Hemorrhagic shock-induced alterations in circulating and bronchoalveolar macrophage nitric oxide production.

Local and systemic host immune functions are markedly altered after trauma. Activated macrophages (M phi) are the major effector cells of protective immunity, mediated in part by nitric oxide (NO). This study was undertaken to determine the effects of hemorrhage (HEM) on M phi cytotoxic function as measured by NO production. Male Sprague-Dawley 350- to 400-g rats were studied. Sham animals only had their carotid artery exposed and ligated. Hemorrhaged animals had carotid artery cannulation followed by HEM to SBP of 40 mmHg, sustained for 45 min, followed by resuscitation with shed blood and crystalloid. Recovered animals were sacrificed (n = 5 each) at 6, 12, 24, and 72 hr after HEM; blood and alveolar M phi were then isolated and examined. A monolayer of adherent M phi was examined for NO production in resting state and after in vitro LPS stimulation. (1) HEM resulted in significantly reduced alveolar M phi yield at 12 and 24 hr. (2) HEM increased NO production by circulating M phi at 24 hr (P < 0.05), while alveolar M phi had significantly increased NO production at all time points after HEM (P < 0.05). (3) LPS stimulation significantly increased NO production in both circulating and alveolar M phi in sham animals and 6 hr after HEM but not at any other times. We therefore conclude that HEM causes early and prolonged activation of NO production by alveolar M phi and delayed and brief activation of circulating M phi. Alveolar and circulating M phi are unable to significantly increase NO production in response to LPS stimulation during the later phases of HEM.(ABSTRACT TRUNCATED AT 250 WORDS)

Improved survival in simulated surgical infection with combined cytokine, antibiotic and immunostimulant therapy.

A study was performed to find an ideal combination and sequence of cytokines, antibiotics and immunorestorative agents to enhance survival from serious infection. The effects of combinations of granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor (TNF) alpha, the immune adjuvant muramyl dipeptide (MDP) and two systemic antibiotics were studied in a validated murine model of surgical infection. A single cotton suture containing absorbed Klebsiella pneumoniae was placed into the thighs of mice to produce local and systemic infection. Control mice received a volume of subcutaneous saline equal to that of the therapeutic agent; only 18 per cent survived 9 days after infection. The survival time of mice treated with any single agent was similar to that of controls. The group given maximal combined therapy (65 mice) received GM-CSF, TNF-alpha, MDP, and ampicillin-sulbactam or cefoxitin for 6 days. The survival rate in this group 9 days after the introduction of infection was 84-90 per cent (P < 0.0001), suggesting that specific combinations of cytokines, immunostimulants and antibiotics may be useful in combating lethal infection.

Pneumonia in the surgical intensive care unit. Immunologic keys to the silent epidemic.

