Florence M. Rollwagen

Killed Campylobacter elicits immune response and protection when administered with an oral adjuvant

The heat-labile toxin (HLT) of enterotoxigenic Escherichia coli (ETEC) is a potent oral adjuvant. We determined whether the ETEC HLT could be mixed with killed campylobacter to induce an immune response protective upon subsequent challenge with live pathogens. Mice were immunized orally three times with 10(9) sonicated campylobacter with or without 25 micrograms of ETEC HLT, and humoral immune responses in intestinal lavage fluids measured by ELISA. Whereas 10(9) live bacteria induced strong intestinal IgA responses, killed bacteria did not unless ETEC HLT was also added. The magnitude of the antibody response was dependent on the amount of antigen given. The ETEC HLT given with bacteria also induced a potent cross-reaction with cholera toxin. The latter had an adjuvant effect in mice similar to that of ETEC HLT. Protection against colonization was studied in mice and rabbits. In contrast to non-immune animals, those given live organisms or sonicated cells mixed with ETEC HLT quickly cleared homologous, but not heterologous, Lior serotypes of Campylobacter upon challenge. These data show for the first time that ETEC HLT can potentiate an immune response to killed campylobacter that promotes a rapid clearance of live pathogens from the intestine.

Induction of early inflammatory gene expression in a murine model of nonresuscitated, fixed-volume hemorrhage.

The etiology of many end-organ problems associated with hemorrhage has been attributed to the inflammatory response to hemorrhage. In a murine model of nonresuscitated, fixed-volume hemorrhage, we sought to elucidate the role that hemorrhagic insult alone plays in the generation of the early inflammatory cascade. Differences could be appreciated as early as 1 h post-hemorrhage, with consistent differences detected by 3 h in all of the major cytokine genes studied. Significant upregulation of IL-1&bgr;, IL-6, TNF-&agr;, and IL-10 mRNA expression was observed in both the liver and lung samples of mice subjected to fixed-volume hemorrhage when compared with sham-hemorrhaged mice. The cyclooxygenase-2 (COX-2) and inducible nitric oxide synthetase (iNOS) genes also were upregulated in the livers and lungs of hemorrhaged mice. Finally, expression of the genes that encode the Toll-like receptors (TLR)-2 and -4 was increased by hemorrhage. Taken collectively, these data demonstrate that the initial inflammatory cascade associated with hemorrhage occurs within hours after the initial hemorrhagic event, and can be associated with significant modulation of expression of key pro- and anti-inflammatory cytokine, enzyme, and TLR genes, suggesting that these may be possible new therapeutic targets.

Effects of mild hypothermia on survival and serum cytokines in uncontrolled hemorrhagic shock in rats.

Previous studies have suggested benefit of mild hypothermia during hemorrhagic shock (HS). This finding needs additional confirmation and investigation into possible mechanisms. Proinflammatory cytokines are mediators of multiple organ failure following traumatic hemorrhagic shock and resuscitation. We hypothesized that mild hypothermia would improve survival from HS and may affect the pro- and anti-inflammatory cytokine response in a rat model of uncontrolled HS. Under light halothane anesthesia, uncontrolled HS was induced by blood withdrawal of 3 mL/100 g over 15 min followed by tail amputation. Hypotensive (limited) fluid resuscitation (to prevent mean arterial pressure [MAP] from decreasing below 40 mmHg) with blood was started at 30 min and continued to 90 min. After hemostasis and resuscitation with initially shed blood and Ringers solution, the rats were observed for 72 h. The animals were randomized into two HS groups (n = 10 each): normothermia (38°C ± 0.5°C) and mild hypothermia (34°C ± 0.5°C) from HS 30 min until resuscitation time (RT) 60 min; and a sham group (n = 3). Venous blood samples were taken at baseline, RT 60 min, and days 1, 2, and 3. Serum interleukin (IL)-1&bgr;, IL-6, IL-10, and tumor necrosis factor (TNF)-&agr; concentrations were quantified by ELISA. Values are expressed as median and interquartile range. Survival time by life table analysis was greater in the hypothermia group (P = 0.04). Survival rates to 72 h were 1 of 10 vs. 6 of 10 in the normothermia vs. hypothermia groups, respectively (P = 0.057). All cytokine concentrations were significantly increased from baseline at RT 60 min in both HS groups, but not in the shams. At RT 60 min, in the normothermia vs. hypothermia groups, respectively, IL-1&bgr; levels were 185 (119–252) vs. 96 (57–135) pg/mL (P = 0.15); IL-6 levels were 2242 (1903–3777) vs. 1746 (585–2480) pg/mL (P = 0.20); TNF-&agr; levels were 97 (81–156) vs. 394 (280–406) pg/mL (P = 0.02); and IL-10 levels were 1.7 (0–13.3) vs. 15.8 (1.9–23.0) pg/mL (P = 0.09). IL-10 remained increased until day 3 in the hypothermia group. High IL-1&bgr; levels (>100 pg/mL) at RT 60 min were associated with death before 72 h (odds ratio 66, C.I. 3.5–1255). We conclude that mild hypothermia improves survival time after uncontrolled HS. Uncontrolled HS induces a robust proinflammatory cytokine response. The unexpected increase in TNF-&agr; with hypothermia deserves further investigation.

