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We have previously reported in dogs after ventricular fibrillation that (1) potassium and calcium levels decrease and magnesium and glucose increase, (2) all values return to control levels by 3 hours, and (3) propranolol blocks the changes in potassium, magnesium, and glucose but not calcium. The purpose of this study was to evaluate the beta 1- and beta 2-selectivity of changes in potassium, magnesium, and glucose and assess the response of the change in calcium to calcium channel blockade. Before initiating ventricular fibrillation, we pretreated dogs with an intravenous solution of either normal saline, metoprolol, ICI 118551 (two doses), or diltiazem. After a 2-minute episode of ventricular fibrillation, dogs were resuscitated. Baseline electrolyte measurements were obtained before ventricular fibrillation and sequentially for 3 hours after fibrillation. The saline-treated control group had a maximal decrease in the serum potassium level of 0.5 +/- 0.2 mEq/L. High-dose ICI 118551 reduced this decrease to 0.3 +/- 0.3 mEq/L (p = 0.055), but the other three groups showed no difference compared with the control group. Magnesium increased in the saline control group by 0.2 +/- 0.1 mEq/L. This increase was partially reduced by high-dose ICI 118551 to 0.1 +/- 0.1 (p = 0.055) but not by the other drugs. Glucose increased to 40 +/- 13 mg/dl in the saline control group. This increase was partially reduced by high-dose ICI 118551 to 23 +/- 6 mg/dl (p = 0.007) but not by the other drugs. Calcium showed a maximal decrease of 0.6 +/- 0.3 mg/dl in the control group. This decrease was not attenuated by any of the drugs.(ABSTRACT TRUNCATED AT 250 WORDS)

In August 1991 the Institute of Medicine released a report entitled "Adverse Effects of Pertussis and Rubella Vaccines" that examined, among other relations, the relation between immunization with the RA 27/3 rubella vaccine strain and chronic arthritis. The committee spent 20 months reviewing a wide range of information sources including case series and individual case reports published in peer-reviewed journals and reported by vaccine manufacturers; unpublished case reports from physicians, parents, and other concerned persons; epidemiological studies; and laboratory studies. There were no animal studies available. The committee found that the evidence is consistent with a causal relation between the RA 27/3 rubella vaccine strain and chronic arthritis in adult women, although the evidence is limited in scope. Proving that rubella vaccination can cause chronic arthritis will require a better understanding of pathogenetic mechanisms and additional well-designed studies. We briefly describe the committee's evaluative methods and present the evidence underlying its conclusion.

Protein-calorie malnutrition predisposes to infection by intracellular pathogens, but the basis for this predisposition is unclear. We studied the effect of protein deprivation on mouse peritoneal macrophage function and on granuloma formation during infection by bacille Calmette-Gueŕin (BCG). Injection of lipopolysaccharide (LPS) to induce inflammation elicited fewer peritoneal cells from mice fed a 2.5% protein diet than from mice fed an isocaloric chow in which protein calories constituted 24% of the total. LPS-elicited macrophages from protein-deprived mice demonstrated a reduction in spreading, total cell protein, cell lactate dehydrogenase, and release of superoxide anion (O2-) in response to stimulation. Priming in vitro by interferon (IFN)-gamma for enhanced release of O2- was also significantly impaired in protein-deprived mice. This defect was reversible by repletion with 24% protein diet for 10 days. Impairment of macrophage function in protein-deprived mice was further evidenced by an impaired capacity to express Ia antigen in response to IFN-gamma and by reduced production of IL-1 activity in response to LPS. Infection by BCG in protein-deprived mice was characterized by impaired granuloma development in liver, lungs, and spleen. Thus, in this model, protein deprivation significantly impaired macrophage activation, as assessed by morphologic, metabolic, and functional criteria. This impairment might compromise immune effector mechanisms dependent on macrophage activation, including rejection of intracellular pathogens.