OBJECTIVE: The authors undertook a prospective study of trauma victims in the intensive care unit (ICU) to investigate the clinical course of pneumonia and the local and systemic immune responses to the pneumonia. SUMMARY BACKGROUND DATA: The silent epidemic of pneumonia has been an "unappreciated killer" in terms of being overlooked in surgical ICUs for the past 5 years, and specifically, the most common major infection after severe trauma. Little is known about the immune response to an acute pulmonary infection. METHODS: The authors studied 50 consecutive, critically ill trauma patients, with a mean injury severity score of 28 +/- 2, who developed pneumonia while ventilated mechanically. Patients were observed clinically, and specific immunologic parameters, including major histocompatibility antigen HLA-DR, complement receptor (CR3), and Fc receptor (FcRIII), were measured in circulating and local alveolar leukocytes for up to 30 days. Eleven patients provided unique clinical data via bronchoscopy for unilateral pneumonia, with collection of bronchoalveolar lavage (BAL) fluid from both the infected and uninfected sides. RESULTS: Patients developed clinical pneumonia 5.3 +/- 0.4 days after admission to the ICU. At diagnosis, mean temperature was 101.4 F, white blood cell count was 16,000/mm3, arterial oxygen tension was 104 +/- 14, fraction of inspired oxygen was 0.47, and positive end-expiratory pressure was 5. Thirty patients (Group A) recovered relatively promptly; 20 patients had prolonged illnesses (Group B), 15 of whom ultimately survived, and five of whom died. Patients with poor outcomes had greater leukocytosis (p < 0.05) and temperature elevation (p < 0.05) after 5 days of pneumonia. Immunologically, peripheral leukocyte expression of HLA-DR, FcRIII, and CR3 was equivalent in both groups. However, the expression of all three antigens on local alveolar leukocytes was decreased to a greater extent in the poor outcome group compared to the good outcome group, evident before any clinical differentiation between the two outcome groups. CONCLUSIONS: Pneumonia prolonged duration of mechanical ventilation, ICU and hospital stay, and overall infectious morbidity. Although immune suppression has been recognized as a result of initial injury, the development of pneumonia coincided with the nadir of immune function. Poor outcome patients were clinically identifiable 5 days after pneumonia and immunologically identifiable within 2 days. Moreover, there was localized suppression of pulmonary leukocytes at the site of the infiltrate compared to the uninfected lobes. This same alteration was noted in experimental Klebsiella pneumoniae pneumonia. This evidence suggests that there is active immune participation within the respiratory system. It also suggests that there are predispositions to pulmonary infections, and it may allow immune modulation targeted to pulmonary leukocytes to hasten clinical recovery and minimize pulmonary dysfunction.

Effect of topical recombinant TGF-beta on healing of partial thickness injuries.

Peptide growth factors produced by platelets, macrophages, epidermal, and dermal cells may play key roles in regulating healing of partial-thickness skin wounds. We examined the effects of recombinant transforming growth factor beta (TGF-beta) on cultures of epidermal and dermal cells in vitro and on healing of partial-thickness injuries in vivo. Increasing concentrations of TGF-beta (0.1, 1, and 10 ng/ml) progressively inhibited serum-stimulated DNA synthesis by up to 95% in cultures of adult human keratinocytes during 48 hr of exposure to TGF-beta. In contrast, TGF-beta (10 and 100 ng/ml) in serum-free media stimulated DNA synthesis by up to 80% compared to serum-free control cultures of adult human dermal fibroblasts. To evaluate the effects of TGF-beta on healing of partial-thickness injuries in vivo, wounds (20 x 20 x 0.6 mm) were created on the dorsal thoracolumbar region of adult pigs by an electrokeratome and were treated daily for 5 days after injury with vehicle or vehicle containing 0.1 or 1 microgram/ml TGF-beta and covered with occlusive dressing. Computerized planimetry of wound photographs demonstrated that TGF-beta treatment stimulated statistically significantly increases in the area of regenerated epidermis compared to wounds treated with saline vehicle on Days 3, 4, 5, and 7 after injury probably due to TGF-beta increasing the rate of epidermal cell migration. In addition, morphometry of biopsy specimens showed that TGF-beta treatment stimulated statistically significant increases in the cross-sectional depths of regenerated dermis compared to wounds treated with saline or Silvadene vehicles on Days 5, 6, and 8 after injury.(ABSTRACT TRUNCATED AT 250 WORDS)

An experimental assessment of the effect of blood transfusion on susceptibility to bacterial infection.

Clinical investigations in patients who have undergone transplantation and patients with cancer and infection suggest that blood transfusions have an immunosuppressive effect. To investigate the impact on responses to infection, an experimental transfusion model was developed in the rat with allogeneic or syngeneic transfusions. Animals were given either a moderate or severe bacterial challenge, both simulating a clinical surgical infection, immediately after transfusion or 1 week later; hypotension and hemodilution were especially avoided. Blood transfusion adversely affected the animals' capacity to resist immediate moderate challenge, but not a later one. The effect was maximized by allogeneic transfusion but was not demonstrable after severe bacterial challenge, whether immediate or late.

Protective effects of recombinant human tumour necrosis factor alpha and interferon gamma against surgically simulated wound infection in mice.