Can specific biological signals be digitized

At the request of the United States Defense Advanced Research Projects Agency, we attempted to replicate the data of Professor Jacques Benveniste that digital signals recorded on a computer disc produce specific biological effects. The hypothesis was that a digitized thrombin inhibitor signal would inhibit the fibrinogen‐thrombin coagulation pathway. Because of the controversies associated with previous research of Prof. Benveniste, we developed a system for the management of social controversy in science that incorporated an expert in social communication and conflict management. The social management approach was an adaptation of interactional communication theory, for management of areas that interfere with the conduct of good science. This process allowed us to successfully complete a coordinated effort by a multidisciplinary team, including Prof. Benveniste, a hematologist, engineer, skeptic, statistician, neuroscientist and conflict management expert. Our team found no replicable effects from digital signals.— Jonas, W. B., Ives, J. A., Rollwagen, F., Denman, D. W., Hintz K., Hammer, M., Crawford, C., Henry, K. Can specific biological signals be digitized? FASEB J. 20, 23–28 (2006)

Modulation of mucosal immunity against Campylobacter jejuni by orally administered cytokines.

The effect of oral recombinant interleukin (rIL) treatment on the course of Campylobacter jejuni infection and the development of mucosal immunity in mice was investigated. rIL-2, rIL-5, and rIL-6 were administered to mice at 24 and 6 h before infection and at 0, 24, and 48 h after infection with C. jejuni HC, and the subsequent development of an immune response and intestinal colonization resistance were determined. In this model, orally administered cytokines retained their biological activities with no apparent side effects. Following infection, initial bacterial counts in fecal samples collected from cytokine-treated and untreated mice were similar. However, within 48 h of infection a greater than 3-log-unit reduction in the number of C. jejuni shed in the feces was found for rIL-6-treated animals. Colonization levels were similarly reduced in rIL-5-treated mice, although the rate of clearance was somewhat slower. In contrast, rIL-2 treatment had no significant effect on colonization levels compared with that in controls. Oral rIL-6 treatment was also associated with enhanced intestinal and systemic Campylobacter-specific immunoglobulin A responses compared with those observed in either rIL-5- or rIL-2-treated animals. Upon rechallenge, initial colonization in all cytokine-treated groups was approximately 2 log units lower than that in controls. However, local infection was controlled only in rIL-2-treated mice over time. rIL-5 and rIL-6 treatment had only a marginal effect on colonization resistance following rechallenge. On the basis of these results, it appears that rIL-5 or rIL-6 may function to modulate the induction and/or expression of anti-C. jejuni immunity through different mechanisms.

Microvascular Effects of Oral Interleukin-6 on Ischemia/Reperfusion in the Murine Small Intestine

Oral administration of interleukin-6 (IL-6) has been shown to reduce hemorrhage-induced bacterial translocation from the gut in mice and rats. To examine the intestinal microvasculature, mice were given the electron-dense tracer horseradish peroxidase (HRP) after hemorrhage and IL-6 or vehicle administration. In normal mice and in those hemorrhaged and given IL-6, the electron-dense marker, administered intravenously, could be found in intestinal capillaries and between mucosal epithelial cells, suggesting that the microvasculature was patent. In mice given saline after shock, however, no marker was present in the gut, suggesting that the intestinal microvasculature was unable to deliver the marker to the epithelia. When mice were given HRP intralumenally (il) the tracer was able to penetrate between intestinal epithelial cells only in mice given vehicle after hemorrhage. This finding suggests that hemorrhaged mice were susceptible to sepsis and endotoxic shock from the leaky gut. In normal and IL-6-treated mice, the tracer was unable to pass from the lumen between mucosal epithelial cells, because the presence of an intact zonula occludens prevented passage. Functional studies supported the electron microscopy findings. Bacteria were cultured from the livers of mice fed vehicle after hemorrhage, but not from those fed IL-6. These data support the conclusions that parts of the intestinal microvasculature remain diminished after hemorrhage and resuscitation and that oral IL-6 restores this circulation.

