The ascidian Styela plicata hemocytes as a potential biomarker of marine pollution: In vitro effects of seawater and organic mercury.

Toxic metals, such as mercury, contribute substantially to anthropogenic pollution in many estuarine environments. Animals living in those environments, particularly invertebrate filter feeders like tunicates, can be used as bioindicators. In an attempt to identify cellular markers for revealing pollution, this study examined in vitro the effects of different concentrations of methyl mercury on Styela plicata hemocytes. The harvested hemocytes from S. plicata that were exposed to the metal had a significant mortality, cellular count and morphometric alterations. These findings provided evidence of MeHg immunotoxic effects on S. plicata, resulting in hemocyte death and morphological changes induced by cytoskeleton alterations. Thus, a morphometric cellular parameter, such as spreading ability, was used as a complementary method for differentiation between hemocytes treated with a marine solution (as a negative control) and hemocytes incubated with methylmercury and/or Sicilian seawater samples.

Do electrospray mass spectra of surfactants mirror their aggregation state in solution?

One important feature in the gas phase chemistry of surfactants is to ascertain whether their aggregates produced by electrospray ionization reflect those formed in the starting solution. With this aim, we have performed ESI-MS, ESI-MS/MS and ER-MS spectra of bis(2-ethylhexyl)sulfosuccinate (AOTNa) solutions in different solvents, i.e. water, water/methanol, methanol and n-hexane. The results clearly indicate that, notwithstanding the strongly different aggregation state in solution (direct micelles in water and in water/methanol, molecular dispersion in methanol and reverse micelles in n-hexane) and marked effects of the solvent polarity on the total ionic current, the surfactant aggregates in gas phase show identical structural features. Analogous conclusions can be drawn analyzing the infrared multiple photon dissociation (IRMPD) spectra of AOTNa solutions in water/methanol and n-hexane. Moreover, according to the idea that gas phase can be considered an apolar environment par excellence, data consistently suggest a reverse micelle-like aggregation. Some peculiarities of the mechanisms leading to aggregate formation through electrospray ionization of surfactant solutions in solvent media with different polarity have been also discussed.

Biological activities of a recombinant adenovirus p53 (SCH 58500) administered by hepatic arterial infusion in a Phase 1 colorectal cancer trial.

The major focus of intrahepatic arterial (IHA) administration of adenoviruses (Ad) has been on safety. Currently, there is little published data on the biological responses to Ad when administered via this route. As part of a Phase I study, we evaluated biological responses to a replication-defective adenovirus encoding the p53 transgene (SCH 58500) when administered by hepatic arterial infusion to patients with primarily colorectal cancer metastatic to the liver. In analyzing biological responses to the Ad vector, we found that both total and neutralizing Ad antibodies increased weeks after SCH 58500 infusion. The fold increase in antibody titers was not dependent on SCH 58500 dosage. The proinflammatory cytokine interleukin-6 (IL-6) transiently peaked within 6 h of dosing. The cytokine sTNF-R2 showed elevation by 24 h post-treatment, and fold increases were directly related to SCH 58500 doses. Cytokines TNF-alpha, IL-1beta, and sTNF-R1 showed no increased levels over 24 h. Predose antibody levels did not appear to predict transduction, nor did serum Ad neutralizing factor (SNF). Delivery of SCH 58500 to tumor tissue occurred, though we found distribution more predominantly in liver tissues, as opposed to tumors. RT-PCR showed significantly higher expression levels (P<0.0001, ANOVA) for adenovirus type 2 and 5 receptor (CAR) in liver tissues, suggesting a correlation with transduction. Evidence of tumor-specific apoptotic activity was provided by laser scanning cytometry, which determined a coincidence of elevated nuclear p53 protein expression with apoptosis in patient tissue. IHA administration of a replication defective adenovirus is a feasible mode of delivery, allowing for exogenous transfer of the p53 gene into target tissues, with evidence of functional p53. Limited and transient inflammatory responses to the drug occurred, but pre-existing immunity to Ad did not preclude SCH 58500 delivery.

Evaluation of standard and state of the art analytical technology-bioassays.

