The laparoscopic retrieval of an orthopedic fixation pin from the liver with repair of an associated diaphragmatic laceration.

We report the successful removal of a shoulder fixation pin from the right lobe of the liver with intracorporeal repair of a diaphragmatic laceration. An expeditious workup and urgent operative intervention were required. We adhered to the principles of room setup, optical correctness, establishment of the triangle of success, appropriate instrument entry and convergence angles, two-handed surgical skills, and competence in intracorporeal suturing techniques that were all required for successful completion of the case. We also present a review of the significant literature.

Chronic pathophysiologic elevation of corticosterone after thermal injury or thermal injury and burn wound infection adversely affects body mass, lymphocyte numbers, and outcome.

The purpose of the present studies was to investigate the effect of glucocorticoids on catabolism and lymphocyte numbers in a rat model of thermal injury or thermal injury plus burn wound infection. Thermal injury alone caused only an acute increase in plasma corticosterone concentrations. Furthermore, body weight declined moderately (5%), and lymphocyte numbers in lymph nodes draining the burn wound and blood increased markedly, whereas splenic lymphocyte numbers declined by about 60%. By contrast uninjured rats subjected to chronic elevation of corticosterone by corticosterone pellet implantation showed large decreases in body weight and lymphocyte numbers in all tissues examined. The combination of injury and chronic corticosterone elevation resulted in body weight and lymphocyte changes intermediate between injury alone and corticosterone treatment alone. Chronic elevation of corticosterone for 4 days before burn wound infection significantly decreased survival time and survival. Burn wound infection immediately after injury caused chronic elevation of endogenous plasma corticosterone and body weight and numeric lymphocyte changes that were remarkably similar to those of uninjured rats treated with corticosterone. Finally, the glucocorticoid receptor antagonist RU 486 significantly increased survival time in thermally injured, burn wound-infected rats. These results lend support to a hypothesis that chronic elevation of plasma cortisol concentrations as observed in patients with burns may be deleterious.

Myocardial reperfusion injury. Platelet-activating factor stimulates polymorphonuclear leukocyte hydrogen peroxide production during myocardial reperfusion.

To study the roles of platelet-activating factor, polymorphonuclear leukocytes, and oxygen free radicals in myocardial reperfusion injury, we subjected 10 sheep to 90 minutes of mid-left anterior descending coronary artery followed by 6 hours of reperfusion. Stainings with gentian violet and tetratriphenyl ammonium chloride demonstrated 20% +/- 3% of the left ventricular mass at risk for ischemia, of which 75% +/- 10% underwent infarction. Coronary sinus blood was assayed for platelet-activating factor and neutrophil hydrogen peroxide production before and during coronary occlusion and during reperfusion. Platelet-activating factor was isolated by column chromatography and lipid extraction and quantified by radioimmunoassay. Neutrophil hydrogen peroxide production was measured by a 2',7'-dichlorofluorescein flow-cytometric assay. Platelet-activating factor was elevated to 899 +/- 210 pg/ml at 15 minutes of reperfusion, compared with the preocclusion level of 271 +/- 55 pg/ml and coronary occlusion level of 359 +/- 64 pg/ml (p less than 0.05; analysis of variance). Neutrophil hydrogen peroxide production, measured on a relative fluorescence scale, was also elevated to a level of 141 +/- 27 at 1 hour of reperfusion, compared with the preocclusion level of 103 +/- 6 and the coronary occlusion level of 114 +/- 13 (p less than 0.01; analysis of variance). Both of these parameters returned toward baselines at the end of 6 hours of reperfusion. Histologic examination revealed infiltration of polymorphonuclear leukocytes into the interstitium of the reperfused myocardium. Neutrophils isolated from unoperated and healthy sheep demonstrated a graded dose response in hydrogen peroxide production when stimulated by purified platelet-activating factor in vitro. These findings suggest that platelet-activating factor is released in the coronary circulation and is a mediator of oxygen free radical production in polymorphonuclear leukocytes during myocardial reperfusion.

