Alveolar fluid neutrophil elastase activity in the adult respiratory distress syndrome is complexed to alpha-2-macroglobulin.

We characterized the elastase and antielastase activity of the alveolar fluid of seven patients with the adult respiratory distress syndrome (ARDS) and thirteen normal volunteers. Alpha-1-antitrypsin (A1AT) concentrations were 60-fold higher in ARDS as compared to normal lavage fluid (2,140 +/- 498 nM; 36.1 +/- 4.2 nM, respectively). ARDS fluid antineutrophil elastase activity was also considerably higher than that of normals (979 +/- 204 nM; 31.3 +/- 2.9 nM, respectively). Despite the antineutrophil elastase excess, 5 of 7 ARDS lavage samples contained elastase activity (mean, 6.1 +/- 2.4 pM) as assayed using low-molecular-mass substrate, while only 1 of 13 normal subjects had detectable elastase activity (0.2 pM) (P less than 0.01, compared with ARDS). That this activity was due to alpha-2-macroglobulin (A2MG)-complexed neutrophil elastase was evidenced by (a) the Sephadex G-75 elution profile; (b) the inactivity against insoluble [3H]elastin; (c) the inhibitory profile with phenylmethylsulfonyl fluoride, methoxy-succinyl-alanyl-alanyl-prolyl-valyl-chloromethylketone, ethylene diamine tetraacetic acid, and A1AT; and (d) the immobilization by A2MG antibody bound to polystyrene plates. Furthermore, in agreement with the predicted affinity of A1AT and A2MG for neutrophil elastase, the ratio of A2MG to A1AT in the fluid (0.57%) coincided with the ratio of the A2MG- to A1AT-complexed elastase (0.36%). These findings suggest that the net lung protease-antiprotease balance in ARDS is shifted largely in favor of the antiproteases (chiefly A1AT), and that the antiproteases, A1AT and A2MG, have similar affinities for neutrophil elastase in vivo.

Sandwich ELISA formats designed to detect 17 kDa IL-1 beta significantly underestimate 35 kDa IL-1 beta.

The IL-1 beta precursor (proIL-1 beta) represents a significant component of total IL-1 beta production in certain cell types such as keratinocytes, fibroblasts and alveolar macrophages. It has been presumed that immunodetection systems for the mature 17 kDa IL-1 beta can be used interchangeably for the 35 kDa intracellular proIL-1 beta. However, during attempts to purify alveolar macrophage proIL-1 beta, we found that conventional enzyme-linked immunoassays (ELISAs) (using antibodies directed against the 17 kDa mature IL-1 beta) underestimated the amounts of 35 kDa proIL-1 beta by at least ten-fold compared to detection by Western blot techniques. This difference was due to the fact that ELISAs, with an antigen capture format (i.e., that use more than one epitope), can more readily see these distinct epitopes on mature or partially processed IL-1 beta than on the proIL-1 beta molecule. This problem does not occur with the Western blot technique, either because only one antibody is needed and hence there is no stearic blockade of a second epitope or because it denatures 35 kDa proIL-1 beta during the immobilization step, presumably better exposing epitopes as expressed on mature 17 kDa IL-1 beta. The problem with the ELISA can be partially corrected by proteolytic removal of the aminoterminus of 35 kDa proIL-1 beta with neutrophil elastase. More accurate determinations of proIL-1 beta by ELISA can be made by using 35 kDa proIL-1 beta as the reference standard (when the 35 kDa proIL-1 beta is free of molecular weight IL-1 beta). These data suggest that there are conformational differences between the carboxyterminus of 35 kDa proIL-1 beta and mature 17 kDa IL-1 beta which may affect immunodetection when using antibodies directed against mature 17 kDa IL-1 beta.

Effect of dimethylthiourea on the neutrophil myeloperoxidase pathway.