The development of biological assays for assessing potency is a critical component for monitoring the quality of therapeutic biologicals. Traditional cell-based bioassays, which are the most widely used, are typically based on a terminal cellular response such as cell proliferation or inhibition. While these assays can be very user-friendly, results often take days and sensitivity is sometimes not sufficient for the needs of the development programme. Recent improvements in analytical technology have led to new approaches in bioassay development. Many of these assays exploit cell signalling pathways far upstream from a terminal cellular response. Bioassays based on a cell signal are much more rapid, sensitive, and indicate stability better than their predecessors. Many of these newer assays are "hybrid" assays which combine the receptor signalling of traditional bioassays with the sensitivity of detection found in immunoassays. One such method, the Kinase Receptor Activation Assay (KIRA), works through the detection of receptor phosphorylation following analyte stimulation. Validations of newer technology assays, such as KIRA, require an individualized strategy due to their unique attributes. A thorough assessment of robustness should be paramount in the validation of these assays. Several examples of new technology platforms for bioassays are also discussed.

Bioassays for the measurement of neutralizing antibodies in serum (SNFs).

Bioassays to measure serum neutralizing factor (SNF) activity against therapeutic biological products are an important tool for assessment of the immune status of patients on a dosing regimen. Because of the large diversity of bioanalytical methodology currently available to measure neutralization of a biological response, approaches to assay development and validation can create a considerable challenge. Assay design can take on many forms depending on the biological response to be monitored and the severity of complicating serum effects. In addition, the manner in which assay results are reported should have clinical relevance, (e.g. related back to the amount of analyte neutralized per volume of serum, etc.). Although ICH/FDA assay validation guidelines exist, they were not created with biotechnology-based bioassays in mind. Therefore, a thorough assessment of the analytical method and proper interpretation of current guidelines is critical for a meaningful validation effort. Several assay development strategies and approaches to assay validation will be discussed.

Wastewater reuse in Italy.

In many parts of Italy, particularly in the South, it has become ever more difficult to meet the water demand. The recent years of drought and the constant increase of water demand for the civil sector have made irrigation supply more problematic. Wastewater reuse could represent a viable solution to meet water demand. The focus of this paper is on the regulation problems, hampering the development of wastewater reuse for irrigation, and on the potentials for reuse, particularly in Southern Italy. Planned exploitation of municipal wastewater could help meeting the irrigation water demand particularly in Southern Italy, where farmers have been practising uncontrolled wastewater reuse for a long time. In Northern and Central Italy, where available water resources generally meet water needs for different purposes, wastewater reuse could play an important role in controlling the pollution of water bodies. Despite the fact that Italian legislation is extremely strict and outdated, for several years in some regions, such as Sicily, wastewater reuse systems have been in operation; furthermore, several projects of wastewater reuse are currently in progress.

Development and validation of sensitive assays to quantitate gene expression after p53 gene therapy and paclitaxel chemotherapy using in vivo dosing in tumor xenograft models.

SCH58500 (ACN53) is a replication-deficient, type 5 adenovirus (Ad) expressing human wild-type p53 tumor suppressor. It is currently undergoing clinical trials as a cancer therapeutic. Many SCH58500 clinical trials incorporate an arm comparing traditional chemotherapy against chemotherapy combined with SCH58500. Paclitaxel was chosen for combination therapy in the preclinical study reported here due to its extensive use as a first-line therapy in ovarian cancer, its synergy with SCH58500 in preclinical cancer models, and its activation of p53-independent apoptosis, which might result in a "lowered threshold" for tumor cell death. SCID mice bearing human tumor xenografts were dosed with intratumoral vehicle, control Ad vector, or SCH58500, with or without paclitaxel. Real-time quantitative reverse transcriptase polymerase chain reaction assays were developed and validated to quantitate expression of p53, the p53 downstream effector gene p21, and the apoptosis-related genes, bax, bcl-2, and survivin. Protein expression was confirmed using immunohistochemical assays for p53 and p21. Only tumors injected with SCH58500 had detectable levels of exogenous p53 DNA and mRNA. After SCH58500 treatment, 3-11-fold elevations of p21 expression were observed in tumor xenografts containing nonfunctional p53 (MDA-MB-468, MDA-MB-231, MIAPaCa2, DU-145, and SK-OV-3), but no change in p21 mRNA in wild-type p53 PA-1 tumors. Immunohistochemical assays confirmed induction of p21 protein in MDAMB-468 and SK-OV-3 cells, but not in PA-1 cells. Ad vector alone or paclitaxel alone had no effect on p21 mRNA levels in most tumors. However, paclitaxel suppressed p21 expression induced by SCH58500 4-fold in DU-145 and SK-OV-3 tumors. Paclitaxel also affected expression of the housekeeping gene gapdh. There was no consistent pattern to the changes in bax, bcl-2, or survivin after SCH58500 treatment with or without paclitaxel between tumor types, although there were consistent responses within individual tumor lines. The mRNA ratios for bax/bcl-2 and bax/survivin were also not informative across tumor types. Of the genes examined, only p21 gave a predictable response 24 hours after p53 gene therapy and therefore, p21 expression may be useful for confirming SCH58500 activity in human tumor biopsies.