Tumour necrosis factor alpha and interferon gamma have both been shown to have immunoregulatory properties, and to be able to influence, several microbial infections. This study showed that tumour necrosis factor was effective in modifying surgically simulated wound infections when administered both as prophylaxis and as therapy. Two models were used; one was an intramuscular bacterial challenge, and the other involved the use of a bacteria-laden thigh suture. The test bacterium for both models was Klebsiella pneumoniae, a common surgical pathogen in our surgical service. Interferon gamma was an effective biological response modifier in these models. Tumour necrosis factor was more potent than interferon gamma and there was no additive or synergistic effect with interferon gamma. This indicates potentially different mechanisms of action for these two cytokines.

Reversal of Adriamycin-impaired wound healing by transforming growth factor-beta.

Impaired wound healing remains an important clinical problem. Treatment with systemic Adriamycin (doxorubicin) is known to impair wound healing in patients, and it has been used to produce animal models of impaired healing. The results of previous studies have shown that local treatment of incisions in normal rats with transforming growth factor beta (TGF-beta) or epidermal growth factor (EGF) stimulated early increases in tensile strength of surgical incisions in normal rats. We investigated the effects of locally applied, biosynthetic TGF-beta or EGF on the tensile strength of standardized incisions in rats treated with Adriamycin. Systemic Adriamycin treatment (8 milligrams per kilogram) produced significant decreases in wound tear strength (WTS) and wound tear energy (WTE) when compared with that of normal rats at seven and ten days (p less than 0.01). A single dose of TGF-beta (2 micrograms) in a collagen vehicle stimulated a reversal of this wound healing impairment at ten days (p less than 0.05), returning the WTS and WTE to near normal levels. A single dose of EGF (50 micrograms) in hyaluronic acid failed to increase tensile strength, probably because of formulation of EGF in a vehicle that does not prolong its release in incisions. These results suggest that exogenous growth factors may be clinically useful in stimulating healing in incisions in healing impaired conditions.

Evaluation of copovithane as a nonspecific immunomodulator in surgically simulated sepsis.

Copovithane (CPV), a synthetic polymer, has been shown to have antitumor activity and also to reduce mortality in experimental murine peritonitis. The purpose of this study was to compare CPV with muramyl dipeptide (MDP), an immunomodulator of proven efficacy in simulated surgical infection. Six groups of CBA/J mice were compared; they received intramuscular injections of normal saline (controls), MDP (100 micrograms), or CPV (100, 200, and 400 mg/kg) 24 h prior to bacterial challenge. The challenge consisted of a Klebsiella-impregnated thigh suture. The first experiment assessed survival after bacterial challenge. The MDP and the CPV groups both had median survival times of 3 days, significantly longer than the control group (1 day, p less than .05). In the second experiment, animals were sacrificed at 6, 24, and 48 h following bacterial challenge, and blood and infected muscle were taken for quantitative bacteriology. At 6 h, there was no difference between groups. Both the MDP and CPV groups had significantly (p less than .05) lower blood bacterial counts than the control group at 24 and 48 h. Both the MDP and CVP groups had significantly lower local bacterial recovery than controls at 48 h (p less than .05), and local bacterial recovery of the MDP group was significantly lower than the control group at 24 h (p less than .05). CPV improved survival and reduced local and systemic bacterial recovery compared with controls. Although the effect of CPV was similar to MDP in this model, it consistently was of lower magnitude and had a narrow dose range.

The natural history of murine intra-abdominal abscess formation.

We determined the natural history of experimental abscess formation and had a secondary interest in the effect of muramyl dipeptide. Swiss-Webster mice were injected intraperitoneally with autoclaved mouse fecal suspension and either Bacteroides fragilis (10(8) colony-forming units [cfu]/mL) alone or Escherichia coli (10(4) cfu/mL), enterococcus (10(3) cfu/mL), and B fragilis (10(5) cfu/mL) after pretreatment with muramyl dipeptide or saline solution. All deaths occurred within 48 hours of injection and surviving mice, including those bearing abscesses, appeared to be healthy throughout the study. The number of mice with abscesses and the number of abscesses per group were at their maximum at two to four weeks. Groups with live bacteria had a substantial reduction in the number of abscesses between eight and 26 weeks, compared with two- and four-week values. Manual rupture of palpably large eight-week-old abscesses in 21 mice produced only one death and at autopsy two weeks later, all of the mice showed multiple smaller abscesses. Abscess formation appeared to be beneficial and the natural history of such may include spontaneous resolution without mortality.