The sulfur-centered compound dimethylthiourea (DMTU) affords antioxidant protection in animal models of acute lung injury, an effect that has been attributed to its OH. scavenging properties. Although DMTU can also react with H2O2 in certain experimental systems, the effect of DMTU on the neutrophil myeloperoxidase (MPO) pathway has not been studied. DMTU (1-10 mM) completely blocked stable oxidants and hypochlorous acid formation by phorbol myristate acetate- and zymosan-stimulated neutrophils. DMTU also provided complete inhibition when incubated with cell-free supernatants after the formation of the MPO products. DMTU prevented the oxidative inactivation of alpha 1-antitrypsin by neutrophil-stable oxidants. Evidence that DMTU was oxidized by the MPO products was obtained by titration of oxidized DMTU with reduced glutathione. Surprisingly, supernatants from cells incubated with DMTU (10 mM) consumed two- to threefold higher amounts of reduced glutathione than supernatants from cells incubated with taurine (15 mM). Metabolic studies with stimulated neutrophils and experiments with the MPO enzyme system in a cell-free system suggested that DMTU acts by scavenging the products of the MPO pathway rather than by blocking H2O2 production in the intact cell. These findings demonstrate that DMTU blocks the neutrophil MPO pathway in addition to its known ability to scavenge other reactive O2 species. The capacity of DMTU to scavenge MPO products may explain some of its protective effects in acute lung injury.

Effect of O2 partial pressure on the myeloperoxidase pathway of neutrophils.

Neutrophils recruited to different tissues undergo respiratory burst activity at widely different PO2 levels. The present study investigated the in vitro effects of PO2 on neutrophil oxidative metabolism. When neutrophils were stimulated with either zymosan or phorbol myristate acetate (PMA) under different PO2's (0-700 Torr), hexose monophosphate shunt activity, H2O2, and hydroxyl radical (OH.) production were directly related to the level of PO2. Neutrophils functioned surprisingly well at PO2's as low as 10 Torr, where metabolic burst activity was prolonged and usually exceeded 50% of maximal values. The production of neutrophil stable oxidants and hypochlorous acid (HOCl) by zymosan-stimulated neutrophils was also directly related to PO2. In contrast, the production of stable oxidants and HOCl by PMA-stimulated neutrophils was significantly higher at 10 Torr compared with 700 Torr. The decrease in stable oxidant production by PMA-stimulated neutrophils at elevated PO2's was explained by both increased destruction of stable oxidant products and by decreased availability of the precursor HOCl. Superoxide dismutase and the OH. scavenger benzoate partially prevented the fall in stable oxidants at elevated PO2's. Measurements of stable oxidants in PMA-stimulated supernates generated at 10 and 700 Torr correlated with the ability of these supernates to decrease the elastase inhibitory capacity of the serum antiprotease alpha 1-antitrypsin. These findings suggest that different PO2's alter the magnitude and pattern of neutrophil oxidative metabolism.

Effect of TNF alpha production inhibitors BRL 61063 and pentoxifylline on the response of rats to poly I:C.

BRL 61063 is a novel xanthine phosphodiesterase (PDE) type IV inhibitor with selective inhibitory activity for tumor necrosis factor (TNF) alpha production. This compound inhibits TNF alpha production by activated human blood monocytes in vitro and in animal models of endotoxemia and influenza infection. Inhibition of TNF alpha may be beneficial in many diseases; however, little is known about potential adverse effects of such inhibition on host defense. In an ex vivo study, we examined the effect of BRL 61,063 on the microbicidal and tumoricidal activity of pulmonary lavage cells during a local inflammatory response in rats challenged with Poly I:C. Pentoxifylline, a PDE inhibitor which also blocks TNF alpha production, was used for comparison. Treatment with BRL 61063 or pentoxifylline did not block the inflammatory response to Poly I:C or the activation of bronchoalveolar lavage (BAL) cells but reduced the level of tumoricidal activity attained. At the dosages used, pentoxifylline was more inhibitory than BRL 61063. Drug treatment did not prevent further stimulation of tumoricidal activity by LPS in vitro. LPS-stimulated cells from BRL 61063-treated rats reached a level of activation similar to the control group while the LPS-stimulated activity of BAL cells from pentoxifylline treated rats remained lower than control. Although pentoxifylline was more inhibitory for tumoricidal activity than BRL 61063, the latter was a more potent inhibitor of TNF alpha release as measured in vivo in LPS-challenged rats. This finding indicates that TNF alpha is not the main mediator involved in the activation of pulmonary macrophage tumoricidal function. Treatment with either BRL 61063 or pentoxifylline had little or no effect on the Poly I:C-induced candidacidal activity of BAL cells indicating that these compounds are unlikely to compromise non-specific host defense against infection.

Practical aspects of including functional endpoints in developmental toxicity studies. Case study: immune function in HuCD4 transgenic mice exposed to anti-CD4 MAb in utero.