Design, characterization, and structure of a biologically active single-chain mutant of human IFN-gamma.

A mutant form of human interferon-gamma (IFN-gamma SC1) that binds one IFN-gamma receptor alpha chain (IFN-gamma R alpha) has been designed and characterized. IFN-gamma SC1 was derived by linking the two peptide chains of the IFN-gamma dimer by a seven-residue linker and changing His111 in the first chain to an aspartic acid residue. Isothermal titration calorimetry shows that IFN-gamma SC1 forms a 1:1 complex with its high-affinity receptor (IFN-gamma R alpha) with an affinity of 27(+/- 9) nM. The crystal structure of IFN-gamma SC1 has been determined at 2.9 A resolution from crystals grown in 1.4 M citrate solutions at pH 7.6. Comparison of the wild-type receptor-binding domain and the Asp111-containing domain of IFN-gamma SC1 show that they are structurally equivalent but have very different electrostatic surface potentials. As a result, surface charge rather than structural changes is likely responsible for the inability of the His111-->Asp domain of to bind IFN-gamma R alpha. The AB loops of IFN-gamma SC1 adopt conformations similar to the ordered loops of IFN-gamma observed in the crystal structure of the IFN-gamma/IFN-gamma R alpha complex. Thus, IFN-gamma R alpha binding does not result in a large conformational change in the AB loop as previously suggested. The structure also reveals the final six C-terminal amino acid residues of IFN-gamma SC1 (residues 253-258) that have not been observed in any other reported IFN-gamma structures. Despite binding to only one IFN-gamma R alpha, IFN-gamma SC1 is biologically active in cell proliferation, MHC class I induction, and anti-viral assays. This suggests that one domain of IFN-gamma is sufficient to recruit IFN-gamma R alpha and IFN-gamma R beta into a complex competent for eliciting biological activity. The current data are consistent with the main role of the IFN-gamma dimer being to decrease the dissociation constant of IFN-gamma for its cellular receptors.

Design and analysis of an engineered human interleukin-10 monomer.

A monomeric form of human interleukin 10 (IL-10M1) has been engineered for detailed structure-function studies on IL-10 and its receptor complexes. Wild type IL-10 (wtIL-10) is a domain swapped dimer whose structural integrity depends on the intertwining of two peptide chains. wtIL-10 was converted to a monomeric isomer by inserting 6 amino acids into the loop connecting the swapped secondary structural elements. Characterization of IL-10M1 by mass spectroscopy, size exclusion chromatography, cross-linking, and circular dichroism shows that IL-10M1 is a stable alpha-helical monomer at physiological pH whose three-dimensional structure closely resembles one domain of wtIL-10. As previously reported, incubation of wtIL-10 with a soluble form of the IL-10Ralpha (sIL-10Ralpha) generates a complex that consists of 2 wtIL-10 molecules and 4 sIL-10Ralphas. In contrast, IL-10M1 forms a 1:1 complex with the sIL-10Ralpha. Characterization of the interaction using isothermal titration calorimetry confirmed the 1:1 stoichiometry and yielded a dissociation constant of 30 nm with an apparent binding enthalpy of -12.2 kcal/mol. Despite forming a 1:1 complex, IL-10M1 is biologically active in cellular proliferation assays. These results indicate that the 1:1 interaction between IL-10M1 and IL-10Ralpha is sufficient for recruiting the signal transducing receptor chain (IL-10Rbeta) into the signaling complex and eliciting IL-10 cellular responses.

The use of laser scanning cytometry to assess depth of penetration of adenovirus p53 gene therapy in human xenograft biopsies.

