Collective consciousness and its pathologies: understanding the failure of AIDS control and treatment in the United States.

We address themes of distributed cognition by extending recent formal developments in the theory of individual consciousness. While single minds appear biologically limited to one dynamic structure of linked cognitive submodules instantiating consciousness, organizations, by contrast, can support several, sometimes many, such constructs simultaneously, although these usually operate relatively slowly. System behavior remains, however, constrained not only by culture, but by a developmental path dependence generated by organizational history, in the context of market selection pressures. Such highly parallel multitasking--essentially an institutional collective consciousness--while capable of reducing inattentional blindness and the consequences of failures within individual workspaces, does not eliminate them, and introduces new characteristic malfunctions involving the distortion of information sent between workspaces and the possibility of pathological resilience--dysfunctional institutional lock-in. Consequently, organizations remain subject to canonical and idiosyncratic failures analogous to, but more complicated than, those afflicting individuals. Remediation is made difficult by the manner in which pathological externalities can write images of themselves onto both institutional function and corrective intervention. The perspective is applied to the failure of AIDS control and treatment in the United States.

Injury induces alterations in T-cell NFkappaB and AP-1 activation.

The immune dysfunction that occurs after severe injury involves major changes in T-cell-mediated immunity resulting in suppressed T-helper 1 (Th1) type responses and increased or persistent T-helper 2 (Th2) type cytokine production. Since little is known about what signaling pathways are responsible for this injury-induced phenotypic shift in T-cells, we undertook this study to address the molecular basis for injury effects on T-helper cell subset cytokine expression. Experiments were designed to test whether diminished IL-2 gene expression after thermal injury coincided with changes in the induction of IL-2 gene regulatory transcription factors. Electrophoretic mobility shift assays (EMSA) were used to screen for nuclear expression of changes of the IL-2 gene transcription factors. Our findings revealed that changes in mitogen-stimulated T-cell AP-1 and NFkappaB factor activation correlated directly with defective mitogen-induced IL-2 mRNA expression. We determined that there was a loss of nuclear AP-1 activation and changes in NFkappaB factor activation at 9 days after injury. T-cell nuclear extracts prepared from sham injured mice showed induction of NFkappaB2 (p52) and RelA (p65) containing NFkappaB EMSA complexes, while we detected no RelA or NFkappaB2 in EMSA complexes using T-cell nuclear extracts prepared from burn injured mice. Instead, these NFkappaB EMSA complexes contained mostly NFkappaB1 (p50). Western immunoblot analysis confirmed defective nuclear RelA translocation. Taken together, these results indicate that T-cell NFkappaB and AP-1 activation pathways may be involved in the injury-induced changes in T-cell cytokine production and the immune deviation that occurs after injury.

Injury induces deficient interleukin-12 production, but interleukin-12 therapy after injury restores resistance to infection.

OBJECTIVE: To assess at serial intervals the production of interleukin-12 (IL-12) by monocytes/macrophages from the peripheral blood of injured patients and control subjects, and using a mouse model to confirm human findings and explore the effectiveness of low-dose IL-12 therapy in restoring resistance to infection after injury. SUMMARY BACKGROUND DATA: Serious injury is associated with loss of function of the T helper 1 lymphocyte phenotype, but little is known about IL-12 production in injured patients. The authors previously reported that early, moderate-dose IL-12 therapy in a mouse model of burn injury restored resistance to a later infectious challenge (cecal ligation and puncture, CLP). However, the efficacy of clinically relevant low-dose IL-12 therapy carried out to or beyond the time of septic challenge remains to be tested. METHODS: Peripheral blood mononuclear cells (PBMCs) and adherent cells were obtained from 27 patients with major burns or traumatic injury and 18 healthy persons and were studied at serial intervals for IL-12 production stimulated by bacterial lipopolysacharide (LPS). PBMCs from 18 of the same patients were studied for IL-10 production as well. IL-12 production by adherent cells from the spleens of burn or sham burn mice was studied at serial intervals after injury to confirm the human findings. Low-dose IL-12 or vehicle was given every other day to groups of burn and sham burn mice, which were then challenged with CLP on day 10, and survival was determined. Finally, spleens were harvested from burn or sham burn animals receiving low-dose IL-12 or vehicle after CLP. After splenic cellularity was determined by hemocytometer, splenocytes were cultured and production of tumor necrosis factor-alpha, interferon-gamma, and IL-10 were assessed by immunoassay. RESULTS: Adherent cells from patients' PBMCs produced significantly less IL-12 than normal PBMCs after injury, reaching a nadir 8 to 14 days after injury. Stimulation of whole PBMCs by LPS indicated that at 8 to 14 days after injury, IL-12 production by PBMCs was significantly lower and IL-10 production was significantly higher than that of PBMCs from healthy persons. Low-dose IL-12 therapy significantly increased survival after CLP. Splenocytes from burn mice treated with IL-12 had significantly increased production of TNF-alpha and IF-beta, both before and after CLP, when compared with vehicle-treated burn animals. IL-10 production by bum splenocytes remained high after IL-12 treatment. Splenic cellularity increased after IL-12 treatment in burn mice. CONCLUSION: The capacity to produce IL-12 by adherent cells of the monocyte/macrophage lineage is significantly reduced after serious injury in humans and in a mouse burn model. In humans, there is a reciprocal relation between diminished IL-12 production and increased IL-10 production at approximately 1 week after injury. Low-dose IL-12 therapy in the mouse burn model markedly increased survival after a septic challenge, even when treatment was carried beyond the onset of sepsis. Low-dose IL-12 treatment in the mouse increased production of proinflammatory mediators important in host defense and at the same time maintained or increased production of IL-10, an important antiinflammatory cytokine.