Pathophysiologic glucocorticoid elevations promote bacterial translocation after thermal injury.

Thermal injury results in transient elevations of plasma glucocorticoids and promotes translocation of bacteria from the gut to the mesenteric lymph nodes (MLN) in rats. Translocated organisms are quickly cleared following uncomplicated thermal injury. However, subsequent burn wound infection, in temporal association with sustained pathophysiologic elevations of plasma corticosterone, results in the continued presence of enteric bacteria in the MLN. To study the role of sustained pathophysiologic steroid elevations in the mediation of this prolonged bacterial translocation, Wistar rats were randomly placed in groups receiving one of the following: (i) a 30% total body surface area scald injury with placement of a subcutaneous corticosterone pellet, (ii) a 30% total body surface area scald and a sham pellet implantation, (iii) a sham burn and a corticosterone pellet implantation, or (iv) a sham burn and a sham pellet implantation. The animals were sacrificed on days 1 and 4 after injury, and cultures of the MLN, as well as the liver and spleen, were taken. Implantation of corticosterone pellets resulted in sustained elevations of plasma corticosterone compared with controls not receiving corticosterone pellets, similar to results seen in association with injury and infection. These pathophysiologic elevations were associated with the prolonged presence of organisms in the MLN (90% of burned rats with implanted corticosterone pellets versus 25% of rats with uncomplicated burns on postburn day 4; P less than 0.01), but only in the presence of burn injury. Pathophysiologic glucocorticoid elevations did not lead to progression of translocation to the viscera or blood. Thus, the pathophysiologic glucocorticoid response contributes to the translocation of enteric bacteria and their prolonged presence in the MLN after systemic injury.

Lymphocytes. Window to the future.

The surgical sciences have made significant advances in our understanding of the immunological alterations associated with transplantation, trauma, oncology, and wound healing. Immune surveillance, regulation, and effector cell functions have been demonstrated to play integral roles in these disease states. Immune manipulation may be an effective therapeutic modality with even greater potential for the future. This study highlights recent progress in our understanding of immunomodulatory interventions that exploit manipulations in lymphocyte function in transplantation.

Peripheral blood leukocyte kinetics following in vivo lipopolysaccharide (LPS) administration to normal human subjects. Influence of elicited hormones and cytokines.

Lipopolysaccharide (LPS, endotoxin) administration to human subjects elicits significant elevations in plasma cachectin/TNF, epinephrine, and cortisol. This study examined the temporal relationship between changes in blood leukocyte subsets and plasma mediators following endotoxin administration to normal human subjects. A five-minute intravenous infusion of purified LPS (20 units/kg Escherichia coli) was administered to 12 healthy volunteers. Blood samples were obtained at varying intervals after infusion and analyzed for differential cell counts and lymphocyte subsets (CD2, CD3, CD4, CD8, CD20, and HLA-DR) by flow microfluorimetry, and also assayed for plasma cachectin/TNF, epinephrine, and cortisol. Plasma cachectin/TNF was significantly elevated at 75 and 90 minutes after infusion with a peak concentration of 261 +/- 115 pg/ml noted 75 minutes after infusion. A significant plasma epinephrine elevation of 181 +/- 75 pg/ml was demonstrated one hour after infusion, while significant elevations in plasma cortisol were noted from one to five hours after infusion with a peak level of 34 +/- 3 micrograms/dl three hours after infusion. A profound monocytopenia (p less than 0.01) was noted one hour after infusion. Temporally associated with the rise in plasma cortisol was a reversal of the early granulocytopenia to a significant granulocytosis (p less than 0.01 versus preinfusion mean), whereas a marked lymphocytopenia (p less than 0.01) was observed from one to six hours after infusion. During the period of hypercortisolemia, CD2, CD3, and CD4 lymphocyte percentages were decreased (p less than 0.01) while CD20 and HLA-DR lymphocyte percentages were increased (p less than 0.01). There was a small percentage decrease in CD8 lymphocytes from one to 24 hours after infusion (p less than 0.01), although relative to the one-hour nadir, there was a significant rise in the percentage during the time of elevated plasma cortisol concentrations. A six-hour infusion of epinephrine (30 ng/kg/min) administered to six healthy volunteers resulted in a monocytosis (p less than 0.05) and granulocytosis (p less than 0.01) without a change in lymphocyte number or lymphocyte subset percentage. Previous reports have shown that in vivo corticosteroid infusion causes a prominent granulocytosis, monocytopenia, and lymphocytopenia with a decrease in the percentages of CD3 and CD4 lymphocytes. The peripheral blood leukocyte dynamics documented in the current study are similar to patterns observed following in vivo corticosteroid administration. This study suggests that the acute adrenocortical response to endotoxemia primarily mediates the subsequent changes in leukocyte subsets.

