John F. Valente

Blunt carotid artery injuries

BACKGROUND Blunt carotid artery trauma remains a rare but potentially devastating injury. Early detection and treatment remain the goals of management. Our objective was to identify patients sustaining blunt carotid injuries at a regional trauma center and report on the incidence, demographics, diagnostic workup, management, and outcome. STUDY DESIGN A retrospective chart review was performed of patients sustaining blunt carotid artery injury between 1990 and 1996. RESULTS Twenty patients were identified during the 7-year period. All patients suffered blunt trauma, with motor vehicle accidents being the most common mechanism, and the internal carotid the most frequently injured vessel. Associated injuries were present in all patients, with head (65%) or chest (65%) injuries being the most common. The combination of head and chest trauma (45%) was found to be associated with a 14-fold increase in the likelihood of carotid injury. Cerebral angiography was diagnostic in all patients and the majority were treated nonoperatively with anticoagulation. Twenty percent of patients were discharged with a normal neurologic exam, while 45% left with a significant neurologic deficit. Overall mortality was 5%. CONCLUSIONS Blunt carotid injuries are rare but are associated with significant morbidity and mortality. The combination of craniofacial and chest wounds should raise the index of suspicion for blunt carotid injury. Anticoagulation was associated with the least morbidity.

Late hepatic artery thrombosis in liver allograft recipients is associated with intrahepatic biliary necrosis.

Hepatic artery thrombosis (HAT) after liver transplantation is a potentially life-threatening complication that occurs in 2-25% of patients, depending on several risk factors and the patient population studied. Arterial thrombosis occurring early after liver transplantation is associated with acute fulminant hepatic failure, biliary tract necrosis and leaks, or relapsing bacteremia and is associated with a high rate of graft loss and patient mortality. The onset of late posttransplant HAT (after 6 months) has been thought to have a more benign and often asymptomatic course. The reasons for the differences between the manifestations of early and late HAT are not well understood. We reviewed the adult liver transplant experience at the University of Cincinnati and found four patients with late HAT, three of whom developed severe intrahepatic biliary necrosis. Two patients were successfully retransplanted and 1 patient who refused retransplantation died. One patient had mild, transient graft damage due to gradual arterial stenosis and the development of arterial collaterals prior to thrombosis. Late HAT has a significant potential for irreversible graft damage requiring retransplantation. Screening for the development of hepatic artery stenosis prior to late thrombosis may be worthwhile.

THERMAL INJURY INDUCES THE DEVELOPMENT OF INFLAMMATORY MACROPHAGES FROM NONADHERENT BONE MARROW CELLS

The normal course of hematopoiesis is controlled by growth factors and cytokines and, therefore, should be susceptible to alterations induced by systemic mediator release such as that seen following thermal injury. We hypothesized that a brief exposure of developing macrophages to the postthermal injury state would result in functionally altered progeny. We measured the production of inflammatory mediators by rat, bone-marrow macrophage precursors harvested 24 h following a 30% TBSA burn after subsequent maturation in a controlled, in vitro environment. Interleukin (IL)-6, tumor necrosis factor (TNF), and prostaglandin (PG) E2 levels in response to 24 h stimulation with lipopolysaccharide (LPS) were measured following 4 or 8 days of incubation with IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), or both. Flow cytometric analysis showed that bone marrow cells harvested from burn and sham animals cultured in GM-CSF developed principally into macrophages (His48−, R21A6A+, CD11b+. Unstimulated cells produced negligent levels of cytokines and PGE2. Stimulated burn-derived cells released greater amounts of IL-6 and TNF at 4 or 8 days of culture depending on the conditions. Elevated PGE2 release was noted in all GM-CSF containing cultures, with burn-derived cells showing a trend towards reduced prostaglandin release. Detection of mRNA for cytokines after LPS stimulation showed no change in IL-6 or TNF transcripts. A short exposure to the systemic effects of thermal injury preprogramed macrophage progenitor cells with the propensity to develop into inflammatory macrophages, secreting higher levels of TNF and IL-6. This shift towards proinflammatory functions in these cells suggests they could be a source of enhanced inflammatory mediator release at 4 or more days post thermal injury.

Dietary omega-3 and omega-9 fatty acids uniquely enhance allograft survival in cyclosporine-treated and donor-specific transfusion-treated rats.