The effects of injury on the adaptive immune response.

For more than thirty years it has been apparent that serious injury in humans and experimental animals is associated with a decrease in immune functions dependent upon T cells, the principal cells involved in initiating adaptive immune responses. This review focuses on more recent evidence that T helper cell function is altered after serious injury with loss of T helper 1 function and cytokine production and with preservation of T helper 2 function and an increased production of T helper 2 cytokines. Emphasis is placed on the importance of interactions between the innate and adaptive immune systems in the perturbed immune responses seen following injury. Immunomodulatory strategies are mentioned that have had success in animal models in ameliorating the diminished resistance to infection commonly seen after major traumatic or thermal injury. Finally, it is emphasized that immunomodulatory treatments that are successful in preventing infection may be contraindicated once infection is manifest.

Major injury induces increased production of interleukin-10 by cells of the immune system with a negative impact on resistance to infection.

OBJECTIVE: The purpose of this study was to compare the production of interleukin-10 (IL-10) by peripheral blood mononuclear cells (PBMC) from injured patients and control subjects to determine the responsible cell types and to relate IL-10 production to the occurrence of sepsis. A mouse model of burn injury was used to confirm the human findings and to assess the importance of IL-10 in the lowered resistance to infection after injury. SUMMARY BACKGROUND DATA: Severe injury is associated with depressed immune responses. Although IL-10 is known to inhibit several aspects of immune reactivity, the role of IL-10 in postinjury immune suppression remains controversial. METHODS: Peripheral blood mononuclear cells from 14 burn and 12 trauma patients and 16 healthy individuals were studied at serial intervals for IL-10 production stimulated by a T-cell mitogen, phytohemagglutinin, and by bacterial lipopolysaccharide. To determine the source of IL-10, CD4+ and CD8+ lymphocyte subsets were obtained by selective depletion of PBMC with antibody-coated magnetic beads and were stimulated by anti-CD3 antibody to induce IL-10 secretion. In addition, IL-10 production by patients' PBMC in the first 10 days after injury was assessed for correlation with subsequent septic events. Anti-CD3-stimulated IL-10 production also was determined for CD4- and CD8-enriched lymphocyte subsets obtained by antibody and complement depletion of splenocytes harvested from groups of burn and sham burn mice at day 10 after injury, the time of maximal susceptibility to a septic challenge, cecal ligation and puncture (CLP). Finally, to test the importance of IL-10 in immune suppression in vivo, groups of burn and sham burn mice were treated with anti-IL-10 monoclonal antibody or control immunoglobulin G (IgG) on days 1 and 3 postinjury and were observed for survival after CLP on day 10. RESULTS: Patients' PBMC produced significantly more IL-10 than did controls' PBMC 7 to 14 days after injury. Patients' CD4+ (T-helper) but not CD8+ (T-cytotoxic) lymphocytes also showed increased IL-10 production versus those of control subjects early after injury. Increased PBMC IL-10 production in the first 10 days postinjury correlated significantly (p < 0.05) with subsequent septic events. Burn mouse CD4-enriched but not CD8-enriched splenocytes produced more IL-10 than did sham burn splenocyte subsets on day 10 after injury. Burn mice treated with anti-IL-10 antibody but not with control IgG had significantly increased survival after CLP. CONCLUSION: Serious injury in humans and in a mouse burn model is followed by increased stimulated production of IL-10 by cells of the immune system. The CD4+ T-helper cells appear to be a major source of IL-10 after injury. In injured patients, increased IL-10 production is correlated with subsequent septic events, and in the burn mouse, IL-10 appears to induce decreased resistance to infection.

Is circulating endotoxin the trigger for the systemic inflammatory response syndrome seen after injury?