Modulation of infection by gamma interferon treatment following trauma.

Gamma interferon (IFN-gamma) has been shown to be able to modulate several microbiol infections, perhaps as a result of the immunoregulatory properties of this interferon. The present study was designed to determine the efficacy of IFN-gamma treatment in a mouse model of infection that simulates clinical conditions occurring following abdominal trauma. In this model, mice were first infected intraperitoneally with Escherichia coli and then underwent immediate surgical laparotomy. Finally the mice were secondarily infected intramuscularly with Klebsiella pneumoniae. Groups of CBA/J mice received either IFN-gamma or RPMI 1640 medium (controls) subcutaneously. IFN-gamma was administered daily at a dose of 7,500 U, commencing 1 h postlaparotomy and continuing until the second bacterial challenge. Mice treated with IFN-gamma survived significantly longer than controls. The Ia antigen expression of peripheral blood monocytes was severely reduced in animals for 3 days after laparotomy and for 5 days after laparotomy and infection. This drop in Ia antigen expression was prevented in animals treated with IFN-gamma. These data indicate that IFN-gamma had a beneficial effect in a model simulating bacterial infection after trauma and that maintenance of Ia antigen expression in interferon-treated mice may have contributed to the observed therapeutic effect.

Modulation of Klebsiella pneumoniae infection of mice by interferon-gamma.

The aim of this study was to determine the efficacy of interferon-gamma (IFN-gamma) as prophylaxis and therapy in a wound infection model with a 'surgical' pathogen. The bacterial challenge consisted of intramuscular injections of Klebsiella pneumoniae (10(3) organisms in 0.1 ml). Groups of 12 CBA/J mice had either IFN-gamma or RPMI-1640 medium (controls) injected subcutaneously. Mice pretreated with IFN-gamma in a dose of 7,500 or 750 units per day, followed by infection and 2 days additional IFN-gamma treatment, survived significantly longer than controls or mice treated with 150 units of IFN-gamma per day. Significantly greater survival than controls was seen with only 5 or 3 days pretreatment with IFN-gamma but not with 1 day pretreatment. Administration of IFN-gamma to the opposite hind leg from the one receiving bacterial challenge was as effective as same leg treatment. When IFN-gamma therapy was commenced 1 h after bacterial challenge and continued for 7 days, 13 of 60 mice survived, which was significantly greater than four of 60 surviving controls. These effects may be secondary to IFN-gamma's immunoregulatory effects rather than by involving any antiviral properties.

Effect of interferon-gamma treatment on the course of a burn wound infection.

Interferon-gamma (IFN-gamma) has been shown to have immunoregulatory properties and is able to modulate resistance to several microbial infections. This study was designed to determine the efficacy of IFN-gamma treatment in a murine burn wound infection model. Bacterial challenge consisted of Klebsiella pneumoniae (10(8) organisms/ml) or Pseudomonas aeruginosa (10(8) organisms/ml), applied topically immediately after burning. Groups of CBA/J mice received either IFN-gamma or RPMI-1640 medium (controls) subcutaneously. IFN-gamma was administered daily at a dose of 7,500 units for 5 days prior to bacterial challenge. Burn without bacterial challenge produced no mortality. Mice treated with IFN-gamma survived significantly longer than controls when the bacterial challenge was K. pneumoniae. There was no difference in survival when bacterial challenge was P. aeruginosa. The Ia antigen expression of peripheral blood mononuclear cells was severely reduced for 3 days post-burn. This drop was prevented on day 3 post-burn in mice treated with IFN-gamma. These data indicate that interferon was effective treatment in a murine model of Klebsiella burn wound infection and was associated with maintenance of Ia antigen expression that may have contributed to the action of the IFN-gamma.