Walking track analysis is an investigative technique that allows a researcher to assess objectively the functional capacity of the limb in the rat sciatic nerve model. This study has shown that paint and paper is a better method for imaging the rat foot plantar surface than other methods described in the literature. The block printing paint has the distinct advantage of keeping radial diffusion error to a minimum. Paint more correctly images the important anatomical landmarks of the rat plantar surface. There is better traction (less slippage) rendering the prints easier to interpret. The authors conclude that the paint and paper is a superior method to the other methods currently available.

Addition of chemical oxidants to cells in culture has been shown to induce binding of low-molecular-weight thiols to reactive sulfhydryls on proteins in a process termed S-thiolation. We found that stimulation of the respiratory burst in mouse macrophages, with release of O2-, resulted in S-thiolation of several proteins, most prominently three with molecular weights of 74, 33, and 22 kDa. One protein (28 kDa) was S-thiolated without addition of an exogenous stimulus. Exposure of cells to concentrations of hydrogen peroxide like those released in the respiratory burst induced S-thiolation of these same proteins. S-thiolation and release of O2- began at approximately the same time. Stimulation of LPS-elicited macrophages induced prominent S-thiolation of three different proteins (38, 30, and 21 kDa). Under the conditions of these experiments, there was no detectable increase in glutathione disulfide and a negligible decrease in glutathione, which suggests that S-thiolation can occur without significant perturbation of the glutathione peroxidase/reductase cycle. S-thiolation of proteins could help protect the macrophage against the autoxidative damage associated with the respiratory burst. Modification of specific proteins by S-thiolation might serve to modulate cellular metabolic events.

Neutrophils possess a classical Ca2+, phosphatidyl serine (PS) and diglyceride (DG)-dependent protein kinase C (beta-PKC) which was translocatable from cytosol to membrane in response to elevated Ca2+ in the physiologic range or to pretreatment with phorbol myristate acetate (PMA). The translocatable beta-PKC was purified from neutrophil membranes prepared in the presence of Ca2+, eluted with EGTA and subjected to hydroxyapatite chromatography. An 80-kDa protein possessing Ca/DG/PS-dependent histone phosphorylating activity was recognized by a monoclonal antibody to beta-PKC but not to alpha-PKC or gamma-PKC. A cytosolic kinase activity remaining after Ca(2+)-induced translocation of beta-PKC was dependent on PS and DG but did not require Ca2+. This novel Ca(2+)-independent, PS/DG-dependent kinase, termed nPKC, eluted from hydroxyapatite between alpha-PKC and beta-PKC, ran as a 76-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was reactive to a polyclonal consensus antibody but not to monoclonal antibodies to alpha-PKC, beta-PKC, or gamma-PKC. Long chain fatty acyl-CoA, but not the corresponding free fatty acids, inhibited nPKC in the 1-10 microM range. The chemotactic peptide fMet-Leu-Phe triggered prompt but transient increases in neutrophil long chain fatty acid acyl-CoA, suggesting that nPKC is regulated by fatty acyl-CoA as well as DG during neutrophil activation. Purified beta-PKC phosphorylated a number of cytosolic proteins in a Ca(2+)-dependent manner, including a major 47-kDa cytosolic protein, which may be implicated in superoxide anion generation. In contrast, nPKC did not phosphorylate the 47-kDa protein, but phosphorylated numerous cytosolic proteins in a Ca(2+)-independent manner, including a 66-kDa protein which was not phosphorylated by beta-PKC. Differences in location, substrate specificity, and cofactor dependence between nPKC and beta-PKC suggest these kinases may play selective roles in the activation sequence of the neutrophil.