OBJECTIVE: Patients with severe traumatic or burn injury and a mouse model of burn injury were studied early after injury to determine the relation of plasma endotoxin (lipopolysaccharide [LPS]) to the production of proinflammatory cytokines and subsequent resistance to infection. SUMMARY BACKGROUND DATA: Elevated levels of plasma LPS have been reported in patients after serious injury. It has been suggested that circulating LPS may be a trigger for increased proinflammatory cytokine production and may play a role in the septic syndromes seen in a substantial portion of such patients. Yet, despite multiple reports of leakage of LPS from the gut and bacterial translocation after injury in animal models, there is little direct evidence linking circulating LPS with production of inflammatory mediators. METHODS: The authors studied serial samples of peripheral blood from 10 patients with 25% to 50% surface area burns and 8 trauma patients (injury Severity Score, 25-57). Patients were compared with 18 healthy volunteers. The study was focused on the first 10 days after injury before the onset of sepsis or the systemic inflammatory response syndrome. Plasma samples were assayed for LPS, and adherent cells from the blood were studied for basal and LPS-stimulated production of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and interleukin-6 (IL-6). The correlation of increased plasma LPS with TNF-alpha production was studied as was the association of increased plasma LPS and increased TNF-alpha production with subsequent septic complications. We also studied a mouse model of 25% burn injury. Burn mice were compared with sham burn control subjects. Plasma samples were assayed at serial intervals for LPS, and adherent cells from the spleens were studied for basal- and LPS-stimulated production of TNF-alpha, IL-1 beta, and IL-6. Expression of the messenger RNAs for IL-1 beta and TNF-alpha also was measured. The relation of increased TNF-alpha production with mortality from a septic challenge, cecal ligation and puncture (CLP), was determined. Finally, the effect of administration of LPS to normal mice on subsequent mortality after CLP and on TNF-alpha production was studied. RESULTS: Elevated plasma LPS (> 1 pg/mL) was seen in 11 of the 18 patients within 10 days of injury and in no normal control subjects. In this period, patients as compared with control subjects showed increased stimulated production of TNF-alpha, IL-1 beta, and IL-6. Increased TNF-alpha production was not correlated with elevated plasma LPS in the same patients. Neither increased plasma LPS nor increased TNF-alpha production early after injury was correlated with subsequent development of systemic inflammatory response syndrome or sepsis in the patients. Burn mice, as compared with sham burn control subjects, showed elevated plasma LPS levels chiefly in the first 3 days after injury. Increased stimulated production of proinflammatory cytokines by adherent splenocytes from the burn mice also was seen at multiple intervals after injury and did not correlate with mortality from CLP. Increased production of TNF-alpha and IL-1 beta was associated with increased expression of messenger RNAs for these cytokines. Finally, two doses of 1 ng LPS administered 24 hours apart to normal mice had no effect on mortality from CLP performed 7 days later nor on the production of TNF-alpha at the time of CLP. CONCLUSIONS: These findings call into question the idea that circulating LPS is the trigger for increased proinflammatory cytokine production, systemic inflammatory response syndrome, and septic complications in injured patients.

Interleukin-12 treatment restores normal resistance to bacterial challenge after burn injury.

BACKGROUND: Preliminary studies in this laboratory have shown that treatment with interleukin-12 (IL-12), a cytokine that induces expression of the T-helper-1 lymphocyte phenotype, in an animal model of burn injury increased survival after a septic challenge. The purpose of this study was to define the efficacy of IL-12 therapy and to explore its mechanism of action. METHODS: Adult male A/J mice were subjected to 25% full-thickness scald or sham burn. Starting on day 3 after burn, groups of mice received five daily injections of IL-12, interferon-gamma (IFN-gamma), or saline solution. Some animals received anti-IFN-gamma monoclonal antibody. At day 10 most animals underwent cecal ligation and puncture (CLP) and were observed for survival. Some animals were killed at day 10, and CD4-enriched splenocytes were stimulated with anti-CD3 antibody or concanavalin A and were studied for cytokine production and mRNA expression. RESULTS: IL-12 treatment, 25 ng daily for 5 days, increased survival of the burn group after CLP to that of the sham burn control group. Anti-IFN-gamma antibody, 500 micrograms, administered 1 day before IL-12 treatment, reduced the efficacy of IL-12. IFN-gamma treatment, 7000 units, moderately increased survival. IL-12 had no effect on survival of the sham burn group. At the time of CLP IL-12 therapy had induced a marked decrease in CD4+ lymphocyte IL-4 and a moderate increase in IFN-gamma production and mRNA expression without affecting IL-2. CONCLUSIONS: IL-12 is the most effective therapy so far tested in this burn plus CLP model. It acts at least in part through IFN-gamma. However, IFN-gamma therapy was not as effective as IL-12.

Dosage and timing of anti-TNF-alpha antibody treatment determine its effect of resistance to sepsis after injury.