Keliximab is a human-cynomolgus monkey chimeric (Primatized) monoclonal antibody with specificity for human and chimpanzee CD4. As the preclinical safety assessment of biopharmaceuticals requires evaluation in pharmacologically responsive species, comprehensive toxicology studies, including reproductive toxicity, of this antibody were conducted in a human CD4 transgenic mouse model. The reproductive toxicology studies included a pre- and postnatal development study that incorporated immunotoxicological evaluation of offspring (F1) mice. The potential effects of exposure to treating maternal mice (F0) with keliximab during pregnancy and lactation on offspring viability, physical growth, neurobehavioral development, reproductive function, lymphoid tissue morphological structure, lymphocyte subsets and host resistance to Candida albicans infection were assessed. The results showed no impairment of these functions. The use of F1 transgenic mice in study with keliximab provides an example of a novel practical approach to assess developmental immunotoxicity within a study of pre- and postnatal development designed in accordance with ICH Guidelines.

Preclinical development of keliximab, a Primatized anti-CD4 monoclonal antibody, in human CD4 transgenic mice: characterization of the model and safety studies.

The preclinical safety assessment of biopharmaceuticals necessitates that studies be conducted in species in which the products are pharmacologically active. Monoclonal antibodies are a promising class of biopharmaceuticals for many disease indications; however, by design, these agents tend to have limited species cross-reactivity and tend to only be active in primates. Keliximab is a human-cynomolgus monkey chimeric (Primatized) monoclonal antibody with specificity for human and chimpanzee CD4. In order to conduct a comprehensive preclinical safety assessment of this antibody to support chronic treatment of rheumatoid arthritis in patients, a human CD4 transgenic mouse was used for chronic and reproductive toxicity studies and for genotoxic studies. In addition, immunotoxicity studies were conducted in these mice with Candida albicans, Pneumocystis carinii and B16 melanoma cells to assess the effects of keliximab on host resistance to infection and immunosurveillance to neoplasia. The results of these studies found keliximab to be well tolerated with the only effects observed being related to its pharmacologic activity on CD4+ T lymphocytes. The use of transgenic mice expressing human proteins provides a useful alternative to studies in chimpanzees with biopharmaceutical agents having limited species cross-reactivity.

Alveolar macrophages differ from blood monocytes in human IL-1 beta release. Quantitation by enzyme-linked immunoassay.

Fresh human alveolar macrophages and blood monocytes were stimulated with LPS and assessed for their ability to produce and release antigenic IL-1 beta. Using a sensitive and specific ELISA for IL-1 beta, monocytes released 13.3 +/- 3.1 ng/10(6) cells compared to 3.5 +/- 0.8 ng/10(6) cells for alveolar macrophages (p less than 0.01). To investigate the reason for this difference in IL-1 beta release, monocytes were compared to alveolar macrophages for total IL-1 beta production (i.e., the amount released plus that detected in the lysates). Monocytes produced a total of 19.0 +/- 3.2 ng/10(6) cells whereas alveolar macrophages produced 24.8 +/- 5.6 ng/10(6) cells (p = 0.37). The relative increase in alveolar macrophage intracellular IL-1 beta was confirmed by Western blot analysis of cell lysates. Thus, the limitation in IL-1 release from alveolar macrophages appears to be due to a decrease in the processing and release of the IL-1 beta precursor. In addition, TNF production studies demonstrated that the limitation in IL-1 release was not a generalized defect. In contrast to the IL-1 beta data, when TNF was measured from monocytes and macrophages, monocytes released only 14.6 +/- 3.4 ng/10(6), whereas macrophages released 101 +/- 30 ng/10(6) (p less than 0.02). In this same context, when fresh monocytes were allowed to mature in vitro they took on monokine production characteristics similar to alveolar macrophages. In vitro matured monocytes had a greater than 20-fold decrease in their ability to release IL-1 beta and a 6- to 8-fold increase in their ability to release TNF. Taken together, these studies suggest that IL-1 beta release is limited in mature mononuclear phagocytes as compared to fresh blood monocytes, and furthermore, that IL-1 beta regulation differs significantly from that of TNF-alpha.

ELISA detection of IL-1 beta in human sera needs independent confirmation. False positives in hospitalized patients.