SCH58500 is an agent for gene therapy of cancer, consisting of a replication-deficient type 5 adenovirus (Ad5) expressing the human p53 tumor suppressor gene (Ad5/p53). An important question about the use of Ad5/p53 gene therapy is how to achieve the therapeutically effective delivery of an Ad5/p53 vector to the tumor. We wanted to determine the effective depth of penetration of an Ad5/p53 vector by dosing the vector in an experimental human xenograft/SCID model. To assess depth of penetration, we developed a novel methodology for scanning tissue sections by laser scanning cytometry (LSC). SCID mice were given intraperitoneal injections of either p53(null) SK-OV-3 human ovarian tumor cells or p53(mut) DU-145 human prostate tumor cells to establish xenograft solid tumors. Mice were then dosed once or twice at 24-hour intervals by intraperitoneal injection with SCH58500 (Ad5/p53), an adenovirus construct expressing beta-galactosidase (Ad5/beta-gal), or a buffer control. Additional groups of mice received a single intraperitoneal dose of 10 mg/kg paclitaxel either alone or coadministered with Ad5/p53. Twenty-four hours after each last dose, the human solid tumor xenograft and relevant mouse tissue were removed from each mouse for the analysis of Ad5/p53 penetration. Immunohistochemistry (IHC) for beta-galactosidase protein revealed a depth of penetration of between 1 and 10 cells from the tumor surface. In some mice, hepatocytes in the periportal regions of liver lobules were also positive, indicating systemic absorption of adenovirus from the peritoneal cavity. IHC staining for p53 and p21 proteins in SK-OV-3 solid tumor xenografts revealed similar Ad/p53 penetration. LSC was used to map and quantitate apoptosis in both tumor and liver tissue biopsies, with over 450,000 nuclei from liver tissue and 150,000 nuclei from tumor tissue being evaluated. LSC analysis demonstrated a high level of apoptosis in the tumors that had been removed from Ad5/p53-dosed mice (12.7-19.7%). This level of apoptosis was significantly higher (P < 0.05) than was observed for liver tissues taken from Ad5/p53-dosed mice (2.7-8.0%) or tumor tissues taken from either Ad5/beta-gal-dosed mice (3.0-6.4%) or buffer control-dosed mice (3.0-5.3%). Scan bit maps from the extensive LSC analyses confirmed that apoptosis was present to about the same depth (1-10 cells) as had been identified by IHC for beta-galactosidase, p53, and p21 proteins. Paclitaxel coadministered with Ad5/p53 had no effect on Ad5 penetration into solid tumors in vivo as measured by IHC for p53 or p21 protein. However, the combination therapy did cause an elevation in the number of tumor cells undergoing apoptosis.

Effect of Sch 55700, a humanized monoclonal antibody to human interleukin-5, on eosinophilic responses and bronchial hyperreactivity.

This report describes the development and the biology of Sch 55700, a humanized monoclonal antibody to human IL-5 (hIL-5). Sch 55700 was synthesized using CDR (complementarity determining regions) grafting technology by incorporating the antigen recognition sites for hIL-5 onto consensus regions of a human IgG4 framework. In vitro, Sch 55700 displays high affinity (Kd = 20 pmol/l) binding to hIL-5, inhibits the binding of hIL-5 to Ba/F3 cells (IC50 = 0.5 nmol/l) and blocks IL-5 mediated proliferation of human erythroleukemic TF-1 cells. In allergic mice, Sch 55700 (0.1-10 mg/kg, i.p. or i.m.) inhibits the influx of eosinophils in the lungs, demonstrates long duration of activity and the anti-inflammatory activity of this compound is additive with oral prednisolone. In allergic guinea pigs, Sch 55700 (0.03-30 mg/kg i.p.) inhibits both the pulmonary eosinophilia and airway hyperresponsiveness and at 30 mg/kg, i.p. inhibited allergic, but not histamine-induced bronchoconstriction. In allergic rabbits, Sch 55700 blocks cutaneous eosinophilia. Sch 55700 (0.1-1 mg/kg i.p.) also blocks the pulmonary eosinophilia and neutrophilia caused by tracheal injection of hIL-5 in guinea pigs. In allergic cynomolgus monkeys, a single dose of Sch 55700 (0.3 mg/kg i.v.) blocks the pulmonary eosinophilia caused by antigen challenge for up to six months. Sch 55700 is, therefore, a potent antibody against IL-5 in vitro and in a variety of species in vivo that could be used to establish the role of IL-5 in human eosinophilic diseases such as asthma.