Changes in lymphocyte number and phenotype in seven lymphoid compartments after thermal injury.

Thermal injury is associated with dysfunction of host defense systems. The present study used flow cytometric immunofluorescence analyses to investigate changes in number and phenotype of lymphocytes in seven different lymphoid compartments at 2, 6, 12, 24, 48, and 60 days after 50% total body-surface area thermal injury in the rat. Relative to sham-injured control rats, at postburn day 2, significant lymphopenia was observed in the peripheral blood along with depletion of lymphocytes from the spleen and thymus. By day 6 after injury, lymphocytes in the bone marrow and cervical lymph nodes decreased significantly while numbers in the spleen and thymus remained depressed. Splenic and cervical node lymphocyte numbers normalized by day 12, the bone marrow and thymus numbers still were significantly lower than control, and a 6.5-fold increase in number of lymphocytes was observed in the nodes draining the burn wound, pooled axillary, brachial, inguinal, and lumbar lymph nodes. At day 24 after injury, the thymus and bone marrow virtually were depleted of lymphocytes, the mesenteric lymph nodes manifested a significant decrease, and lymphocytes in the nodes draining the burn wound continued to increase in number. This same pattern was maintained on day 48, but numbers of lymphocytes in the mesenteric nodes normalized. At day 60 after injury, lymphocyte numbers in all tissues were normalized, but the spleen and nodes draining the burn wound where increased numbers compared to control persisted. Cell-surface phenotyping was performed on all lymphoid tissues at all time intervals to determine the percentages of lymphocytes comprising the following subsets: Ia+ cells (B cells and activated T cells), T cells, T-Helper/Inducer cells (T-H/I), and T-Suppressor/Cytotoxic (T-S/C) cells. Although changes in lymphocyte subset percentages were complex, they could be divided grossly into two phases. First, all compartments showed significant phenotypic changes in the first six days after burn. With the exception of the nodes draining the burn wound and the blood, this was followed by a return towards normal on day 12. The second phase then ensued with significant phenotypic changes again occurring in most tissues from days 24 to 60 after injury. These studies demonstrate that burn injury results in dramatic alterations in lymphocyte numbers and subset percentages in different lymphoid compartments. Immune alterations observed following thermal injury may be due, in part, to a redistribution of the cellular elements responsible for generation of the immune response.

The decrease in peripheral blood CD4+ T cells following thermal injury in humans can be accounted for by a concomitant decrease in suppressor-inducer CD4+ T cells as assessed using anti-CD45R.

Using single- and two-color fluorescence flow cytometry, 10 thermally injured human subjects were assessed over time for both percentages and absolute numbers of lymphocytes comprising peripheral blood lymphocyte subpopulations. The CD3+ lymphocyte percentage decreased significantly in the early postburn period, and this decrease could be accounted for entirely by a concomitant decrease in the CD4+ lymphocyte percentage. Further, the decline in CD4+ percentage was due to a specific decrease in the suppressor-inducer subset of CD4 as defined using anti-CD45R. No change in the helper-effector subset of CD4 was noted. The percentage of CD8+ lymphocytes did not change significantly at any time postburn nor did subsets of CD8 as defined using anti-CD11. Numerical changes in lymphocyte subsets were dominated by a general lymphopenia occurring on Day 4 following injury. However, suppressor-inducer (CD4+/CD45R+) T cells also decreased significantly on postburn Day 1. These results further elucidate phenotypic changes in immunoregulatory subsets following major injury and suggest a possible basis for depressed autologous mixed lymphocyte responsiveness of burn patient T cells, one of the functional immunologic defects associated with severe injury.