The detection of cytokines in human sera has become an accepted index of disease activity in various diseases, including sepsis. However, little attention has been paid to the specificity of these measurements. Using a sensitive sandwich enzyme-linked immunoassay (ELISA), we studied IL-1 beta detectability in sera from 419 serum samples randomly obtained from our clinical laboratory. In initial studies, 6.7% of samples were positive (n = 24, 0.1 to 1.0 ng/ml, and n = 5, 1 to 80 ng/ml). However, attempts to further characterize positive samples revealed that over 90% were falsely positive. For example, samples fractionated on Sephadex G-75 demonstrated IL-1 beta "detectability" near the void volume, and negative samples, spiked with rIL-1 beta, eluted at approximately 17 kD. To determine if this detectability was due to heterophillike antibodies, 23 of 29 "positive" samples were retested in the presence of nonimmune mouse serum. Only 2 of 23 previously positive samples were still positive. Importantly, mouse serum had no effect upon normal human serum spiked with rIL-1 beta. Furthermore, blinded samples sent to a reference laboratory also demonstrated false positive IL-1 beta detection in selected samples. Taken together, these data demonstrate that the presence of nonspecific immunoactivity in sera may confound cytokine assays of human biologic material and suggest that, when possible, a second means of confirming ELISA-positive samples be used.

Colchicine has opposite effects on interleukin-1 beta and tumor necrosis factor-alpha production.

To study the role of microtubules in cytokine production, the effect of the microtubule depolymerizing agent colchicine on lipopolysaccharide endotoxin (LPS)-induced interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) release by blood monocytes and alveolar macrophages were examined. Immunofluorescence microscopy demonstrated that LPS resulted in the appearance of microtubule-containing cytoplasmic appendages and that colchicine, which resulted in microtubule disruption in monocytes, blocked appendage formation. Colchicine resulted in approximately 50% increase in LPS-induced IL-1 beta release and a 50% decrease in LPS-induced TNF-alpha release by human monocytes at all doses of LPS tested. Although colchicine resulted in a statistically significant increase in LPS-stimulated human alveolar macrophage IL-1 beta release, the increase was not as great as that observed with monocytes. Northern blot analysis suggested that the colchicine effect occurs pretranslationally because colchicine caused an increase in LPS-stimulated IL-1 beta mRNA levels and a decrease in TNF-alpha mRNA levels. These results suggest that microtubules contribute to the regulation of endotoxin-stimulated mononuclear phagocyte cytokine production and that this regulation differs significantly between IL-1 beta and TNF-alpha.

Comparison of smoker and nonsmoker lavage fluid for the rate of association with neutrophil elastase.

Cigarette smoking may impair the lung antiprotease screen. To test this hypothesis, the lung lining fluid from 10 smoking and 9 nonsmoking individuals was evaluated for its ability to inhibit neutrophil elastase and porcine pancreatic elastase. To eliminate the possibility of concentration- or purification-induced artifact, unconcentrated bronchoalveolar lavage fluid was used for all experiments. Smokers did not differ significantly from nonsmokers in the antigenic alpha-1-antitrypsin (alpha 1AT) concentrations (0.67 +/- 0.04 versus 0.73 +/- 0.09 nmol alpha 1AT/mg protein), in the neutrophil elastase inhibitory capacity (NEIC) (0.59 +/- 0.03 versus 0.52 +/- 0.05 nmol NEIC/mg protein), or in the porcine pancreatic elastase inhibitory capacity (PPEIC) (0.36 +/- 0.03 versus 0.42 +/- 0.05 nmol PPEIC/mg protein). In contrast, when the PPEIC/NEIC ratio was evaluated, smoker lung lining fluid exhibited a relative defect (0.64 +/- 0.06 smokers versus 0.80 +/- 0.05 nonsmokers, P less than 0.05). In agreement with the smokers' defect in the PPEIC/NEIC ratio, the kinetics of association (Ka) of lung lining fluid for neutrophil elastase was 0.38 +/- 0.3 x 10(7) M-1 s-1 from smokers and 0.58 +/- 0.05 x 10(7) M-1 s-1 from nonsmokers (P less than 0.002). Thus for a given amount of neutrophil elastase, smoker lung lining fluid took approximately 1.5 times longer to inhibit neutrophil elastase. These findings suggest that cigarette smoking is associated with a subtle defect in the antiprotease screen of the lower respiratory tract, recognizable by time-dependent measures of anti-neutrophil elastase function.

Macrophage and monocyte IL-1 beta regulation differs at multiple sites. Messenger RNA expression, translation, and post-translational processing.