Applications for the new electrochemiluminescent (ECL) and biosensor technologies.

Biosensor and electrochemiluminescent (ECL) assays are replacing enzyme-linked immunosorbent assays (ELISAs) at Schering-Plough as immunoassays of choice to monitor cytokine levels and detect anti-cytokine antibody responses during cytokine therapy. These new assays provide increased sensitivity and a better correlation with biological assays. Biosensor assays using the BIACORE 2000 (BIACORE, Uppsala, Sweden) are being adopted to support preclinical and clinical trials for the detection of antibodies capable of binding to IL-10 and IL-4. Significant advantages when using a biosensor assay are that real-time and label-free detection permit increased throughput and direct detection of binding interactions which enables detection of low affinity antibodies that are not detected by ELISA. The ECL assays using the ORIGEN Analyser (IGEN, Gaithersburg, MD) that we have implemented to replace existing ELISAs for quantification of serum IL-10 and serum interferon alfa levels are more sensitive and less subject to matrix effects. Data obtained during the validation of these assays are described.

Characterization of replication-competent adenovirus isolates from large-scale production of a recombinant adenoviral vector.

Replication-deficient adenoviral vectors have been developed for the delivery of DNA sequences encoding a variety of proteins intended for the management of disease through gene therapy. One concern is the occurrence of replication-competent adenovirus (RCA) in the population of replication-deficient adenoviral vectors as a result of recombination or contamination. To address this concern, it is necessary to determine the frequency of occurrence and to fully characterize the molecular structure and biological infectivity of RCA. rAd/p53 is a pIX-deleted p53 gene therapy vector that is designed to lower the RCA occurrence and to deliver the tumor suppressor gene p53 for treatment of various cancers. Multiple preparations of the replication-deficient adenoviral vector rAd/p53 were tested for the presence of RCA, employing a sensitive biological assay. Single plaques from RCA-positive preparations of rAd/p53 were isolated for molecular characterization. All of the RCA isolates displayed a single unique structure that contains the complete E1 sequence of adenovirus type 5 but lacks the p53 sequence. The detailed sequence analysis of the RCA suggests that it is most likely generated as a result of recombination events between the rAd/p53 DNA and the 293 host adenoviral sequence. Results from viral infectivity analysis by flow cytometry demonstrate no substantial difference in infectivity of RCA, rAd/p53, and wild-type adenovirus type 5 in 293 cells.

Characterization of interleukin-10 receptors on human and mouse cells.

Human interleukin (IL)-10 is a pleiotropic cytokine acting on a variety of immune cells. Here we show that the protein can be enzymatically iodinated to high specific radioactivity with retention of biological activity. The radiolabeled ligand binds specifically to its receptor in several mouse and human cell lines, notably human B-lymphoma line JY and mouse mast cell line MC/9. Human IL-10 apparently binds as a dimer to a single class of receptor in both the JY and MC/9 cell lines with a Kd in the 50-200 pM range. Interestingly, mouse IL-10 was capable of blocking binding of human IL-10 to mouse but not human cells. There appears to be at most only a few hundred IL-10 receptors/cell for both mouse and human cell lines examined. Chemical cross-linking of the radioiodinated hIL-10 to JY and MC/9 cells revealed a common protein complex with an apparent molecular mass of about 97 kDa. Additional high molecular weight complexes were detected with JY but not MC/9 cells.

GM-CSF triggers a rapid, glucose dependent extracellular acidification by TF-1 cells: evidence for sodium/proton antiporter and PKC mediated activation of acid production.