Granulocyte contamination of Ficoll-Hypaque preparations of mononuclear cells following thermal injury may lead to substantial overestimation of lymphocyte recovery.

During ongoing flow cytometric studies of burned patient blood leukocytes, it was noted frequently that large numbers of granulocytes were present along with the mononuclear cells at the plasma/Ficoll-Hypaque (F-H) interface following centrifugation over F-H. Since differential WBC counts are not routinely performed on F-H interface cells, it is possible that many previous immunologic studies of burned patients have greatly overestimated numbers of lymphocytes recovered. The present study sought to quantify the extent to which granulocyte contamination of F-H separated cells occurs following burn injury. Blood from 15 thermally injured patients (7-55% total body surface area burn) was studied serially at 24 hr, 48 hr, and weekly thereafter through 6 weeks postburn (PB). Controls were age-matched normals (No. of control bloods = 59). Three-part differential cell counts (lymphocytes, monocytes, and granulocytes) were performed on both F-H interface cells and RBC-lysed whole blood. Counts were performed by light scatter analysis on a flow cytometer. Except at 48 hr, at every time studied through 4 weeks PB, there was significant contamination of F-H interface cells with granulocytes. At 24 hr PB, 41 +/- 9% of the interface cells were granulocytes while at 4 weeks, PB 24 +/- 8% of the interface cells were granulocytes. The data did not support the interpretation that this increase in F-H interface granulocytes was simply reflective of the granulocytosis commonly observed after burn. Thus artificial generation of granulocytosis by addition of extra normal leukocytes to normal blood resulted in complete separation of granulocytes from mononuclear cells following centrifugation over F-H.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibodies directed at human T lymphocyte activation antigens cross react with concanavalin A-stimulated canine and baboon peripheral blood lymphocytes.

A panel of 10 monoclonal antibodies directed at activation antigens on human T lymphocytes were tested for cross reactivity with canine and baboon resting and ConA stimulated peripheral blood lymphocytes. Monoclonal antibodies anti-OKT19, anti-OKT-21, and anti-OKT22 labeled a high percentage of both resting and stimulated canine and baboon cells. Anti-OKT24 labeled activated but not resting baboon lymphocytes and did not label canine lymphocytes. Anti-HLA-DR labeled a small percentage of resting baboon lymphocytes (presumably B cells) and a high percentage of activated baboon and resting and activated canine lymphocytes. Anti-OKT14, anti-OKT20, and anti-OKT23 did not label canine or baboon lymphocytes. Anti-OKT9 did not label baboon lymphocytes, but labeled a low percentage of lymphocytes in one dog. Anti-TAC labeled activated but not resting canine and baboon cells.

Comparison of numerical and phenotypic leukocyte changes during constant hydrocortisone infusion in normal humans with those in thermally injured patients.