Maturation of blood monocytes into macrophages is accompanied by a number of functional changes including decreased IL-1 beta release in response to LPS. This limitation has previously been ascribed to transcriptional regulation. However, in seeming conflict with the observed depression in IL-1 beta mRNA levels, recent work demonstrates increased intracellular IL-1 beta in macrophages. Therefore, the present study sought to explain these differences by comparing IL-1 beta production from autologous alveolar macrophage and blood monocyte pairs at multiple regulatory sites, including endotoxin responsiveness, mRNA expression, protein translation, and post-translational processing. Macrophages did not differ from monocytes in endotoxin sensitivity, but when analyzed by both ELISA and Western blot, were confirmed to have limitations in IL-1 beta release. Gene expression studies demonstrated that at 4 h, macrophage IL-1 beta steady state mRNA levels were 3-fold lower than the monocyte's. However, total IL-1 beta protein production, as measured by [35S]methionine labeling with immunoprecipitation, demonstrated three- to sixfold higher amounts in macrophages at comparable time points. The enhanced protein production in the face of relatively low mRNA levels suggests that macrophages translate IL-1 beta mRNA more efficiently. Furthermore, characterization of IL-1 beta release into supernatants revealed that whereas monocyte release occurred early, represented 5 to 20% of the intracellular amounts, and contained largely processed IL-1 beta, macrophage release was delayed, represented 1 to 5% of the intracellular amounts, and contained primarily unprocessed IL-1 beta. Taken together, these data demonstrate that the limitations in alveolar macrophage IL-1 beta release occur due to slower export and conversion of 35- to 17-kDa protein and are not due to differences in sensitivity to endotoxin or to transcriptional control mechanisms.

Tumor necrosis factor increases the elastolytic potential of adherent neutrophils: a role for hypochlorous acid.

Neutrophils adhered to biologic surfaces exhibit proteolytic cleavage of surface proteins even in the presence of proteinase inhibitors. Such proteolysis is restricted to the pericellular space and appears to require the dual action of proteinases and reactive oxygen species. The present study was designed to investigate the mechanism by which tumor necrosis factor-alpha (TNF) stimulates neutrophil proteolysis. Tissue culture wells were coated with insoluble 3H-labeled elastin substrate. Human blood neutrophils (0.5 to 2.0 x 10(6) cells/ml/well) were incubated in the coated wells for 4 to 18 h at 37 degrees C in the presence of varying concentrations of serum or purified alpha 1-antitrypsin (A1AT). TNF (0 to 1,000 U/ml) was also present in the incubations. Elastin degradation was determined as soluble 3H-elastin fragments released into the supernatants. As previously reported, cells (no TNF) exhibited spontaneous elastolysis even in the presence of 1% serum or 4 microM AlAT. Compared with cells incubated alone (no TNF), TNF increased elastolysis 3-fold in the 4-h incubations and 83% in 18-h incubations. TNF also significantly increased proteolysis when neutrophils were concurrently treated with phorbol myristate acetate or N-formylmethionylleucylphenylalanine. Since TNF is known to prime neutrophils for hypochlorous acid (HOCl) release, the present study hypothesized that the enhancement of proteolysis by TNF was related to increased release of HOCl. First, TNF caused a 4-fold increase in HOCl release by neutrophils adhered to elastin surfaces. Second, the effect of methionine on elastolysis by adherent neutrophils was studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Human whole blood interleukin-1-beta production: kinetics, cell source, and comparison with TNF-alpha.

Because removal of monocytes from their natural milieu may alter their subsequent immune response patterns, we have compared the production of interleukin-1 beta (IL-1-beta) and tumor necrosis factor alpha (TNF-alpha) by cultured human whole blood to that by purified monocytes. IL-1-beta was released in a dose-dependent fashion by whole blood after stimulation with lipopolysaccharide. Immunofluorescence studies indicated that monocytes were the main producers of IL-1-beta in whole blood cultures. On a per monocyte basis, after stimulation with 10 micrograms/ml lipopolysaccharide, much more IL-1-beta was released by cultured whole blood (56.2 +/- 8.3 ng/10(6) monocytes) than by purified mononuclear cells maintained in tissue culture medium (7.1 +/- 2.6 ng/10(6) monocytes). However, maintaining purified mononuclear cells in autologous plasma restored IL-1-beta release to levels observed in whole blood cultures. IL-1-beta release by whole blood peaked at 9 to 12 hours, in contrast to release of TNF-alpha, which peaked at 6 hours. In parallel with protein production, IL-1-beta messenger RNA levels peaked later and were more sustained than TNF-alpha messenger RNA levels. These experiments suggest that plasma augments the stimulatory effect of endotoxin and that IL-1-beta and TNF-alpha have differing kinetics in whole blood.