Pneumococcal pneumonia poses a serious threat to children in developing countries and remains an important disease in the industrialized world. Capsular polysaccharide is the only bacterial factor proven to contribute to pathogenesis. However, the mechanisms responsible for the hypoxemia, toxemia, crisis, and death associated with this common infection are poorly understood. Toxins secreted by the bacteria, byproducts of bacterial breakdown (e.g., pneumolysin or teichoic acid), or constituents of the intense inflammatory response in the lung might play a role in these phenomena. Malnourished children presumably carry less metabolic reserve with which they can resist the exceptional stresses of pneumococcal disease, and underlying parasitic infestation or other chronic infections, through release of potent mediators such as tumor necrosis factor or platelet-activating factor, might prime the mechanisms of host defense for a more fulminant and systemic response to infection. Vaccines that can protect infants are badly needed, and a better understanding must be gained of the mechanisms by which this pathogen continues to cause such devastating disease in the world's children and elderly.

We studied the biochemical basis of phagocytosis of Candida albicans, a serious pathogen, and Candida parapsilosis, which is rarely pathogenic, by human monocytes (Mo) and monocyte-derived macrophages (MDM). Optimal phagocytosis of both species by Mo required the presence of extracellular Ca2+ and opsonization through both the classic and alternative complement pathways. Serum-opsonized Candida were ingested equally by Mo and MDM; unopsonized Candida were phagocytosed only by macrophages, and uptake began slowly. This opsonin-independent phagocytosis required Ca2+ and could be blocked by yeast mannan or mannose-BSA conjugate, suggesting a role for the mannose receptor. Opsonized Candida elicited a vigorous increase in the concentration of [Ca2+]i in Mo and MDM, but no Ca2+ transient was detected in MDM stimulated with unopsonized Candida. Pretreatment of MDM with ionomycin to increase [Ca2+]i had no effect on phagocytosis of unopsonized Candida. Addition of 5 mM EGTA completely inhibited changes in [Ca2+]i in Mo and MDM, suggesting that the Ca2+ transient induced by opsonized Candida is due to an influx of extracellular Ca2+. Differences in pathogenicity between the two Candida species could not be explained by differences in any aspect of phagocytosis. Uptake mediated by the macrophage mannose receptor could play a role in clearance of Candida under opsonin-poor conditions.

We studied the biochemical basis of candidacidal activity by comparing the killing of Candida albicans, a serious pathogen, and Candida parapsilosis, a low-grade pathogen, by human monocytes (Mo) and monocyte-derived macrophages. Mo killed C. parapsilosis significantly better than C. albicans. The two species triggered the respiratory burst and release of myeloperoxidase (MPO) and beta-glucuronidase in Mo to an equivalent extent. In contrast to Mo, macrophages killed both species to an equivalent extent. Mo exhibited a greater candida-stimulated respiratory burst than did monocyte-derived macrophages, and the respiratory burst was required for the killing of both species. C. parapsilosis was killed much more easily than C. albicans by exposure to low concentrations of hypochlorite or monochloramine, MPO-dependent oxidants released by Mo but not macrophages, which lack MPO. With six different Candida strains there was a significant correlation between killing by Mo and susceptibility to hypochlorite (r = 0.926) or monochloramine (r = 0.981) (p less than 0.01 for each). Species differences in resistance to killing by Mo may be related to differences in sensitivity to MPO-derived oxidants, and the ability of C. albicans to resist the effects of these oxidants may be a virulence factor associated with this species.

Protein-energy malnutrition induces immunosuppression that predisposes to sepsis, but the mechanisms are unclear. This study examines the role of the macrophage in host defense in the malnourished state. Swiss-Webster mice (N = 300) were randomly allocated to control (24% casein) or low-protein (2.5% casein) diets for 8 weeks. We studied the ability of two populations of macrophages, peritoneal macrophages, and Kupffer cells to produce superoxide anion after in vivo administration of endotoxin or mycobacterium (bacille Calmette-Guérin). Phorbol diester and Candida albicans were used as stimuli. In another group of mice, we evaluated the ability of interferon gamma to up-regulate superoxide anion release and Candida phagocytosis and killing. Mice under protein-energy malnutrition demonstrated decreased mean body weights, serum protein levels, and cell yields. Superoxide anion production in resident and activated (lipopolysaccharide, interferon gamma, bacille Calmette-Guérin infection) peritoneal macrophages was significantly reduced in the malnourished group. Candida phagocytosis and killing were also both depressed in malnourished mice. Kupffer cells failed to generate superoxide anion in all groups. We conclude that severe protein-energy malnutrition significantly impairs macrophage function, which could diminish response to acute and chronic septic challenges. Interferon gamma up regulated peritoneal macrophage and Kupffer cell microbicidal function, which suggests a therapeutic role for this lymphokine in the malnourished septic host.