RNA interference targeting Akt promotes apoptosis in hypoxia-exposed human neuroblastoma cells

Overactivation of the PI3 kinase/Akt pathway plays an essential role in the development and progression of various tumors. Akt is a key component of this pathway and hyperactivated in different tumors including neuroblastoma and glioma. In the present study, we tested the therapeutic efficacy of siRNA targeting Akt in inducing apoptotic cell death in NBFL cells (a human neuroblastoma cell line) subjected to anoxia/reoxygenation (A/R), a process that has been shown to modulate growth and progression of malignant tumors. We observed that siRNA targeting Akt effectively induced apoptotic cell death in NBFL cells (as determined by TUNEL assay and activated caspase-3 immunoreactivity) under normoxic conditions, an effect that was greatly enhanced under conditions of A/R. These findings underscore the importance of Akt signaling in promoting survival of neuroblastoma cells and may have potential therapeutic applications.

Gut damage during hemorrhagic shock: effects on survival of oral or enteral interleukin-6.

It has been reported that oral interleukin (IL)-6, without deleterious systemic side effects, prevents bacteremia and gut epithelial apoptosis after hemorrhagic shock (HS) in rodents. The goal of this study was to explore potential benefit of oral or enteral IL-6 on the gut and, consequently, on survival in a long-term outcome model of HS in rats. In Study A, 20 rats (control and IL-6, n = 10 per group) were anesthetized by spontaneous breathing of halothane and N2O. The left femoral vein and artery were cannulated. HS was initiated with withdrawal of 3 mL of blood per 100 g body weight over 15 min, and mean arterial pressure was maintained at 40 to 50 mmHg for another 75 min (total HS 90 min) by blood withdrawal or infusion of Ringers solution. At HS 90 min, resuscitation included reinfusion of shed blood and additional Ringers solution to restore normotension for 30 min. After awakening at resuscitation time 30 min, the rats received either 300 units IL-6 or the same volume of vehicle (controls) injected into the stomach via a feeding cannula. In Study B, 20 rats (control and IL-6, n = 10 per group), fasted overnight, were prepared and treated as in Study A, except that HS was initiated with withdrawal of 2 mL blood per 100 g over 10 min, and mean arterial pressure was maintained at 35-40 mmHg. IL-6 rats received 3,000 units IL-6 in 5 mL of normal saline injected directly into the ileum lumen 20 min after induction of shock and again at resuscitation time 60 min. Control rats received normal saline alone. In both studies, survival was observed to 72 h. In Study A, 7 of 10 rats in the control group and 5 of 10 in the IL-6 group survived to 72 h (NS). Macroscopic assessment of gut injury was not different between the two groups. In Study B, 6 of 10 rats survived to 72 h in each group. Frequency of bacteria growth in liver tissue of 72 h survivors was not different between the two groups. IL-6, administered into the stomach or directly injected into the small intestine lumen, did not protect the gut from ischemic injury, nor did it improve survival following severe HS in rats.

Administration of recombinant interleukin-11 improves the hemodynamic functions and decreases third space fluid loss in a porcine model of hemorrhagic shock and resuscitation.

We have previously demonstrated that the administration of recombinant human interleukin-11 (rhIL-11) during resuscitation improves the blood pressure in a rodent model of hemorrhagic shock. The purpose of this study was to determine whether the effects of rhIL-11 could be reproduced in a large animal model and to elucidate the impact of rhIL-11 administration on the intravascular volume status and the degree of third space fluid loss after resuscitation. A 40% blood volume hemorrhage was induced in swine (n = 45, weight of 25-35 kg) followed by a 1-h shock period and resuscitation with 0.9% sodium chloride (three times the shed blood volume). The animals were randomized to receive sham hemorrhage (group I, sham); sham hemorrhage and 50 μg/kg rhIL-11 (group II, sham + IL-11); no drug (group III, saline); or 50 μg/kg rhIL-11 (group IV, IL-11). Blood and urine samples were obtained and analyzed at baseline, at the end of hemorrhaging, and thereafter once every hour. The pleural and peritoneal effusions were precisely quantified by using clinically accepted criteria. The mean arterial pressure (MAP) was higher postresuscitation (PR) in groups I, II, and IV (71.4 ± 7.5 mmHg, 71.0 ± 8.9 mmHg, and 72.9 ± 12.3 mmHg, respectively) than in group III (59.9 ± 10.9 mmHg), and the cardiac output of PR was higher in group IV (3.46 ± 0.56 L/min) than in group III (2.99 ± 0.62 L/min; P < 0.01). The difference in MAP between groups I and II became statistically significant at 40 min after rhIL-11 injection and such a difference persisted for 90 min. After resuscitation, the urine output was higher, and the urine specific gravity and third space fluid loss were lower in group IV (1434 ± 325 mL and 1.0035, 82 ± 21 mL) than in group III (958 ± 390 mL and 1.0053, 125 ± 32 mL; P < 0.05). In a porcine model of hemorrhagic shock, the administration of rhIL-11 at the start of resuscitation significantly improved the cardiac output and blood pressure. This strategy also significantly reduced the extent of third space fluid losses while also having a favorable impact on the intravascular volume status as evidenced by the improved urine output.