The extracellular acidification rate of the human bone marrow cell line, TF-1, increases rapidly in response to a bolus of recombinant granulocyte-macrophage colony stimulating factor (GM-CSF). Extracellular acidification rates were measured using a silicon microphysiometer. This instrument contains micro-flow chambers equipped with potentiometric sensors to monitor pH. The cells are immobilized in a fibrin clot sandwiched between two porous polycarbonate membranes. The membranes are part of a disposable plastic "cell capsule" that fits into the microphysiometer flow chamber. The GM-CSF activated acidification burst is dose dependent and can be neutralized by pretreating the cytokine with anti-GM-CSF antibody. The acidification burst can be resolved kinetically into at least two components. A rapid component of the burst is due to activation of the sodium/proton antiporter as evidenced by its elimination in sodium-free medium and in the presence of amiloride. A slower component of the GM-CSF response is a consequence of increased glycolytic metabolism as demonstrated by its dependence on D-glucose as a medium nutrient. Okadaic acid (a phospho-serine/threonine phosphatase inhibitor), phorbol 12-myristate 13-acetate (PMA, a protein kinase C (PKC) activator), and ionomycin (a calcium ionophore) all produce metabolic bursts in TF-1 cells similar to the GM-CSF response. Pretreatment of TF-1 cells with PMA for 18 h resulted in loss of the GM-CSF acidification response. Although this treatment is reported to destroy protein kinase activity, we demonstrate here that it also down-regulates expression of high-affinity GM-CSF receptors on the surface of TF-1 cells. In addition, GM-CSF driven TF-1 cell proliferation was decreased after the 18 h PMA treatment. Short-term treatment with PMA (1-2 h) again resulted in loss of the GM-CSF acidification response, but without a decrease in expression of high-affinity GM-CSF receptors. Evidence for involvement of PKC in GM-CSF signal transduction was obtained using calphostin C, a specific inhibitor of PKC, which inhibited the GM-CSF metabolic burst at a subtoxic concentration. Genistein and herbimycin A, tyrosine kinase inhibitors, both inhibited the GM-CSF response of TF-1 cells, but only at levels high enough to also inhibit stimulation by PMA. These results indicate that GM-CSF activated extracellular acidification of TF-1 cells is caused by increases in sodium/proton antiporter activity and glycolysis, through protein kinase signalling pathways which can be both activated and down-regulated by PMA.

Effect of age on second messenger generation in neutrophils.

Neutrophils from healthy elderly donors generate significantly less diacylglycerol (DAG) and inositol triphosphate (IP3) than neutrophils from young donors, following stimulation by the chemotactic peptide, formyl-methionyl-leucylphenylalanine (FMLP). The defect in signal transduction occurred at a point proximal to the generation of IP3 and DAG, since the reduction in FMLP-induced superoxide generation was corrected if the intervening signal transduction steps were bypassed, either by priming with a substimulatory dose (1.62 nmol/L) of phorbol myristate acetate (PMA), by ionophore elevation of cytosolic calcium, or by using a stimulatory dose of PMA (1.62 mumol/L). FMLP receptor number and affinity were unaffected by aging. On FMLP activation, neutrophils from old, as compared with young, volunteers showed significantly greater and more long-lasting decreases in the concentrations of phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2). This indicates a reduction with age in the metabolically active precursor pools responsible for the generation of IP3 and DAG. In contrast, aging had little effect on the production of phosphatidic acid (PA), which has recently been suggested to serve as a major activator of the NADPH oxidase. This may explain why the decrease in IP3 and DAG production was not accompanied by a comparable decrement in superoxide generation, which was only 17% lower in the old than in young donor neutrophils. Thus, aging is associated with reductions in the concentration of critically important phosphoinositides, resulting in diminution in the ability to produce key second messengers. Although the aged neutrophil is largely able to compensate for the decrements in signal transduction, its reserve capacity is compromised, making it particularly vulnerable to external insults that also impair function.

Effect of age on phorbol-ester stimulation of human neutrophils.

When stimulated by phorbol 12-myristate 13-acetate (PMA), superoxide generation in neutrophils from old volunteers was modestly lower than neutrophils from young subjects. PMA receptor number and affinity were normal. Protein kinase C (PKC) translocation to the membrane was normal but its activation was reduced. PMA-induced total endogenous phosphorylation and phosphorylation of individual proteins showed no age-related differences as determined by SDS-PAGE analysis. These minimal alterations in neutrophil function contrast with the much more significant decrements in superoxide generation and calcium homeostasis noted when neutrophils from old volunteers are stimulated by chemotactic peptide formyl-methionyl-leucine-phenylalanine (FMLP) (Lipschitz et al., 1988). It is well recognized that phorbol activates the cell through a mechanism that bypasses the membrane-receptor. Taken together with our observations with FMLP, these results point to a membrane-associated deficiency in the signal transduction pathway, most likely through receptor coupling or alterations in membrane lipids. They also demonstrate that there is not an overall reduction of metabolic responses in neutrophils from the elderly.