Modulation of human neutrophil function by endogenous opiate enkephalins.

On the basis of recent evidence, the natural opiate enkephalins, which previously were believed to be confined to the central nervous system, are now known, in fact, to be released from the adrenal glands by sympathetic activation or trauma. To determine if enkephalins (EKs) affect peripheral function, the influence of synthetic leucine and methionine enkephalin (leuEK and metEK) on several relevant functions of human polymorphonuclear leukocytes was evaluated. Initial attempts to detect interaction of leuEK and metEK with neutrophils yielded inconsistent results. Further studies were done using protease-resistant methionine enkephalin-amide (metEKamide). MetEKamide was able to induce degranulation when present at 10(-3) and 10(-4) mmol/L as determined by release of beta-glucuronidase and lysozyme. Using the under-agarose chemotaxis method, treatment with metEKamide resulted in no change of the neutrophil's chemotactic response to an optimal concentration of the chemotactic peptide formyl-methionyl-leucyl-phenyl-alanine (FMLP). However, responsiveness to low levels of FMLP increased in cells treated with 10(-3)-10(-5) mmol/L metaEKamide. This appeared to be a result of increased chemokinesis of the treated cells. Scanning electron microscopic studies of cells exposed to metEKamide revealed that treatment resulted in changes in neutrophil morphology. When metEKamide itself was tested as a potential chemotactic agent, 10(-2) mmol/L metEKamide in an opposing well served to induce chemotaxis. Our results, along with those of recent studies of EKs as immunomodulators of T cell function, suggest that neurohormones can function as regulators of the immune response.

Suppressor T cell activity in lymphoid interstitial pneumonia.

A case of lymphoid interstitial pneumonia was investigated for immunological abnormalities. Suppressor T cells were found. The abnormality in the lymphoid system could be corrected in vitro with levamisole. The patient thereafter developed histiocytic lymphoma and soon died without any response to chemotherapy.

Synthetic immunomodulators for prevention of fatal infections in a burned guinea pig model.

Individuals who have suffered severe trauma, such as burns, have a high incidence of infection associated with impaired host resistance. Nonspecific stimulators of host defense mechanisms, i.e., immunomodulators, may be of benefit in such situations. A small animal model (guinea pigs) was developed to study the efficacy of immunomodulators in burns. Anesthetized animals received a 20% total body surface area, full-thickness, scald burn. There was no mortality associated with this injury, but these animals were highly susceptible to challenge with Pseudomonas aeruginosa strain 1244 by direct injection into the burn wound within 24 hours of injury. This susceptibility persisted about 7 days. The standard model adopted was to injure animals, then challenge with 1 median lethal dose (LD50) of P. aeruginosa 96 hours after injury. Using this model, six synthetic immunomodulators were tested: CP-20,961, CP-46,665, muramyl dipeptide, thymopoietin pentapeptide (TP-5), levamisole, and lithium. Drug administration began 24 hours after injury and ended prior to challenge with P. aeruginosa at 96 hours. CP-20,961, muramyl dipeptide, levamisole, and lithium all had no beneficial effect on survival. A single dosage (0.3 mg/kg, I.V.) of CP-46,665, administered 24 hours postinjury, increased the survival rate from 50% to 85% and mean survival time (MST) from 8.2 days to 12.4 days. TP-5, given in four doses (0.1 mg/kg, I.V. each) every 24 hours, increased the survival rate from 40% to 80% and MST from 6.9 days to 11.6 days. These data show that immunomodulators could be of benefit in burns, but also that not all agents are effective in this particular situation.

Analysis of brain indoleamines and catecholamines associated with burn-induced anorexia.

Guinea pigs subjected to a full-thickness 30% body surface flame burn significantly reduced food intake and lost weight for 5 days thereafter. Comparison of brain monoamine changes in burned, pair-fed control, and freely feeding control groups revealed normal levels of norepinephrine and elevated concentrations of dopamine in burned animals compared to the pair-fed control group. Levels of the serotonin precursor, tryptophan, and metabolite, 5-hydroxyindoleacetic acid, were also increased in burned compared to pair-fed animals, suggesting increased activity of indoleamine neurons. Although brain catecholamine levels were not correlated with food intake, significant negative correlations between food consumption and tryptophan, serotonin, and 5-hydroxyindoleacetic acid were observed. These data suggest that aberrations in indoleamine and possible catecholamine neurotransmitter systems may be involved in the mediation of burn-induced anorexia.

Failure to demonstrate a role for line Ib tumor-associated surface antigen in the etiology of age-dependent polioencephalomyelitis.