Twenty-four to 48 hours after thermal injury, percentages and number of T X mu or T4 lymphocytes decrease with little or no change in T X gamma or T8 cells. Additionally, the plasma hydrocortisone level is extremely elevated. Since administration of hydrocortisone to normal humans also produces a specific decrease in T X mu or T4 lymphocytes, it was hypothesized that burn induced elevations of hydrocortisone were responsible for the decrease in T X mu/T4 cells. In this study, normal humans were administered constant infusions of hydrocortisone for six hours, such that plasma levels were increased to an extent that mimics those 24 to 48 hours after thermal injury. Before, during and after infusion, percentages and numbers of lymphocytes, monocytes, granulocytes and T3, T4, T8, T11, HLA-DR and Leu7 lymphocytes were quantified by flow cytometry. Results were compared with those for patients with burns. The plasma hydrocortisone level rose to 49.0 micrograms per deciliter during infusion, similar to the mean of 47.5 micrograms per deciliter for patients with burns. Infused volunteers showed significant lymphopenia, monocytopenia and granulocytosis. Additionally, there were significant decreases in percentages of T3, T4 and T11 lymphocytes, no significant changes in percentages of T8 or HLA-DR and an increase in percentages of Leu7+ cells. These changes in lymphocyte subsets mimicked those of burn patients. Numbers of T3, T4 and T11 cells significantly decreased during hydrocortisone infusion while numbers of T8, HLA-DR and Leu7 lymphocytes did not change. Burn patients showed decreased numbers of T3 and T4 cells, but this T3/T4 lymphopenia was not as great as during hydrocortisone infusion. These results support the hypothesis that elevation of hydrocortisone is responsible for the lymphocyte phenotypic changes that occur in the early postburn period.

Analysis of the mitogenic effects of toxic shock toxin on human peripheral blood mononuclear cells in vitro.

It has been shown previously that the staphylococcal enterotoxins A and B are T-cell mitogens and also cause inhibition of murine plaque-forming cells generated in vitro. Similarly, toxic shock toxin, a 24,000-MW protein produced by toxic shock-associated strains of Staphylococcus aureus, is mitogenic and inhibits the generation of both murine and rabbit plaque-forming cells. In this study, an analysis of the T-cell response to toxic shock toxin was performed. Human peripheral blood mononuclear cells responded to toxic shock toxin over a broad dosage range (1 ng/ml to 5 micrograms/ml) with maximum proliferation at day 4 (96 hr) of culture. Heat treatment (100 degrees C for 60 min) of toxic shock toxin attenuated its mitogenic effects by only a small amount, and this attenuation could be reversed with increasing concentration of the toxin. By cytofluorography, both untreated and toxic shock toxin-treated small lymphocytes manifested normal percentages of OKT3+, OKT11+, OKT4+, OKT8+, HLA/DR+, and Leu-7+ cells. However, toxic shock toxin-induced blasts were 99% OKT11+ and expressed the receptor for interleukin 2 (89%-100% TAC+). Approximately 85% of the blasts were OKT4+, and 25% of the blasts were OKT8+. Proliferation of purified, double-rosetted T cells was enhanced monotonically by the addition of irradiated "non-T" cells. Irradiated, monocyte-enriched non-T cells were 2.5 times more potent than unfractionated non-T cells in producing quantitatively similar proliferation by toxic shock toxin-stimulated, autologous T cells. In addition, preincubation of non-T cells for 24 hr with toxic shock toxin, followed by extensive washing and irradiation, induced substantial proliferation by unexposed, autologous T cells. These data show that toxic shock toxin is mitogenic for T cells and requires accessory cells for maximal activity. Further, this substance appears to induce both a subset of OKT4+ (Class II MHC-restricted) and OKT8+ (Class I MHC-restricted) blasts.

Flow cytometric analysis of lymphocyte subpopulations after thermal injury in human beings.

Several theories have been advanced in an effort to explain immunologic suppression after thermal injury, that is monocyte production of immunoregulatory prostaglandins, activation of suppressor cells, production of suppressive serum factors and alteration in helper cell function. In the current study, cytofluorometric analysis was performed on peripheral blood mononuclear cells isolated from 30 severely burned individuals using a FACS IV cell sorter. Fluorescein labeled monoclonal antibodies were used to phenotype total T cells (OKT3+), helper cells (OKT-4+), suppressor cells (OKT-8+), monocytes (antimono.2+) and B cells (anti-Ia+). After burn injury, the most striking phenotypic alterations observed were a marked decrease in the number and percentage of total OKT3+ T cells and OKT4+ helper cells. No significant increases were observed in the OKT8+ suppressor cell subpopulation. Monocytes exhibited a transitory increase during the first 48 hours postburn which returned to normal by postburn day 7. The percentage of Ia+ cells were either normal or decreased in number during the course of the injury. An OKT-4 to OKT-8 ratio of less than 1.00 at 24 to 48 hours postburn may represent a reliable predictive index for death by sepsis. These data suggest that the syndrome of burn induced immunologic suppression may be better described as a "burn induced immunodeficiency syndrome," that is characterized by decreased numbers or function of Interleukin-2 producing helper cells, or both.