BACKGROUND Both laboratory and clinical studies have shown that dietary lipids may affect immunologic responses. This study was conducted to compare different classes of long-chain unsaturated fatty acids for their effect on allograft survival in animals receiving a donor-specific transfusion and a short course of low-dose cyclosporine (CsA). METHODS Heterotopic ACI strain cardiac allografts were transplanted to Lewis strain rat recipients given diets with different lipid composition. In experiment 1, animals received CsA for 14 days and different diets were enriched with lipids with high concentrations of omega-3, omega-6, or omega-9 fatty acids. In experiment 2, animals received CsA for only 8 days and different diets were enriched with corn oil (omega-6), canola oil (omega-3 and omega-9), fish oil (omega-3) or a mixture of sunflower oil and fish oil (omega-3 and omega-9). RESULTS In experiment 1, animals receiving the diet with 30% sunflower oil had the best allograft survival (200+/-42 days vs. 53+/-8 days for regular chow plus donor-specific transfusion and CsA, P<0.05). In experiment 2, diets containing canola oil (a mixture of omega-3 and omega-9 fatty acids) were associated with the best survival (P=0.0011 vs. regular chow). CONCLUSION Dietary omega-3 and omega-9 fatty acids both enhanced cardiac allograft survival in a stringent rat strain combination. Canola oil is a convenient oil for administering both alpha-linoleic acid (omega-3) and oleic acid (omega-9) in a palatable form for human consumption. Further investigation of the potential usefulness of lipids in transplant therapy is warranted.

Effect of in vivo infusion of granulocyte colony-stimulating factor on immune function.

As the applications of hematopoietic growth factors increase, their complex impact on host defense and immune responses continues to unfold. The effect of the administration of granulocyte colony-stimulating factor (G-CSF) on bacterial defense, proliferation of lymphocytes, and cytokine production by lymphocytes and peripheral blood mononuclear cells (PBMC) was studied. The effect of G-CSF administration on the phenotype of the cells in the major hematopoietic organs was studied as well. ACI rats were given 10 mg/kg/day G-CSF or vehicle daily for 4 days. Isolated bone marrow neutrophils and enterocytes from treated animals showed a greater bactericidal activity than controls. Proliferation of mitogen-stimulated lymphocytes and PBMC was reduced in G-CSF-treated animals. The production of proinflammatory cytokines, tumor necrosis factor (TNF), and interleukin 6 (IL-6) by lymphocytes and PBMC was reduced by G-CSF pretreatment. G-CSF administration caused an increase in IL-4 (Th2 cytokine) release and a decrease in interferon-gamma (IFN&ggr;, Th1 cytokine) release by mitogen-stimulated lymphocytes. Cytometric analysis of cells in the progenitor cell region indicated a large increase in immature cells in the bone marrow of G-CSF-treated animals compared with sham along with an increase in B cells and a decrease in polymorphonuclear leukocytes (PMNs). In addition, cytometric analysis showed a large increase in PMNs in blood and splenocytes of the treated animals compared with sham. This study confirms and extends previous observations that G-CSF administration has a number of effects that might simultaneously enhance host defense while reducing the risk of developing uncontrolled systemic inflammation. This may also be efficacious in prolonging graft survival and reducing graft vs. host disease.

Arginine, Fish Oil, and Donor-Specific Transfusions Independently Improve Cardiac Allograft Survival in Rats Given Subtherapeutic Doses of Cyclosporin

BACKGROUND Dietary supplementation with a fish oil and arginine-enriched immunoenhancing diet (Impact; Sandoz Nutrition, Minneapolis, MN) in a rat cardiac allograft model using donor-specific transfusion (DST) and cyclosporin (CsA) resulted in significant prolongation of cardiac allograft survival with many animals developing long-term tolerance. This study was done to determine whether arginine or fish oil was the active ingredient. METHODS A standard AIN-76A diet was modified to include either 10% fish oil, 2% arginine, or 5% arginine with or without fish oil. Diets were fed to Lewis strain rats that received Ax C9935 Irish (ACI) heterotopic cardiac allografts beginning on day 1 and continuing indefinitely. A DST (1.0 mL ACI whole blood) was given with 10 mg/kg CsA on day 1 relative to transplant and 2.5 mg/kg/d on days 0 to 6. Groups of animals receiving AIN-76A diet fortified with 2% glycine and animals receiving a DST or DST/CsA and regular laboratory chow served as controls. RESULTS Mean survival times +/- SEM in days were as follows: untreated, 7.1 +/- 0.4; CsA/2% glycine, 8.5 +/- 0.6; DST only, 9.6 +/- 1.1; DST/CsA, 26.6 +/- 6.4; CsA/2% arginine, 25.5 +/- 3.9; DST/CsA/2% arginine, 68.7 +/- 8.9; DST/CsA/5% arginine, 90.1 +/- 31.1; CsA/fish oil, 73.6 +/- 26.1; and DST/CsA/fish oil/5% arginine, 90.1 +/- 31.1. The effect of arginine was slightly dose dependent and was seen best in combination with DST, but the effect of fish oil was not enhanced by DST. CONCLUSIONS Both fish oil and arginine dietary supplementation significantly improved allograft survival but through different mechanisms (DST vs non-DST dependent).

Changes in bone marrow-derived myeloid cells from thermally injured rats reflect changes in the progenitor cell population.