In the preceding paper [Morris et al. (1982), Molec. Immun. 19, 973-982] we demonstrate an associative interaction between the line Ib tumor-associated surface antigen (Ib-TASA) and the Dk/Kk regions of the major histocompatibility complex, i.e. 'altered-self' antigen. We originally hypothesized that age-dependent polioencephalomyelitis (ADPE) occurred as the result of immune recognition of a 'self'-determinant on the 'altered-self' antigen. In this report we used the non-ionic detergent, NP-40, to solubilize Ib cell surface antigens. Although immunization of immunocompetent C58 mice with the soluble NP-40 Ib cell extract afforded protection to lethal tumor challenge, the extract failed to induce ADPE in immunosuppressed mice. Data presented here demonstrate that Ib-TASA is not involved in the etiology of ADPE. The evidence suggests that lactic dehydrogenase virus, which is a silent virus passaged with line Ib leukemia, is the causative agent of the paralytic disease.

Plasma and skeletal muscle amino acids following severe burn injury in patients and experimental animals.

This study describes and analyzes sequential changes in plasma and skeletal muscle free amino acids following severe burn injury. Plasma free amino acids were determined in children (n = 9) with burns averaging 60% total body surface area and were compared with laboratory beagles (n = 44) which received a flame burn totaling 30% of their body surface area. In addition, needle biopsy specimens were obtained from the semitendonosus muscle in the animals to determine free intracellular amino acids. In both patients and animals the amount of total free amino acids in plasma fell following burn, suggesting relative protein deficiency. This drop was primarily due to a 47% drop in nonessential amino acids. However, plasma phenylalanine was consistently higher than normal following burn, and was strongly associated with death and weight loss in both animals and patients, especially when analyzed as a ratio with tyrosine. This finding suggested excessive catabolism, hepatic dysfunction, or both. Plasma levels of several amino acids correlated significantly with weight loss. Alterations in muscle free amino acids generally were similar to plasma amino acids. Exceptions were muscle alanine and glycine which strongly correlated with weight loss. However, the determination of muscle free amino acid profiles did not yield clinically useful information not available from plasma profiles. Plasma levels of liver enzymes suggested progressive hepatic dysfunction. These studies show that the laboratory beagle is a good model for studying the metabolic alterations of amino acids that accompany burn injury, since they mimic humans in many parameters which appear to be most useful with respect to clinical evaluation.

Improved survival in severely burned animals using intravenous Corynebacterium parvum vaccine post injury.

This study was designed to determine the potential benefit or toxicity of an immunomodulator, Corynebacterium parvum vaccine, when it is given after severe burn injury. Forty conditioned beagles received a 33% total body surface 3-degree flame burn and were resuscitated with Ringer's lactate solution (3 ml/kg/% burn). Wounds were treated daily for 10 days with silver sulfadiazine cream. Two days and nine days after burn, 21 of the animals received C. parvum vaccine (10 mg/kg IV) in a saline infusion, while 19 control animals were given only saline infusion according to a double-blind protocol. Serial measurements were made of temperature, weight, food intake, hematocrit, hemoglobin, red blood count, white blood count, differential, platelet count, fibrin degradation products, activated partial thromboplastin time, clot retraction, C3, blood cultures, neutrophil function, monocyte function, opsonic index, Na, K, Cl, BUN, glucose creatinine, total protein albumin, albumin/globulin ratio, alkaline phosphatase, SGPT, and SGOT. During 45 days of observation, only 16% of the saline control dogs survived compared to 47% of the treated animals. Total white counts and neutrophil function were the only values which were significantly better in animals receiving C. parvum. However, their correlation with increased survival was marginal This preclinical trial suggests that C. parvum is an effective immunodulator for prevention of fatal infection following burn injury. There were no demonstrable toxic effects of the material in this study.

Macrophage activation and mobilization in nude mice by Corynebacterium parvum and pyran: a functional and histologic study.

Peritoneal macrophages from both congenitally athymic ("nude") mice and heterozygous littermates were activated by pyran copolymer or by Corynebacterium parvum vaccine. C parvum did not produce an increase in the number of peritoneal macrophages in nude mice, although it did produce a typical splenomegaly. Pyran produced an even greater influx of macrophages in the peritoneum of nude mice, when compared to normal mice, but did not produce splenomegaly in nude mice. Pyran- and C parvum-induced splenomegaly were accompanied by an increase in the apparent T-cell population of germinal centers. These experiments indicate that: 1) Macrophage activation, per se, by either C parvum, is a thymus-independent event; 2) Macrophage mobilization, as determined by organomegaly or PEC number, does not have an obligatory requirement for T-cells (depending on the agent used); 3) Macrophage activation may not always correlate with mobilization; and 4) Mechanisms for attracting and sequestering macrophages in the peritoneum may be different from those of the spleen.