Murine peritoneal macrophages (m phi) respond to unopsonized zymosan with the production of superoxide anion (O2-). We investigated the involvement of phospholipid turnover in the transduction mechanism for this phenomenon. Zymosan-stimulated m phi produced increased amounts of phosphatidic acid (PA); the increase was first detected at 1.5 min and continued for 10 min of incubation. Production of O2- was not detected until between 2 to 4 min after stimulation, and continued to increase through 60 min. Inhibition experiments suggested that these two processes were linked. Theophylline (theo)/dibutyrylcyclic AMP (dbcAMP) and theo/prostaglandin E2 (PGE2) inhibited O2- production at every time point (79% and 80% inhibition, respectively, at 4 min). Corresponding inhibition of PA production was also achieved at every time point (85% by theo/dbcAMP; 67% by theo/PGE2 at 4 min). These results are compatible with a role for phospholipid remodeling in the transduction process associated with the respiratory burst. Results suggest that the phospholipid species could be phosphatidylcholine (PC) as well as phosphatidylinositol (PI).

C4 allotyping 13 homozygous C2-deficient individuals demonstrated 23 of 25 haplotypes to be of the relatively rare type C4A4 B2. This is of the same magnitude as the association of C2Q0 with HLA-DW2/DR2.

By using a kinetic assay, we have examined the role of antibody in the lysis of rabbit erythrocytes (RaRBC) through the alternative complement (C) pathway. Sera from some hypogammaglobulinemic (Hgamma) humans and all agammaglobulinemic chickens tested had subnormal activity in the assay. Heated normal human or chicken sera, but not heated Hgamma sera, restored activity to deficient Hgamma serum and initiated hemolysis in the presence of rabbit serum as C source. Absorption of heated normal human serum with RaRBC, but not with sheep erythrocytes or zymosan, removed its ability to reconstitute deficient Hgamma serum. Normal hemolytic activity could be resotred to Hgamma serum with human IgM, IgG, or colostral IgA, with goat anti-RaRBC IgG, or with an eluate from serum-sensitized RaRBC, but not with myeloma IgA. Restoration of hemolytic activity to Hgamma serum could be achieved in a dose-dependent fashion with the F(ab')2 fragment of IgG. These results suggest that antibody exerts a significant rate-limiting effect on alternative pathway activity in the RaRBC lytic system. This raises the possibility that antibody may be required for efficient alternative pathway activity in vivo and that the pyogenic infections that occur in Hgamma individuals are due to inefficient activation and fixation of C3 through either the classical or alternative pathway.

The adjuvant muramyl dipeptide (MDP) has been shown to affect a number of macrophage functions in vitro. We studied the effect of subcutaneous injection of MDP into mice. Cultured peritoneal macrophages from treated mice displayed increased spreading, total cell protein, and specific activity of beta-glucosaminidase a constituent of macrophage lysosomes, and of lactate dehydrogenase. Generation of superoxide anion (O2-) by MDP-treated macrophages stimulated by contact with phorbol myristate acetate was enhanced by over fivefold to levels achieved by macrophages from bacillus Calmette-Guérin-infected mice. The enhancement in stimulated O2- release was noted by 1 h after injection of MDP, peaked by 3 h, and remained high for at least 48 h. Priming for enhancement of O2- release by MDP was similar in athymic nude mice and in normal littermates, suggesting that mature T lymphocytes are not involved in this MDP effect. Priming for enhanced stimulated O2- release, and morphologic and enzymic changes, were not achieved by injection of the D-D stereoisomer of MDP. Phagocytosis of Candida albicans was only slightly greater by macrophages from mice give MDP, but MDP-stimulated cells killed two times more C. albicans in vitro than did cells from untreated animals. When MDP was given 18 h before, simultaneously with, or 24 h after lethal infectious challenge with C. albicans, treated mice were protected compared with controls. These results suggest that injection of MDP effectively and rapidly activates macrophages in the recipient animal. This agent should serve as an important probe of macrophage physiology and, perhaps ultimately, as a means of enhancing host defense in humans.