Antibody against tumor necrosis factor-alpha (TNF-alpha) has improved survival in certain models of sepsis, but it remains unproven in clinical studies. In most of the successful animal studies, efficacy has been shown in previously healthy animals subjected to a septic challenge. Patients at risk for sepsis, however, may be ill for some time before the sepsis supervenes. This situation has been described as a "two-hit" model of critical illness. We have developed an animal burn-sepsis model which conforms to this "two-hit" concept. We have quantified macrophage TNF-alpha production at different times after the burn (first "hit") and determined the effect of neutralizing antibody against TNF-alpha during this period on survival after subsequent sepsis (second "hit"). The objective of this study was to determine the role of TNF-alpha and the effect of neutralizing antibody against TNF-alpha in a burn-sepsis model. Animals were subjected to a full thickness burn or sham burn. In vitro TNF-alpha production from cultured lipopolysaccharide-stimulated splenic adherent cells was determined at various time points thereafter by enzyme-linked immunosorbent assay. Separate animals were treated with neutralizing antibody against TNF-alpha at different time points after the thermal injury, and survival was determined after septic challenge (cecal ligation and puncture) on Day 10 after the burn. TNF-alpha production from adherent splenocytes was not elevated in the early days after thermal injury, but was significantly enhanced from Day 6 onward compared with sham-burned animals. Nine percent of the burned mice survived septic challenge compared with 69% of the sham-burned control mice (P < 0.001). Therapy with anti-TNF antibody at 1 x 10(4) neutralizing units (n.u.) kg-1 markedly improved outcome if given when TNF-alpha production was elevated at Day 7 after the burn (survival, 36%; P = 0.01) but did not improve survival when administered at Days 0 or 4 or at the time of the septic challenge (Day 10). High doses of antibody (3.2 x 10(5) n.u.kg-1) were not beneficial and may have been detrimental. These results show that neutralizing antibody against TNF-alpha may reduce the susceptibility to infection seen after thermal injury, but the timing of administration of the antibody and the dose of antibody used are critical to the outcome. This should be considered when neutralizing antibody against TNF is used in the clinical setting.

Decreased interleukin-2 production in murine acute pancreatitis: potential for immunomodulation.

BACKGROUND & AIMS: The role of the cytokine interleukin 2 (IL-2) has long been recognized as central to normal immunologic function and defense against infection after burns and trauma, but little effort has been directed towards its role in acute pancreatitis (AP), which also has a high mortality related to sepsis. This study investigated the potential role of IL-2 in mice with diet-induced AP. METHODS: AP was induced in mice by 10 days of feeding a choline-deficient, ethionine-supplemented diet. T-helper (CD4) cells were estimated, and T-cell mitogen-stimulated splenocyte proliferation and IL-2 production in vitro were measured on days 3, 7, and 10. RESULTS: Significant reduction in IL-2 production was found on day 3 (32%; P < 0.05) and day 10 (48%; P < 0.005). Administration of intraperitoneal lipopolysaccharide on day 10 was associated with reduced IL-2 production (P < 0.025) 4 hours later and 90% mortality in animals with AP. In vivo therapy with recombinant IL-2 improved in vitro IL-2 secretion (P < 0.05) and reduced lipopolysaccharide-induced mortality (P = 0.036). CONCLUSIONS: Murine diet-induced AP is associated with impaired immune function and increased susceptibility to sepsis and may be a valuable tool in the investigation of immunomodulation in AP.

Multiple system organ failure may be influenced by macrophage hypoactivation as well as hyperactivation--importance of the double challenge.

OBJECTIVE: To find out if an infective challenge caused by a burn followed by caecal ligation and puncture in mice caused more abnormalities of the immune response than burn alone or caecal ligation and puncture alone. DESIGN: Laboratory study. SETTING: University hospital, USA. MATERIAL: 80 male 7-8 week old A/J mice. INTERVENTIONS: Burn followed 10 days later by caecal ligation and puncture (n = 18), caecal ligation and puncture alone (n = 24), burn alone (n = 20), and controls (n = 18). The mice had their spleens removed on day 11 (n = 28; 6, 8, 8, and 6 in the respective groups), day 12 (n = 26; 6, 8, 6, and 6), and day 13 (n = 26; 6, 8, 6, and 6), and splenocytes and adherent cells were harvested for measurement of prostaglandin E2 (PGE2), interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 6 (IL-6), and tumour necrosis factor alpha (TNF-alpha). MAIN OUTCOME MEASURES: Alterations in the production of the cytokines. RESULTS: After the double challenge (burn followed by caecal ligation and puncture) there were significant reductions in production of TNF-alpha and IL-6 compared with caecal ligation and puncture alone (p < 0.05), burn alone (p < 0.05), and controls (p < 0.05). These findings indicate that activation of macrophages was reduced after infection; production of TNF-alpha, IL-1, and IL-6 by splenocytes stimulated by lipopolysaccharide was reduced. CONCLUSIONS: The differences do not seem big enough to indicate that mortality would be increased after caecal ligation and puncture alone. Only when there has been a previous injury (which resulted in hyperactivation of macrophages followed by a more pronounced hypoactivation) would mortality increase. In view of clinical trials with antiendotoxin and antiTNF antibodies that failed to improve survival in infected patients, we suggest that the mechanisms of the cellular immune response need further clarification.