Different serum requirements for opsonization of two serologically identical strains of Pseudomonas aeruginosa.

Two strains of serotype 2 Pseudomonas aeruginosa and one species of E. coli were compared regarding their opsonic requirements using human sera and polymorphonuclear leukocytes. None of the three organisms tested activated exclusively one complement pathway. Both strains of Pseudomonas had an absolute requirement for antibody for opsonization, but differed in complement requirements. The studies described herein showed significant differences in the mechanism of opsonization of the two strains of Pseudomonas.

Beneficial effects of aggressive protein feeding in severely burned children.

To determine any potential benefit of feeding increased amounts of protein to hypermetbolic burned patients, 18 children with burns averaging 60% total surface area were randomized into two matched groups and studied serially for at least six weeks: the first group was given a normal diet with a balanced nutritional supplement, and the second group was supplemented with milk whey protein. The normal protein group received 87.1% of their desired caloric intake with 16.5% of calories from protein compared to 77.7% of desired caloric intake with 23.0% of calories from protein for the high protein group. Despite a higher caloric intake, the normal protein group had a worse opsonic index compared to the high protein group (0.42 +/- 0.04 vs. 0.62 +/- 0.05, p < 0.0007), lower levels of C3 (1371 +/- 55 vs. 1585 +/- 64 micrograms/ml, p < 0.01), lower levels of IgG (805 +/- 52 vs. 975 +/- 56 micrograms/ml, p < 0.03), lower levels of transferrin (200 +/- 10 vs. 283 +/- 18 mg/dl, p < 0.0001), lower levels of total serum protein (5.5 +/- 0.1 vs. 6.3 +/- 0.2 g/dl, p < 0.005), more bacteremic days (11% vs. 8%, p < 0.005) and worse survival (5/9--56% vs. 9/9--100%, p < 0.03). Patients receiving the high protein diet had significantly higher plasma levels of valine, lysine, threonine, leucine, aginine, isoleucine, proline, serine, asparagine, tryptophane, and tyrosine. Asparagine levels were significantly (p < 0.01) associated with better neutrophil function and opsonic index. Except for phenylalanine, significant associations were found for serum levels of each of the amino acids with concentrations of one or more serum proteins. These studies provide evidence that many immunologic functions are dependent upon optimal availability of specific amino acids, and that routine diets do not provide sufficient protein to satisfy the needs of seriously burned children.

A comparison of immunologic profiles and their influence on bacteremia in surgical patients with a high risk of infection.

Prospective sequential studies of the antibacterial function of neutrophils, lymphocyte responsiveness, opsonic capacity of serum and serum levels of C3(B), properdin, factor B, IgG, and albumin were made in 32 patients with severe burn injury (greater than or equal to 45%), 21 patients with severe multisystem traumatic injury, 20 high-risk, infected patients, and 22 renal transplant patients. Fifty-five episodes of bacteremia occurred in 37 of the 95 patients. Abnormal neutrophil function was clearly associated as a predisposing factor to these episodes, whereas there was no association between bacteremia and low serum levels of C3, IgG, factor B, or properdin. C3, factor B, and IgG usually rose following bacteremia as acute phase proteins, but there was evidence of a consumptive opsoninopathy in 11% of episodes. Defective opsonization was associated with a high risk of bacteremia only when there was a coexisting abnormality of neutrophil function (88% of such patients became bacteremic). None of 27 nonburned patients tested with delayed hypersensitivity antigens responded normally, and there was regularly depression of lymphocyte responsiveness to phytohemagglutinin-A and concanavalin-A in a whole blood assay related to serum immunosuppressive factors, but poor responsiveness was not associated with bacteremia.

Fresh-frozen plasma vs. plasma protein derivative as adjunctive therapy for patients with massive burns.

Twenty patients with burn injuries involving 45% or more total body surface area were randomly allocated to receive either fresh-frozen plasma (plasma), 200 ml/m2/d (11 patients), or an approximately equal amount of plasma protein derivative (Plasmanate) (nine patients) during the first 45 days postburn. To study the potential effects of these two adjunctive therapies on host resistance to infection, measurements were made twice weekly of the antibacterial funciton of neutrophils, the opsonic index (ability to opsonize alternative pathway dependent E. coli 075), C3(B), IgG, properdin, factor B, total protein, and albumin. The average size of burn in the plasma group was 61.5% total and 42% 3 degrees compared with 61% total and 46% 3 degrees in the Plasmanate group. Ten and 18 episodes of bacteremia occurred in the plasma and Plasmanate groups, respectively. Analysis of the results indicates only slightly better support of host resistance when plasma is administered, but this is counterbalanced by the increased risk of viral hepatitis.