Chronic granulomatous disease (CGD) is a diverse disorder characterized by several clinical presentations and a multitude of metabolic defects. The authors summarize the clinical features and current management of CGD, offer a definition of the disease based on molecular defects of the respiratory burst, and present their experience with trimethoprim/sulfamethoxazole prophylaxis.

Enhanced function of the respiratory burst, measured as stimulated release of superoxide anion (O2-) or hydrogen peroxide, characterizes activated macrophages. Activated macrophages undergo a decline in their capacity to release O2- (a deactivation) when placed in culture for 3 days. To better understand the molecular basis for the enhanced respiratory burst of activated macrophages, we explored the mechanisms underlying deactivation of activated mouse peritoneal macrophages. Deactivation was observed when the assay was performed in a physiologic Na+ buffer, and by day 3 of culture, release of O2- from activated macrophages stimulated with phorbol myristate acetate (PMA) was almost identical to that in resident (nonactivated) macrophages. In contrast, when the assay was performed in a buffer in which Na+ was replaced by K+, release of O2- from activated macrophages on day 3 was equal to or greater than that on day 0, suggesting that the enzyme responsible for the respiratory burst was not altered during culture. The number and affinity of PMA receptors were not changed during culture and were not affected by high external K+. Continuous assay of O2- release by coverslip-adherent macrophages in a cuvette indicated that the lag time between addition of stimulus and release of O2- was reduced, and the initial rate of O2- release was enhanced in K+ buffer. The potency of monovalent cations to support O2- release was K+ greater than Rb+ greater than choline+ greater than Cs+ = Na+ greater than Li+, suggesting that characteristics such as ionic radius or molecular size influence this effect, and the effect is not due simply to absence of Na+. Extracellular Ca2+ or Mg2+ was required for the maximal effect of high external K+, and enhancement by high K+ and divalent cations increased progressively during culture. These findings suggest that deactivation is caused primarily by changes in signal transduction from PMA receptors to the respiratory burst enzyme, rather than by changes in these receptors or the enzyme itself, and that signal transduction can differ in different macrophage populations.

Bacterial infections are the most frequent cause of death and an important factor of morbidity in sickle cell disease. A defect in oxidative metabolism of neutrophils from these patients has been reported as a possible cause for these infections. Since normal neutrophil functions are essential in the defense against bacteria, it seemed important to reassess the capacity of neutrophils from patients with sickle cell disease to undergo the metabolic events associated with phagocytic bactericidal activity. Accordingly, neutrophils from patients and controls were compared for their ability to reduce nitroblue tetrazolium dye, to activate the hexose monophosphate shunt, and to generate superoxide anion, hydrogen peroxide, and chemiluminescence. Patients' cells performed normally in each of these assays and, in addition, killed Staphylococcus aureus as well as did cells from controls. Thus, an abnormality of neutrophil oxidative metabolism cannot explain the propensity to bacterial infections in sickle cell disease.