Major injury leads to predominance of the T helper-2 lymphocyte phenotype and diminished interleukin-12 production associated with decreased resistance to infection.

OBJECTIVE: Patients with serious traumatic injury and major burns and an animal model of burn injury were studied to determine the effect of injury on the production of cytokines typical of the T helper-2 lymphocyte phenotype as opposed to the T helper-1 phenotype and on the production of interleukin-12. SUMMARY BACKGROUND DATA: Perturbations of natural and adoptive immunity are related to the increased susceptibility to infection manifested by seriously injured and burn patients. Earlier work has shown that impaired adoptive immunity after injury is characterized by diminished production of interleukin-2 (IL-2), a product of Th lymphocytes. Exposure of naive Th cells to certain antigens and cytokines causes conversion to either the Th-1 or the Th-2 phenotype. Th-1 cells produce IL-2 and interferon-gamma (IFN-tau) and initiate cellular immunity. Th-2 cells secrete interleukin-4 (IL-4) and interleukin-10 (IL-10) and stimulate production of certain antibodies. Conversion to the Th-1 phenotype is facilitated by IL-12, and conversion to the Th-2 phenotype is promoted by IL-4. The authors believed that serious injury might cause conversion of Th cells to the Th-2 as opposed to the Th-1 phenotype rather than generalized Th suppression. METHODS: The authors studied circulating peripheral blood mononuclear cells (PBMC) from 16 major burn and 8 trauma patients on 32 occasions early after injury and from 13 age- and sex-matched healthy individuals for cytokine production after phytohemagglutinin stimulation. Also studied was a mouse model of 20% burn injury known to mimic the immune abnormalities seen in humans with burns. Splenocytes from burn mice, 10 to 12 per group, were studied after activation by concanavalin A or by the bacterial antigen Staphylococcus aureus Cowan strain I for cytokine production and cytokine messenger RNA expression as determined by reverse transcriptase polymerase chain reaction. Burn mice were compared with sham-burn controls and attention was focused on day 10 after burn injury, a time when IL-2 production and resistance to infection are highly suppressed. Finally, burn and sham-burn animals, 20 per group, were treated in vivo with IL-12 (25 ng daily for 5 days) and observed for mortality after septic challenge (cecal ligation and puncture [CLP]) performed on day 10 after injury. RESULTS: Peripheral blood mononuclear cells from burn and trauma patients produced less IFN-tau, the index cytokine of Th-1 cells, than PBMCs from healthy individuals 1 to 14 days after burn injury (SE = 77.6 +/- 16 pg/mL patients vs. 141.3 +/- 35 pg/mL controls, p < 0.05). However, production of IL-4, the index cytokine of Th-2 cells, by patient PBMCs was increased (51.0 +/- 13.0 pg/mL patients vs. 26.9 +/- 2.5 controls, p < 0.05). Splenocytes from mice 10 days after burn injury, when compared with sham-burn controls, showed diminished production of IL-2 (1.04 +/- 0.91 units/mL burns vs. 5.8 +/- 0.55 units/mL controls, p < 0.05) and IFN-tau (1.05 +/- 0.7 units/mL burns vs. 12.0 +/- 8.9 units/mL controls, p < 0.05). However, burn splenocytes produced more IL-4 (2492 +/- 157.0 pg/mL burns vs. 672.0 +/- 22.7 pg/mL controls, p < 0.01) and IL-10 (695.2 +/- 20.8 pg/mL burns vs. 567.0 +/- 16.7 pg/mL controls, p < 0.05). Splenocyte production of IL-12 was also reduced after burn (0.20 +/- 0.035 units/mL) as compared with sham burn (0.46 +/- 0.08 units/mL, p < 0.05). The reduction in IL-2, IFN-tau, and IL-12 production by burn splenocytes was reflected by a tenfold decrease in expression of their respective cytokine mRNAs. In vivo IL-12 treatment of burn animals decreased mortality from CLP on day 10 after injury from 85% to 15% (sham-burn mortality after CLP, 15%, p < 0.05) and increased splenocyte IFN-tau production to supranormal levels. CONCLUSIONS: Serious injury induced diminished production of IL-1 2 and a shift to the Th-2 phenotype with increased production of IL-4 and IL-10, cytokines known to inhibit Th-1 function. The ability of exogenous IL-12 to restore Th-1 cytokine production and resistance to infection suggests a therapeutic role for IL-12 in the immune dysfunction seen after major injury.