A sequential, prospective analysis of immunologic abnormalities and infection following severe thermal injury.

A sequential, prospective analysis of humoral and cellular immune function was performed on 20 burn patients with injuries involving >/=45% total body surface area. Infected patients had significantly worse neutrophil bactericidal activity against Staphylococcus aureus 502A than did noninfected patients. Chemotaxis of neutrophils correlated poorly with infection although chemotaxis was frequently abnormal. The opsonic index of serum was depressed early after the burn but returned to nearly normal values by the fourth to the fourteenth postburn day. There was no difference between infected and noninfected patients. Serum levels of IgG, properdin and C3, while initially low, remained within the normal range after the ninth postburn day in both groups. Factor B levels rose rapidly during the first three weeks after injury to more than double normal levels in many patients. Suggestive evidence for consumption of opsonic protein occurred with five of 19 episodes of bacteremia. The responsiveness of isolated lymphocytes to PHA was normal. However, patients' sera were shown to significantly inhibit the responsiveness of normal lymphocytes to PHA. Analysis of immunologic profiles for individual patients indicates that abnormalities of neutrophil function are the most important acquired defect predisposing patients to the development of bacteremia following major thermal injury; abnormalities of opsonic action play a secondary but important role.

Growth patterns and metastatic behavior of human tumors growing in athymic mice.

The growth characteristics and metastatic behavior of human tumors growing in athymic nude mice were studied. Human tumor cell lines HEp-2 (carcinoma or larynx) and SW480 (colon carcinoma) were transplanted into athymic nude mice of BALB/c origin. Tumor cells (1 x 10(6) and 2 x 10(7)) were given either s.c. or i.p. Following s.c. injection tumors developed rapidly to become easily palpable with 2 weeks forming a s.c. tumor focus surrounded by a thick fibrous capsule. Animals with s.c. transplants were little affected by the growing tumor. At the time they were sacrificed at Day 34 (HEp-2) and 62 (SW480), a large part of the tumor was necrotic. Capsular infiltration and invasion of lymphatic vessels and perineural and perivascular lymphatic spaces were observed. Metastases to regional lymph nodes were seen in animals kept alive for up to 6 months. Following i.p. transplantation, tumors spread widely in the peritoneal cavity, invaded intraabdominal organs, and metastasized to mediastinal lymph nodes and lungs. Fifteen of 26 animals (60%) developed metastases. Necrosis of the i.p. growing tumors was minimal. All animals in this group died as a result of tumor growth.

The effects of malnutrition on variables of host defense in the guinea pig.

Studies were conducted in young guinea pigs to determine the effects of malnutrition on selected variables of host resistance. Malnutrition was produced differently in two experiments. In the first the quantity of a standard, normal diet was reduced progressively so that test groups were fed 25% less each week over a 4 week period. Control groups were fed ad libitum. In the subsequent experiment, animals were fed defined guinea pig diets containing 5%, 30%, and 60% casein, respectively, which were similar in caloric content, vitamins, and minerals. Measurements of phagocytic bactericidal activity, serum opsonization, serum IgG and C3 levels, and mitogenic response of lymphocytes were made at weekly intervals. Results obtained from both experiments were comparable. There was a significant decline in phagocyte function by the third week in malnourished animals while the numbers of phagocytes per milliliter of peritoneal washings were similar to controls at all time periods. A depression of serum opsonization was observed when animals became moribund even though serum IgG levels remained unchanged. Serum C3 levels in malnourished animals were significantly lower than controls. Mitogenic response of lymphocytes to phytohemagglutinin was 85% lower in the 5% casein group after the third week. These results indicated that a marasmus-like condition and protein malnutrition depress critical functions of resistance.

Whole blood vs. packed red cells for resuscitation of hemorrhagic shock: an examination of host defense parameters in dogs.

Whole blood vs. packed red cells for resuscitation of acute blood loss in dogs was compared with respect to a number of variables of host defense. Dogs subjected to a controlled blood loss equal to 8% of their body weight exhibited significant depressions in serum protein, C3, IgG, and total opsonic activity when resuscitated with packed red cells in saline. No such depression in serum components or activity was observed in identically bled dogs resuscitated with whole blood. There were no differences in the rate of clearance of intravenously injected bacteria indicating an intact reticuloendothelial system (RES) function in both groups. These experiments suggest that whole blood may be preferable to packed cells for resuscitation of acute hemorrhagic shock when the effect on resistance of infection is considered.