Tumor necrosis factor primes neutrophils for hypochlorous acid production.

Tumor necrosis factor (TNF) has a weak direct effect on neutrophil oxidative metabolism and primes neutrophils for oxidant release in response to other stimuli. We examined the effect of recombinant human TNF alpha (rTNF alpha) on production of hypochlorous acid (HOCl) by human neutrophils. TNF alone, even at concentrations of 1,000 U/ml, did not stimulate HOCl production. In contrast, rTNF alpha, in a dose-dependent manner, primed neutrophils for HOCl production in response to the weak agent unopsonized zymosan. rTNF alpha concentrations as low as 10 U/ml resulted in a fivefold increase in HOCl in this system. rTNF alpha-primed cells also exhibited increased phagocytosis. Priming in this model system occurred regardless of whether cells were preincubated with rTNF alpha before addition of zymosan or coincubated with both rTNF alpha and zymosan. rTNF alpha priming for HOCl production could not be washed away and required a lag period of approximately 10 min. rTNF alpha priming was not dependent on extracellular Ca2+ and Mg2+. Preincubation experiments demonstrated that rTNF alpha priming was not inhibited by the microfilament blocker cytochalasin B. Although the mechanism remains unclear, these findings demonstrate that rTNF alpha has an important priming effect on the neutrophil myeloperoxidase pathway.

Comparative pharmacodynamics of keliximab and clenoliximab in transgenic mice bearing human CD4.

Keliximab and clenoliximab are monkey/human chimeric CD4 monoclonal antibodies (mAbs) of the IgG1 and IgG4 isotypes, respectively. The pharmacokinetics (PK) and pharmacodynamics (PD) of these mAbs were evaluated in transgenic mice bearing human CD4 molecules on their T cells after a single i.v. administration at three dose levels (5-125 mg/kg). The PK of keliximab and clenoliximab were similar, dose-dependent, and adequately described by a two-compartment model with saturable elimination from both compartments. The enumeration of circulating CD4(+) T cells and density of CD4 on their surface were determined as the PD effects. An indirect response model was proposed to characterize the PD effects. With the increase in mAb dose, the maximum intensity (R(max)) of PD effects was increased, and the time to reach R(max) shifted to later times. At all three dose levels, keliximab caused a relatively rapid decline in the number of circulating CD4(+) T cells, which then recovered gradually. In contrast, clenoliximab at the lowest dose (5 mg/kg) did not produce a significant effect on CD4(+) T cell counts compared with the placebo group. At high doses, clenoliximab caused a significant decrease in the number of CD4(+) T cells. Keliximab appeared to be more potent and efficient in depleting CD4(+) T cells. Both mAbs produced similar down-modulation of CD4 at corresponding dose levels. The findings of this study are consistent with the results of a recent clinical trial that emphasize the importance of this transgenic mouse model for evaluating PK/PD to support clinical development of anti-human CD4 mAbs.

Flow cytometry in the preclinical development of biopharmaceuticals.

Novel biomarkers are often required in the preclinical development of biopharmaceuticals in order to characterize pharmacologic and toxicologic effects and to establish pharmacodynamic and pharmacokinetic relationships. Flow cytometry is uniquely suited for measurement of these biomarkers. Large numbers of single cells in a heterogeneous population can be rapidly identified and characterized with high accuracy and reproducibility. Cells are not damaged by the detection system and can be subsequently sorted for further morphologic or functional analysis. The availability of clinical instruments and a wide range of fluorescent probes have made this technology applicable for use in toxicologic clinical pathology. Flow cytometry has played an integral role in the development of a monoclonal antibody to human CD4 (keliximab, IDEC-CE9.1, SB 210396). Lymphocyte subset analysis and assays for expression, coating, and modulation of human CD4 were used for sequential assessment of the pharmacologic activity of keliximab in transgenic mice expressing human CD4.

Tumor necrosis factor infusions in humans prime neutrophils for hypochlorous acid production.