Granulocyte-macrophage colony-stimulating factor modulates immune function and improves survival after experimental thermal injury.

Thermal injury is associated with reduced colony-stimulating activity, which correlates with increased susceptibility to infection. To assess the effect of therapeutic administration of granulocyte-macrophage colony-stimulating factor (GM-CSF), 8-week old anaesthetized mice were subjected to either a 20 per cent body surface burn or a sham burn. Animals were subsequently treated with either vehicle or a range of doses of GM-CSF (10-1000 ng) with or without indomethacin (5 micrograms). Sepsis was induced by caecal ligation and puncture on day 10 after injury. Survival was significantly better in animals treated with 200 ng GM-CSF on days 5-9 after the burn. Concanavalin A-stimulated T cell proliferation and interleukin (IL) 2 production were significantly depressed after burn injury. In vivo therapy with 200 ng GM-CSF, however, led to a significant improvement in both of these parameters of T cell function. These data suggest that GM-CSF has a potential therapeutic role in the prevention of death from burn sepsis and appears to act, at least in part, by restoring defective T cell proliferation and IL-2 production.

Long-term immunotherapeutic intervention with pentoxifylline in a mouse model of thermal injury and infection.

Major thermal or traumatic injury often results in abnormalities of immune function, and these abnormalities contribute to the increased susceptibility to infection observed in these patients. Abnormalities of T-cell function, including decreased proliferation and secretion of cytokines are observed following major injury and, conversely, there is markedly increased monokine production. Thus, therapy of this syndrome might logically be aimed at modulating the immune system to upregulate T-cell function and downregulate monocyte hyperactivation. Pentoxifylline (PTX), a methylxanthine derivative, has been shown to be therapeutically effective in several animal models. The purpose of this study was to evaluate PTX and its effect on cytokine production in a mouse model of thermal injury and to study its effect on survival after septic challenge. The results show that PTX therapy after injury can restore T-cell production of IL-2 and downregulate the hyperactive macrophage secretion of proinflammatory cytokines. However, improvement in survival resulting from this therapy following thermal injury and septic challenge depends on timing of dosage.

Pretreatment with enteral cholestyramine prevents suppression of the cellular immune system after partial hepatectomy.

OBJECTIVE: The authors tested the hypothesis that the beneficial effects of the endotoxin-binding agent cholestyramine on the postoperative course in rats that had undergone a partial hepatectomy was the result of improvement of cellular immune functions. SUMMARY BACKGROUND DATA: Major liver resection is associated with severe postoperative complications and a high incidence of systemic infections. Gut-derived endotoxins previously were shown to be involved in the pathogenic processes after partial hepatectomy in rats. In addition, enteral cholestyramine improved postoperative survival, but how its beneficial effects are mediated is not clear. METHODS: Rats that were force-fed for 7 days with either cholestyramine (150 mg/day) or 0.9% saline (equal volume) were randomized to undergo a partial hepatectomy or a sham operation. After 24 hours, the rats were killed and splenic mononuclear cells were tested in vitro for mitogenic responses and cytokine production. RESULTS: Proliferative responses of splenic B and T lymphocytes and lipopolysaccharide-stimulated production of tumor necrosis factor and interleukin-1 by splenocytes were lower in rats after partial hepatectomy than in sham-operated animals. An increased concanavalin A-stimulated production of interleukin-2 also was found after partial hepatectomy compared with sham levels. Pretreatment with enteral cholestyramine preserved cellular proliferative responsiveness of both B and T cells, and restored cytokine production by splenocytes to sham levels. CONCLUSION: Prophylactic treatment with enteral cholestyramine preserved cellular immune functions after partial hepatectomy in the rat, which may explain its beneficial effects on the postoperative course. Furthermore, the authors' results are consistent with the hypothesis that endotoxemia is involved in the pathogenesis of the cellular immune derangements after partial hepatectomy.

Interleukin-2 receptor expression and function following thermal injury.

