAM 56 (for detection of local macrophages), and antibodies against macrophage inhibiting factor (MIF)-related proteins 8 and 14 (MRP 8 and 14). RESULTS: The rise between preoperative and postoperative evaluations was significant for each variable (p < 0.05). With one single exception (IL-6 between laparoscopic cholecystectomy and other operations), the one way analysis of variance (ANOVA) showed no significant differences among the three groups in the detectable increases in staining. Linear regression analysis showed no correlation between length of operation and increases in immunohistochemically detected inflammatory variables. CONCLUSION: Minimally invasive surgery does not necessarily mean minimal peritoneal damage. The immunohistochemical evaluation of the local cellular response may provide additional objective criteria for the grading of operative trauma.

Analysis of the new indirect revascularization method by determining objective parameters of clinical chemistry, histo-chemistry and histology.

OBJECTIVE: This experimental study was initiated to determine whether transmyocardial laser revascularization (TMLR) after acute myocardial ischemia may improve clinical chemistry and diminish the amount of necrosis. In addition, the influence of TMLR on healthy myocardium was analyzed. METHODS: The prolonged short-term effectiveness of TMLR was evaluated in 44 open-chest anesthetized pigs with (n = 21) or without (n = 23) the setting of acute myocardial ischemia (observation period 6 h): seven pigs served as controls (thoracotomy only). An additional seven pigs had left anterior descending artery (LAD) occlusion only (ischemia group). A subsequent 14 pigs were treated by TMLR (CO2) prior to LAD occlusion: Seven pigs received one laser channel/cm2 (group 1) and in seven pigs two channels/cm2 in the LAD territory (group 2) were performed. In addition, 16 pigs underwent TMLR without ischemia: Eight pigs received one channel/cm2 (group 3) and eight pigs two channels/cm2 (group 4). Clinical chemistry, histo-chemical assessment and histology were performed. RESULTS: TMLR limits the expansion of the myocardial infarction zone: laser group 2 indicated a significant smaller area of necrosis in the area at risk (ischemic group (31%) vs. laser group 1 (19%), P = ns; laser group 2 (7%) vs. ischemic group, P < 0.01; laser group 1 vs. 2, P < 0.01). The amount of the area of necrosis and ischemia of laser groups 3 and 4 compared with control did not differ significantly (P = ns). Preventive creation of microchannels before ischemia did not diminish ischemic parameters (P = ns). The myocardial water content-measurements (MWC) in the ischemia, laser 1 and 2 groups did not show any difference at the end of the experiment, except higher values of laser group 2 (P < 0.05). Laser groups 3 and 4 revealed significantly higher MWC values compared with control (P < 0.001). CONCLUSIONS: This prolonged acute study demonstrates that CO2-TMLR significantly reduces the amount of necrosis in the area at risk, but does not reduce cardiac ischemic markers. In healthy myocardium, TMLR significantly increases myocardial water content and ischemic parameters and induces small ischemic and very small necrotic areas surrounding open laser channels. Generally, the elevated cardiac enzymes and proteins are mainly attributed to the expected rise caused by vaporization of myocardial tissue in all laser groups.

[Vaccine development for P. aeruginosa: based on antigen cloning of clinical vaccines][. / Impfstoffentwicklung gegen P. aeruginosa. Von der Antigenklonierung zur klinischen Vakzine.

For the development of a clinical vaccine against P. aeruginosa infections we cloned the genes for the main outer membrane proteins F and I of P. aeruginosa and characterized protective epitopes by monoclonal antibodies. A recombinant hybrid protein (OprFaa190-342-OprIaa21-83) was expressed in E. coli which represents the protective epitopes. The vaccine showed to be highly protective in mice against experimental P. aeruginosa infections. A phase I trial in human volunteers showed that the vaccine is well tolerated and that high antibody titers against P. aeruginosa were induced.

G-CSF during Escherichia coli versus Staphylococcus aureus pneumonia in rats has fundamentally different and opposite effects.

We investigated if bacteria type alters outcome with prophylactic granulocyte colony stimulating factor (G-CSF) therapy during pneumonia. Rats received G-CSF or placebo daily for 6 d and after the third dose were intrabronchially inoculated with either Escherichia coli or Staphylococcus aureus. Without G-CSF, E. coli and S. aureus produced similar (p = NS) mortality rates (36 versus 38%) and serial changes in mean circulating neutrophil counts (CNC), but differing mean (+/- SE) tumor necrosis factor (TNF) levels (E. coli, 259 +/- 104 versus S. aureus, 51 +/- 17 pg/ml, p = 0.01). G-CSF prior to bacteria increased mean CNC more than six times compared with placebo (p = 0.001). However, with G-CSF in the first 6 h after E. coli, there was a greater than 20-fold decrease in mean (+/- SE) CNC (x 10(3)/ mm3) to below placebo (0.5 +/- 0.1 versus 0.8 +/- 0.1), whereas with G-CSF after S. aureus, there was only a fivefold decrease in mean CNC and CNC were greater than placebo (1.8 +/- 0.2 versus 0.8 +/- 0.1) (E. coli versus S. aureus decrease in CNC with G-CSF, p = 0.001). With E. coli, G-CSF worsened oxygenation and increased bacteremia and mortality, whereas with S. aureus, G-CSF improved oxygenation and decreased bacteremia and mortality (G-CSF therapy, E. coli versus S. aureus, p = 0.03, 0.05, and 0.001, respectively). Thus, during S. aureus pneumonia with low TNF levels, G-CSF increased CNC and bacterial clearance, resulting in less pulmonary injury and decreased death. During E. coli pneumonia with high TNF levels, G-CSF paradoxically decreased CNC, resulting in impaired bacterial clearance and worsened pulmonary injury and death. Bacterial species and the associated inflammatory mediator response can alter outcome with prophylactic G-CSF therapy during pneumonia.