Autologous and allogeneic mixed-lymphocyte responses following thermal injury in man: the immunomodulatory effects of interleukin 1, interleukin 2, and a prostaglandin inhibitor, WY-18251.

A group of 30 burn patients with 36-87% total body surface area (TBSA) burns was studied at 24-48 hr postburn. These included studies of (1) autologous and allogeneic mixed-lymphocyte reactions (MLR); (2) the immunoregulatory influence of mitomycin C-treated T cells, non-T cells, and unfractionated peripheral blood lymphocytes (PBL) on allogeneic MLR; and (3) correlation between the proportions of T-cell subsets defined with monoclonal antibodies (OKT4 and OKT8) and autologous MLR. Studies concerning adherent cell production of thromboxane, prostaglandin E2, and prostaglandin F2a and the immunomodulatory effects of Interleukin 1 (IL-1), Interleukin 2 (IL-2), and a prostaglandin inhibitor, WY-18251, on autologous MLR are presented. The autologous mixed-lymphocyte reaction was depressed in 60% of the burn patients tested. This depressed response correlated closely to the extent of third-degree injury (P less than 0.025) and to TBSA injury greater than 60% (P less than 0.025). A linear correlation was observed between the depression in autologous MLR and a decrease in both the percentage of OKT4+ T cells and the OKT4+/OKT8+ ratio. The response of T cells from burn patients in allogeneic MLR was normal. Age, sex, TBSA of the burn, and size of second-degree burn did not correlate with the abnormalities observed in MLR. Mitomycin C-treated mononuclear cells, purified T cells, or non-T cells from burned patients did not demonstrate any suppressive influence on MLR in normals. Monocyte number and arachidonic acid metabolism were investigated. In addition to increased numbers of monocytes following thermal injury, adherent cells produced increased quantities of thromboxane, prostaglandin E2, and prostaglandin F2a. The effects of Interleukin 1, Interleukin 2, and a prostaglandin inhibitor, WY-18251, were studied in autologous MLR (AMLR) of burned and normal patients. Interleukin 1 and WY-18251 did not induce any significant changes in proliferation in burned patients or normal controls. When compared to cultures without exogenous IL-2, an increase in AMLR was observed following the addition of IL-2 to burn patient cultures at Day 6 and Day 7 of culture. Although the addition of IL-2 did increase proliferation in AMLR of normal controls at Day 6 and Day 7, the enhancement observed for the burn patient cultures represented a restoration to the level of normal control cultures without IL-2. A dose-dependent increase in AMLR was observed in T cells isolated from normal and burned patients in the presence of purified Interleukin 2.(ABSTRACT TRUNCATED AT 400 WORDS)

T-cell subpopulations following thermal injury.

In this report, we have enumerated T-cell subsets as defined by receptors for immunoglobulin isotypes at 24 to 48 hours postburn. T cells possessing surface receptors for the Fc fragment of IgM represent a population of cells with helper activity, T mu cells, and T cells possessing surface receptors for the Fc fragment of IgG represent a population of cells with suppressor-cytotoxic function, T gamma cells. Significant alterations in the number and proportion of regulatory T cells was observed early in the course of severe burn trauma. A decrease in total T cells was accompanied by a drop in IgM-Fc binding lymphocytes, helper cells, in all burns greater than 20 per cent total body surface area. An increase in IgG-Fc binding lymphocytes was observed only in those patients who had sustained the most severe injuries, greater than 60 per cent total body surface area burn or greater than 25 per cent third-degree burn. These changes correlated linearly with the amount of full thickness injury, and the ratio of T mu:T gamma cells was significant at 24 to 48 hours postburn in predicting the risk of septic death.