BACKGROUND/OBJECTIVE: Serious traumatic or thermal injury is associated with depression of cellular immunity, including the failure of T-lymphocyte proliferation in response to stimulation that depends both on production of interleukin-2 (IL-2) and on expression of functional IL-2 receptors (IL-2R). While decreased IL-2 production following thermal injury is undisputed, the status of IL-2R expression and function in this setting is controversial; therefore, we sought to investigate this issue. DESIGN: A total of 220 male A/J mice (n = 22 per group) were subjected to a 20% scald burn injury or sham burn, killed 4, 7, 10, 14, or 21 days later, and splenocytes harvested. In vitro parameters of both IL-2R expression and function were measured. RESULTS: On day 7, splenic lymphocyte proliferation and IL-2 production in response to mitogenic stimulation were both suppressed following burn injury to 50% and 60% of controls, respectively. Northern blot analysis revealed normal IL-2R p55 messenger RNA expression in response to mitogenic stimulation on days 7, 10, and 14 in thermally injured animals. Phenotypic IL-2R p55 expression in concanavalin A-stimulated CD3+ cells was unchanged following burn injury. Binding of fluorescein-labeled IL-2 to cell membranes was increased in burned animals at days 10 and 14. The addition of IL-2 to cultures of spleen cells from burned mice consistently restored the mitogenic response to that of the controls. CONCLUSIONS: Thermal injury in this model does not result in either quantitative or functional suppression of IL-2R. Suppression of T-cell activation and proliferation, seen following thermal injury, appears primarily related to abnormal IL-2 production.

The role of cyclic adenosine monophosphate in the suppression of cellular immunity after thermal injury.

BACKGROUND AND OBJECTIVE: Cyclic adenosine monophosphate (cAMP) is an intracellular second messenger that is known to convey inhibitory signals for T-cell proliferation and function. We investigated the association between this molecule and the profound immunosuppression that accompanies thermal injury. DESIGN: Mice were randomized into two groups: one group was subjected to a 20% full-thickness scald burn; the second to a sham burn (control). The mice were killed on days 4, 7, or 10 after the burn injury and splenocytes were pooled and cultured for 15 minutes in the presence or absence of prostaglandin E2 (PGE2). RESULTS: Levels of cAMP in splenocytes were significantly elevated on day 7 after burn in the burn group compared with the sham controls (P < .05, Wilcoxon Rank Sum Test). Incubation of splenocytes with PGE2 resulted in significantly greater levels of intracellular cAMP in cells from the burn group compared with controls on days 4, 7, and 10. Incubation of normal splenocytes with dibutyryl cAMP in the presence of concanavalin A significantly decreased cell proliferation and the production of interleukin-2. The decrease in interleukin-2 production was evident at the level of messenger RNA expression. Stimulation of splenocytes with a combination of phorbol ester and calcium ionophore, bypassing all membrane-associated events prior to protein kinase C activation, reversed the inhibitory effects of dibutyryl cAMP. Incubation of splenocytes from burned animals with H-8, a selective inhibitor of cAMP-dependent protein kinases, restored the proliferative response to that of sham controls on days 4, 7, and 10 after thermal injury. CONCLUSIONS: These data indicate that elevated levels of intracellular cAMP, combined with an increased production of cAMP in response to circulating PGE2, may play a fundamental role in suppression of the immune response following thermal injury and that cAMP exerts its immunomodulatory effects prior to protein kinase C activation.

Altered gene transcription after burn injury results in depressed T-lymphocyte activation.

OBJECTIVE: Patients with major burns and an animal model of burn injury were studied to determine the mechanism of depressed interleukin-2 (IL-2) production after thermal injury and to determine the effect of such injury on IL-2 receptor (IL-2R) expression and function. SUMMARY BACKGROUND DATA: Major burn injury is known to diminish resistance to infection by altering cytokine production and prostanoic secretion and by inhibiting T-lymphocyte activation. T-cell activation requires production of regulatory cytokines, principally IL-2, and expression of the appropriate cytokine receptors. Depressed IL-2 production after major burn injury is undisputed, although the molecular mechanisms remain undefined; the effect of burn injury on IL-2R expression and function currently is controversial. METHODS: The authors studied serial samples of peripheral blood mononuclear cells (PBMC) from 11 patients with 25% to 95% surface area burns and 7 age-matched volunteer control subjects. Peripheral blood mononuclear cells were stimulated by the T-cell mitogen phytohemagglutinin (PHA), and IL-2 production and mRNA expression by Northern blot were determined. Expression and function of IL-2R were determined by monoclonal antibodies to the p55 and p75 chains of the IL-2R, binding of fluorescein-labeled IL-2, and response to exogenous recombinant IL-2. We also studied a mouse model of 20% burn injury known to mimic the immune abnormalities seen in humans with burns. Splenocytes from mice with burns (20-22 per group) were studied for IL-2 production and IL-2 mRNA expression after stimulation with the T-cell mitogen concanavalin A (ConA) and compared with sham burn control subjects. Kinetics of mRNA expression after ConA stimulation also were determined and a nuclear run-on assay performed to determine IL-2 gene transcription. The mRNA expression was determined for the proto-oncogenes c-jun and c-fos, whose protein products join to form transcription factor AP1, which is necessary for activation of the IL-2 promoter. Splenocytes from mice with burns after ConA stimulation also were studied for expression and function of the IL-2R. RESULTS: Peripheral blood mononuclear cells from burn patients compared with healthy control subjects showed diminished (p < 0.05) IL-2 production and mRNA expression 4 to 10 days after burn injury. Burn PBMC demonstrated normal expression of IL-2R, p55, and p75 chains 0 to 7, 8 to 20, and 21 to 37 days after burn injury, normal IL-2R binding of fluorescein-labeled IL-2, and a normal proliferative response to PHA in the presence of exogenous recombinant IL-2. Splenocytes from mice 7 days after burn injury showed diminished production (p < 0.05) of IL-2 and IL-2 mRNA expression after ConA stimulation as compared with sham burn control subjects. Kinetics of mRNA expression after ConA stimulation were the same for burn and control mice, indicating that reduced IL-2 mRNA expression was not caused by altered mRNA degradation. A nuclear run-on assay confirmed decreased IL-2 gene transcription in burn splenocytes. Burn splenocytes showed normal expression of mRNA for c-jun but diminished expression of mRNA for c-fos. Finally, splenocytes from mice with burns after ConA stimulation showed normal expression and function of the IL-2R 7, 10, 14, and 21 days after burn injury. CONCLUSIONS: These human and animal studies indicate that major burn injury depresses T-cell activation at the level of IL-2 gene transcription at least in part by inhibiting c-fos expression, whereas IL-2R expression and function remain normal and T-cell proliferation can be restored to normal levels by exogenous IL-2.