Five cancer patients undergoing intravenous infusions of human recombinant tumor necrosis factor (TNF)alpha were evaluated for the effects these infusions had on the priming of circulating neutrophils for hypochlorous acid (HOCl) production. These patients were also studied for changes in temperature, circulating white blood cell counts, blood pressure, and spontaneous monocyte interleukin 1 beta (IL-1 beta) and TNF production. As predicted by previous in vitro studies, patient neutrophils increased their HOCl production to unopsonized zymosan from a baseline of 29.2 +/- 5.9 nmol I- oxidized/4 x 10(6) cells to a peak of 64.2 +/- 9.8 nmol I- oxidized/4 x 10(6) cells at 4 h after TNF infusion (P less than 0.01). Similar increases were also seen at 4 h with phorbol myristic acetate and opsonized zymosan as the stimuli. The priming effect could be reproduced in neutrophils from a normal individual by incubating them with the 30-min serum samples from the infused patients. The ability of this serum to prime neutrophils was completely blocked by a monoclonal anti-TNF alpha-antibody but not by an anti-IL-1 beta antibody. In addition to the priming of their neutrophils, patients also experienced fever, marked hypotension, and an initial fall, followed by rebound to an elevation, in circulating white blood cell counts. The TNF infusions did not produce detectable circulating IL-1 beta nor did they induce significant production of TNF or IL-1 beta by circulating blood monocytes. These studies confirm the role of TNF in producing the signs of sepsis such as hypotension, fever, and leukopenia followed by leukocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)

The pharmacokinetics, antigenicity, and fusion-inhibition activity of RSHZ19, a humanized monoclonal antibody to respiratory syncytial virus, in healthy volunteers.

Single ascending doses of RSHZ19 (also known as SB 209763), a humanized monoclonal antibody (MAb) directed to the fusion protein of respiratory syncytial virus, were administered to healthy men to evaluate the safety, pharmacokinetics, antigenicity, and fusion inhibition (FI) activity of RSHZ19. Doses of RSHZ19 (0.025-10.0 mg/kg) or placebo were infused over 30 min, and subjects were followed for 10 weeks. Plasma concentrations of RSHZ19 and RSHZ19-specific antibodies were determined by ELISAs. FI titers were used to evaluate the ability of plasma to inhibit virus-induced fusion of VERO cells previously infected with RS Long strain virus. Twenty-six subjects, mean age 24, completed the study. RSHZ19 was safe and well tolerated, and no subject developed antibodies to RSHZ19 during follow-up. RSHZ19 had low plasma clearance and a half-life of approximately 23 days, similar to native IgG. Increases in FI titers relative to pretreatment levels were seen 24 h after MAb administration in all 4 subjects given 10 mg/kg and in 2 of 4 given 5 mg/kg.

Single-organism model of host defense against infection: a novel immunotoxicologic approach to evaluate immunomodulatory drugs.

The immunotoxicologic effects of drugs on host defense have been studied widely using various animal models of infection. Here we describe a new approach to testing host defense by using a single organism (Candida albicans) in CBA/J mice. The model is configured to test 3 effector systems via different routes of inoculation to stimulate different effector arms of the immune response. Nonspecific immunity was evaluated by C. albicans colony-forming unit (CFU) count from the spleen at 2 hr (uptake) and > or = 22 hr (clearance) following intravenous inoculation. Cell-mediated immunity was assessed by CFU count from an intramuscular injection site 6 days postinoculation. Humoral immunity was assessed by anti-Candida antibody titer, following multiple subcutaneous immunizations with C. albicans. Finally, overall immunity was evaluated following intravenous injection using survival as the endpoint. Histopathological, immunohistochemical, and electron microscopic evaluation of selected tissues revealed the involvement of the expected cell types in the different effector systems. Several immunomodulatory drugs--dexamethasone, cyclosporine, liposomal muramyltripeptide phosphatidylethanolamine, and SK&F 105685--were evaluated in the C. albicans model. Dexamethasone impaired host defense against C. albicans by suppressing all endpoints measured. Similarly, cyclosporine showed broad immunosuppressive activity, with the exception of yeast uptake from the spleen. In contrast, muramyl tripeptide-phosphatidylethanolamine enhanced all but cell-mediated immunity to C. albicans. SK&F 105685 displayed both stimulatory and inhibitory effects on immune responses to the infection. Our studies demonstrate that a single organism-based approach can be a useful method for evaluating the immunological hazards of drugs on host resistance to infection.