Nuclear magnetic resonance studies in D2O (greater than 90%) with glutamic pyruvate transaminase (GTP) (2.6.1.2) demonstrate that this enzyme catalyzes the rapid exchange of both the alpha and beta hydrogens of L-alanine, the exchange of only one alpha hydrogen of glycine, and the beta hydrogens of pyruvate and fluoropyruvate. When the beta hydrogens of L-alanine undergo the enzyme-catalyzed exchange, the product may have 1, 2 or 3 of beta hydrogens exchanged. The exchange is stimulated by the addition of catalytic amounts of copartner of transaminations reaction. A mechanism is proposed for an extension of the conjugated system to include the alpha and beta carbons to explain the labilization of the beta hydrogens.

Contact between human neutrophils and aggregated immunoglobulin G bound to micropore filters has been studied as a model of the pathogenesis of tissue damage in immune complex disease. Contact with this surface, as well as with plain filters and polystyrene petri dishes, induced neutrophils to elaborate superoxide anion and hydrogen peroxide and to generate chemiluminescence, which has been attributed to singlet oxygen. Pretreatment of the cells with cytochalasin B decreased these activities but increased release of lysosomal beta-glucuronidase, suggesting that degranulation and the burst of oxygen metabolism that characterizes phagocytes are independently regulated functions. Toxic oxygen metabolites released from neutrophils are highly reactive and could mediate tissue injury at sites of inflammation.

We explored the relationship between phagocytosis and the triggering of oxidative metabolism using resident, lipopolysaccharide (LPS)-elicited, and bacille Calmette-Guérin (BCG)-activated murine peritoneal macrophages. Sheep erythrocytes (E) coated with IgG [E(IgG)], E coated with IgM and complement [E(IgM)C], and E treated with 1% glutaraldehyde (GE) were used as stimuli. All three types of macrophages released superoxide anion (O2-) during phagocytosis of E(IgG). All macrophage types phagocytosed E(IgM)C and GE but none were stimulated to release O2- during phagocytosis of these particles. Vigorous consumption of oxygen was also stimulated by the ingestion of E(IgG) but not by ingestion of E(IgM)C or GE. E(IgM)C did not scavenge the O2- released from macrophages during phagocytosis of E(IgG) or during exposure to phorbol myristate acetate, and further addition of IgG anti-E antibody to E(IgM)C or GE permitted optimal stimulation of macrophage O2- release by these particles. The capacity of macrophages to ingest E(IgM)C and GE without stimulating the respiratory burst raises the possibility that clearance of particulate matter not opsonized with specific IgG might be achieved without stimulation of the release of toxic oxygen metabolites, and, therefore, without the risk of oxidative damage to the phagocytic cell or surrounding tissue.

Human granulocytes accumulate 67Ga when incubated under anoxic conditions and exclude the isotope when oxygenated. When incorporated, 67Ga is associated with the lysosomal fraction of the granulocytes. The ability of granulocytes to exclude the isotope except under hypoxic conditions may explain 67Ga localization in abscesses and some tumor masses.

After in vitro exposure to lipopolysaccharide (LPS) or muramyl dipeptide (MDP), cultured resident mouse peritoneal macrophages were primed to display enhanced generation of superoxide anion (O2-) in response to stimulation by phorbol myristate acetate (PMA) or opsonized zymosan. Priming with LPS (1 microgram/ml) produced a sevenfold enhancement of PMA-stimulated O2- generation; priming was detected within 30 min and persisted for at least 4 d. Exposure to MDP (1 muM) primed the macrophages to double their O2- release; the response was first observed after 4 h and persisted for at least 3 d. The priming response was not observed with stereoisomers of MDP, which are inactive as adjuvants. LPS and MDP appeared to work directly on the macrophages rather than indirectly by interacting with adherent lymphocytes: (a) Addition of nonadherent cell populations that contained lymphocytes had no effect on the response. (b) The response was normal with cells from nude mice, which lack mature T lymphocytes. (c) Macrophages from C3H/HeJ mice, whose B lymphocytes fail to respond to LPS, were weak in their response to priming LPS; the addition of normal (C3Heb/FeJ) nonadherent cells had no effect on this weak response. (d) The macrophage-like cell line J774.1 also showed enhanced O2--generating capacity after a 4-h exposure to LPS or MDP. The O2--generating capacity of macrophages primed with LPS in vitro was equivalent to that previously observed with cells elicited in vivo by injection of LPS or activated by infection with Bacille Calmette-Guérin. The data suggest that previous exposure to bacterial products could prime macrophages to respond with increased production of toxic oxygen metabolites on contact with invading microorganisms or tumor cells.