Does the route of feeding modify the inflammatory response?

OBJECTIVE: The authors compared the responses to endotoxin in enterally and parenterally fed human volunteers. BACKGROUND: Recent investigations have reported that the response to endotoxin in humans is greater in individuals who receive parenteral nutrition rather than enteral feeding. It was proposed that this difference was related to gut barrier dysfunction during intravenous nutrition. To evaluate this hypothesis, the authors analyzed the responses of human subjects to an intravenously administered bolus of endotoxin after enteral or parenteral nutrition. METHODS: Fifteen randomly selected healthy volunteers were studied during two separate investigations; ten studies were performed in ten subjects who received enteral nutrition, and nine studies were carried out in five additional subjects who received parenteral nutrition. After 2 days of enteral feedings or 7 days of parenteral feedings, endotoxin was administered by intravenous injection; temperature, symptom score, and duration then were measured serially. Blood samples were obtained for leukocyte and platelet count, and plasma concentrations of corticotrophin, cortisol, epinephrine, norepinephrine, tumor necrosis factor, and interleukin-6. Mononuclear cell response to phytohemagglutinin was determined at 0, 4, and 24 hours. RESULTS: In the parenteral group, a diminished response was observed in platelet count and plasma interleukin-6 levels compared with volunteers who received enteral nutrition. The duration of symptoms tended to be reduced in the parenterally fed group, although this did not achieve significance. Other responses were not significantly different between the two groups. CONCLUSION: The responses to endotoxin in human subjects who received parenteral nutrition were similar compared with subjects who received enteral nutrition, although platelet count and plasma interleukin-6 concentration were diminished.

Induced hyporesponsiveness in rat Kupffer cells is not specific for lipopolysaccharide.

The phenomenon of lipopolysaccharide (LPS)-induced hyporesponsiveness has been reported to occur in macrophage cell lines and primary cells. Hyporesponsiveness was evidenced by a diminution or lack of production of tumour necrosis factor-alpha (TNF-alpha) after sequential doses of LPS. In order to characterize the hyporesponsive state in Kupffer cells, the production of TNF-alpha was quantified after varying the concentration of a primary low dose of LPS prior to a challenge with a high, normally stimulatory dose of LPS. The kinetics of establishment of the hyporesponsive state and the effect of varying the bacterial serotype and genus of the challenge dose were determined. The specificity of the hyporesponsive state for LPS was examined. Our results demonstrate that complete hyporesponsiveness with no detectable production of TNF-alpha (< 30 pg/ml) was achieved after a primary dose > or = 10 ng/ml. Establishment of the hyporesponsive state took place within 6 hr. Induction of hyporesponsiveness was not dependent upon the serotype or genus of the challenge dose of LPS and was not specific for LPS. Complete hyporesponsiveness was induced after a primary dose (10 micrograms/ml) of the Gram-positive bacterium Corynebacterium parvum (Cp) and was evident upon challenge with 100 micrograms/ml Cp. The data indicate that the mechanisms by which LPS and Cp induce hyporesponsiveness are not identical in that a primary dose of LPS (10 ng/ml) induced only partial hyporesponsiveness upon challenge with Cp (100 micrograms/ml). These studies improve our understanding of Kupffer cell function.