Bone marrow progenitor cells develop into mature tissue myeloid cells under the influence of colony-stimulating factors. Cytokines that are elevated post-thermal injury have been shown to influence this process. We hypothesize that thermal injury alters myelopoiesis at the level of the progenitor cell. These differences should be visible after in vitro cultures that include colony-stimulating factors. Prior to culture, bone marrow at postburn day 1 (PBD1) was assessed for cell surface markers and the levels of myeloid progenitors. After culture in granulocyte/macrophage-stimulating colony-stimulating factor, the cell surface markers of the cultured cells were determined. PBD1 marrow from thermally injured rats had more progenitor cells responsive to granulocyte/macrophage-stimulating colony-stimulating factor than did sham. Cultured PBD1 marrow produced more CD90(br) MY(br) CD45(dim) CD4(-) MHCII(-) CD11b(dim) eosinophils than did sham. Cultured bone marrow from thermally injured animals produces myeloid cells with an altered phenotype. Similar changes in myelopoiesis may take place in vivo.

The ability of endotoxin-stimulated enterocytes to produce bactericidal factors

Objective Bactericidal peptides, specifically defensins, are produced by polymorphonuclear cells. Intestinal epithelial cells also produce bactericidal peptides, perhaps as part of their barrier function, to the greatest load of endogenous bacteria present in the body. We sought to determine whether and under what conditions intestinal cell lines could produce bactericidal compounds. Design Laboratory investigation. Setting Children’s burn hospital. Subjects Caco-2, IEC-6, and HT-29 cell lines. Interventions Three different enterocyte lines were cultured for 1 day ± lipopolysaccharide (1 or 10 &mgr;g/mL), and their supernatants were tested for bactericidal activity. Also, reverse transcription-polymerase chain reaction of Caco-2 cells was performed to assess the expression of defensin-6 mRNA. Measurements and Main Results After culture, enterocytes all were found to release one or more soluble factors with bactericidal activity (as measured fluorometrically by using a metabolizable dye) when stimulated by lipopolysaccharide (1 &mgr;g/mL). The bactericidal activity of these culture supernatants was saturated by increased bacterial load, additive to the effects of normal human peripheral blood polymorphonuclear cells, and was reduced by serial supernatant dilution. Enterocyte stimulation with larger amounts of lipopolysaccharide (10 &mgr;g/mL) resulted in greater bactericidal activity. After supernatant fractionation based on molecular weight, the bactericidal effect was best retained in the <10-kDa fraction. In addition, the expression of mRNA for defensin-6, a bactericidal peptide produced by neutrophils, was seen in Caco-2 cells. Conclusion Enterocytes are shown to produce a soluble, low molecular weight, bactericidal compound in response to endotoxin stimulation. The expression of defensin-6 mRNA in Caco-2 cells suggests that intestinal cells may release defensins as bactericidal peptides. This experimental system provides an in vitro model to study the activity and production of bactericidal factors by enterocytes.

Dietary Amino Acids as New and Novel Agents to Enhance Allograft Survival

Dietary supplementation with arginine was previously found to enhance cardiac allograft survival in rats when given with a donor-specific transfusion and a short low-dose course of cyclosporine. This study was performed to determine further the role of amino acid supplementation in prolonging allograft survival. Standard isocaloric, isonitrogenous diets were modified to contain 2 or 4% of energy from arginine, 2 or 4% from glutamine, 4% from glycine or the following combinations: 2% arginine with 2% glutamine, 2% arginine with 4% glutamine, or 1% arginine with 2% glutamine. These diets were started along with a donor-specific transfusion and a 7-d course of cyclosporine the day before cardiac transplantation from an ACI to Lewis strain rat. Median survival times in days for the groups were as follows: control without amino acids, 19.0; 2% arginine, 68.0; 4% arginine, 35.5; 2% glutamine, 28.5; 4% glutamine, 53.5; 4% glycine, 31.5; 2% arginine with 2% glutamine, 39.5; 2% arginine with 4% glutamine, 42.5 and 1% arginine with 2% glutamine, 35.5. Each experimental diet except 2% glutamine and 4% glycine significantly enhanced allograft survival (P < 0.05) with the 2% arginine diet being the best (91.6 +/- 32.3 d [mean +/- SEM] versus 20.1 +/- 3.2 d for control). It is concluded that both arginine and glutamine enhance the immunosuppressive effects of donor-specific transfusion and cyclosporine.

IMMUNOBIOLOGY OF RENAL TRANSPLANTATION

The clinical application of our knowledge of the immune barriers to transplantation has advanced allo-organ replacement therapy to the level of routine practice, while simultaneously engendering a critical shortage in available donors. Recent work in xenotransplantation addresses this need. The current understanding of the immune barriers to transplantation has evolved to consider alternate responses to alloantigen, namely acceptance. The delineation and application of recent discoveries in T cell costimulatory events, antigen presentation, and differential T lymphocyte responses are opening pathways towards the development of tolerogenic protocols for use in clinical transplantation. This article presents a review of transplant immunobiology with special attention to antigen presentation and T-cell activation as phases of the immune response relevant to the discussion of transplant tolerance.