Human blood monocytes, peritoneal macrophages and milk macrophages, when initially isolated, displayed a vigorous release of superoxide anion (0-2), following stimulation with phorbol myristate acetate (PMA). This result contrasts with behavior of mouse peritoneal macrophages, which produce a weak 0-2 response, unless the macrophages are activated by infection or are elicted by injection of inflammatory agents. Treatment of mouse cells in vitro with bacterial products such as lipopolysaccharide (LPS) or muramyl dipeptide (MDP) also "primes" them for a high 0-2 response. Exposure of human macrophages to LPS or MDP for 16 h failed to enhance their 0-2 response. Thus the human cells appeared to be already "primed" for a vigorous oxygen radical response. In this respect, human cells resembled mouse peritoneal macrophages that have been "activated" by infection. These results suggest that MDP or other macrophage "activators" may offer less protection against infection in normal humans than they do in mice.

Lidocaine is used extensively in coronary care units, yet the effect of lidocaine infusions on neutrophil function has not been known. Lidocaine and other local anesthetics impair leukocyte antibacterial functions when added in vitro. We found that lidocaine added to human neutrophils in vitro markedly impaired the release of superoxide anion (O2-) and the granule enzymes lysozyme and myeloperoxidase after stimulation by phorbol myristate acetate or opsonized zymosan. We then measured production of O2- during stimulation of neutrophils from eight normal subjects, five coronary artery disease patients not receiving lidocaine and 13 coronary artery disease patients receiving lidocaine infusions for at least 12 hr. Release of O2- by cells from lidocaine-treated patients (14.2 +/- 3.8 nmol/2.5 X 10(6) neutrophils per 15 min) was significantly lower than from cells of the normal subjects (78.4 +/- 7.2 nmol; P less than .001) and the coronary patients not receiving lidocaine (70.6 +/- 4.0 nmol; P less than .001). Bactericidal assays at a high concentration (2 mg/ml) of lidocaine demonstrated slight reductions in 2 hr killing rates for Escherichia coli (70% with lidocaine vs. 95% control). Inhibition by lidocaine of the release of toxic oxygen metabolites from neutrophils could potentially reduce infarct size in patients with acute myocardial infarction; but as there is only a slightly reduced ability to kill bacteria, increased susceptibility to infections is unlikely although it cannot be excluded.

Human neutrophils were isolated from peripheral blood by four methods: 1) Ficoll-Hypaque gradients and erythrocyte lysis, 2) plasma-Percoll gradients, 3) a "lipopolysaccharide (LPS)-free" method yielding 85% neutrophils, and 4) by centrifugation of cells prepared by Method 3 through a plasma-Percoll gradient to produce pure neutrophils. The use of the Ficoll-Hypaque method resulted in spontaneous change of cell shape, enhanced formyl-methionyl-leucyl-phenylalanine (FMLP)-stimulated release of superoxide anion, increased release of lysosomal enzymes upon subsequent FMLP stimulation, and reduced chemotactic responsiveness, by comparison with the other methods. These effects were not due to erythrocyte lysis by NH4C1 but were reproduced by exposure of neutrophils prepared by the "LPS-free" method or the use of plasma-Percoll gradients to 10-100 ng/ml LPS. Neutrophil change of shape and stimulated O-2 production were particularly sensitive markers of these effects. The effects of trace concentrations of LPS in the modulation of neutrophil function may have relevance to the pathophysiology of endotoxemia and its resultant tissue injury.

Inflammatory macrophages elicited from the peritoneal cavity of mice injected with endotoxin can avidly ingest E opsonized with IgG antibody (EIgG) or with IgM antibody and C (EIgMC). However, only ingestion of EIgG is associated with activation of the respiratory burst and release of superoxide anion. We compared the endogenous phosphorylation of proteins from macrophages stimulated by interaction with EIgG or EIgMC on the premise that proteins phosphorylated after stimulation by EIgG but not EIgMC could play a role in activating the enzyme (oxidase) responsible for the respiratory burst. Proteins were separated by one-dimensional and two-dimensional electrophoresis in polyacrylamide gels. We found that proteins with approximate Mr of 20 kDa, 23 kDa, 46 kDa, 48 kDa (three proteins), 67 kDa, and 130 kDa were more heavily phosphorylated after EIgG stimulation than after EIgMC stimulation. Exposure to PMA, which activates the respiratory burst oxidase, induced phosphorylation of the 23-kDa, 48-kDa group, and 130-kDa proteins that were phosphorylated after stimulation by EIgG. Activity of protein kinase C was found to be significantly increased in the particulate fraction of macrophages stimulated by EIgG but not in the particulate fraction of EIgMC-stimulated cells. These data are compatible with the hypotheses that phosphorylation of specific cellular proteins, especially with a Mr of approximately 48 kDa, is involved in activation of the respiratory burst oxidase, and that function of protein kinase C also plays a part in this activation process.

Mouse macrophages can be primed by exposure in vitro to the bacterial products lipopolysaccharide and muramyl dipeptide (MDP) or in vivo by injection of MDP, so that they produce more of the bactericidal agent superoxide anion (O2-) when stimulated by phagocytosis or by contact with phorbol myristate acetate (PMA). Because little is known about the physiology of human tissue macrophages, we examined release of O2- by milk macrophages obtained from 45 normal women for the ability to undergo priming for greater O2- release. In samples from the same individuals, PMA-stimulated O2- release was similar from colostrum (0 to 3 days postpartum) or from transitional milk (5 to 8 days). Release of O2- by milk macrophages was almost identical to that by blood monocytes from the same women. Milk macrophages phagocytized and killed Candida albicans relatively effectively. Incubation with lipopolysaccharide activated the macrophages in that they were primed for greater PMA-stimulated O2- release. Incubation with the adjuvant MDP or its analog 6-O-(2-tetradecylhexadecanoyl)-MDP did not prime, but incubation with a second analog, 6-O-(stearoyl)-MDP, primed the macrophage for greater O2- release. These results indicated that human tissue macrophages can be primed for greater oxidative response by exposure to bacterial products. Potential exists for the therapeutic use of such immunomodulating agents in the enhancement of host defense.

Whether and how lidocaine reduced infarct size in a canine model of ischemia and reperfusion was investigated. Twenty dogs underwent a 90-min left anterior descending artery ligation and 300 min of reperfusion. Infarct size was measured by triphenyl tetrazolium chloride and the region at risk by 99Tc-labeled albumin microspheres injected during ischemia. In 10 dogs, lidocaine (70 micrograms/kg/min i.v.) was infused 90 min prior to and during ischemia and reperfusion, while 10 dogs were untreated. The ratio of infarct to risk area was 35.2 +/- 3.4% (SEM) in lidocaine dogs vs. 48.5 +/- 5.3% in untreated dogs (p less than 0.05). Lidocaine did not reduce neutrophil accumulation in ischemic and reperfused myocardium at 5 h of reperfusion, inhibit stimulated neutrophil superoxide production, or scavenge superoxide in vitro. However, during early reperfusion, lidocaine reduced coronary sinus levels of a lipid peroxidation product (conjugated dienes). Thus, clinically relevant lidocaine infusion rates reduced myocardial infarct size when given prior to and during ischemia and reperfusion. This protective effect may be due to lidocaine's membrane stabilizing effects, which could have protected the myocardial cell membrane from lipid peroxidation.