Philip J. Spagnuolo

Thromboxane A2 Mediates Augmented Polymorphonuclear Leukocyte Adhesiveness

We examined the role of prostaglandins and thromboxanes as mediators of plasma-dependent increased polymorphonuclear leukocyte adhesiveness induced by Escherichia coli lipopolysaccharide. The cyclo-oxygenase inhibitors-indomethacin and d,l-6-chloro-alpha-methyl-carbozole-2-acetic acid (R020-5720)-reduced lipopolysaccharide-induced adherence of polymorphonuclear leukocytes by 74 and 62%, respectively. In addition, inhibitors of thromboxane synthetase-imidazole, 9,11-azoprosta-5,13-dienoic acid, and 1-benzylimidazole-suppressed the stimulation of adherence by 31, 66, and 83%, respectively. Exogenous prostaglandins E(1), E(2), and F(2)alpha did not increase polymorphonuclear leukocyte adherence, nor were they detected in significant quantities in supernates of polymorphonuclear leukocytes exposed to lipopolysaccharide. However, inhibitors of both cyclo-oxygenase and thromboxane synthetase reduced increases in adherence induced by arachidonic acid (10 mug/ml), suggesting that lipopolysaccharide-mediated increases in adherence were due to an arachidonic acid product other than prostaglandin E(2) or F(2)alpha. 8,11,14-Eicosatrienoic acid, a precursor of monoenoic prostaglandins, did not enhance polymorphonuclear leukocyte adhesiveness. We next demonstrated lipopolysaccharide-stimulated generation, by polymorphonuclear leukocytes, of a labile, low molecular weight, dialyzable substance capable of enhancing the adherence of unstimulated leukocytes. In parallel experiments, a 10-fold increase in immunoreactive thromboxane B(2) over basal levels was detected after exposure of leukocytes to lipopolysaccharide. The inhibition of lipopolysaccharide enhancement of adherence by specific rabbit antibodies to thromboxane B(2) strongly supported a primary role for thromboxane A(2) as the mediator of the observed increases in adherence. Lipopolysaccharide-stimulated purified platelets did not increase leukocyte adherence, whereas thrombin-stimulated platelets did increase adherence. These studies suggest that lipopolysaccharide stimulates polymorphonuclear leukocytes to produce thromboxane A(2), which enhances their adhesiveness to nylon.

Suppression of lymphocyte responses by tuberculous plasma and mycobacterial arabinogalactan. Monocyte dependence and indomethacin reversibility

During tuberculosis, exposure of monocytes to circulating factors may induce the suppressor activity observed in some anergic patients. To explore this possibility, we examined the effects of plasma pooled from 28 untreated tuberculosis (TB) patients and the mycobacterial cell wall polysaccharide D-arabino-D-galactan (AG) on the in vitro function of peripheral blood mononuclear cells (PBMC) from healthy donors. In the [3H] thymidine incorporation assay, stimulated responses of PBMC incubated in culture medium supplemented with TB plasma or co-cultured with 3.0 microgram/ml AG were depressed significantly when compared with control responses. Cytotoxicity and altered kinetics of stimulated DNA synthesis did not contribute to the observed suppression. TB plasma and AG-induced suppression of the PBMC response to purified protein derivative was monocyte dependent and indomethacin reversible. In addition, TB plasma and AG directly inhibited the phytohemagglutinin-stimulated responses of T lymphocytes. In a quantitative assay of monocyte attachment to plastic, both TB plasma and AG significantly increased monocyte adherence from basal levels. These effects on monocyte adherence were reversed with indomethacin or antibody to mycobacterial polysaccharide. In addition, TB plasma passed over an immunoabsorbent column of Sepharose-linked antibody to mycobacterial polysaccharide was depleted of the suppressive and monocyte-adherence augmenting factors. 3.0 microgram/ml AG stimulated a fivefold increase in prostaglandin E2 production by cultured mononuclear cells. Our data suggest that AG circulating alone or bound in immune complexes may account for the observed effects of TB plasma. Similar in vivo exposure may contribute to the cell-mediated suppression of lymphocyte responses in tuberculosis.

Haemophilus influenzae meningitis: The spectrum of disease in adults

Fifteen cases of Haemophilus influenzae (HI) meningitis in adults occurring of Cleveland during the last 11 years are presented. The majority of patients had factors predisposing to infection such as otitis, pneumonia, diabetes or alcoholism. In addition, 7 of the 15 patients developed meningitis at various intervals following head trauma and neurosurgery, and 3 patients required dural repairs for CSF rhinorrhea. The diagnosis of meningitis may be difficult to establish resulting in delay in appropriate therapy in some cases. Nuchal rigidity was absent frequently; CSF lymphocytosis can be seen initially. The CSF Gram stain may be negative or the pleomorphic nature of the organism on Gram-stain may make distinction from other gram-negative organisms difficult. The majority of patients had meningitis due to non-Type B HI in contrast to previous reports of this illness in children and adults. One of our patients had beta-lactamase producing HI isolated from CSF. We believe that chloramphenicol should be included in the initial empiric therapy for adults with meningitis and gram-negative coccobacillary rods on Gram-stain or negative CSF Gram-stains.

Neutrophil adhesion in the elderly : inhibitory effects of plasma from elderly patients

Neutrophil (PMN) adherence is a critical component of host defense against infection. We questioned whether abnormalities of PMN adherence may be responsible, in part, for the increased susceptibility to infection in the elderly. We examined the adherence of 51Cr-labeled PMN from 18 elderly (65-95 years) and 18 younger subjects (18-40 years) to gelatin-coated plastic (gel) and bovine aortic endothelial monolayers (BAEC). There was no difference in unstimulated or baseline adherence of elderly or control PMN to either gel or BAEC substrates. N-Formyl-methionyleucylphenylalanine (FMLP), phorbol myristate acetate (PMA), and calcium ionophore A23187 (CI) significantly increased adherence of elderly PMN to gel and BAEC by 204 and 140% for FMLP, 271 and 263% for PMA, and 211 and 150% for CI, respectively. No differences were observed in the increment in stimulated adherence between young and elderly PMN. In contrast, in 5 of 18 subjects, incubation of elderly or young PMN with 10% elderly plasma resulted in greater than 25% inhibition in baseline adherence to BAEC compared to their sex-matched controls. The effect of elderly plasma was specific for BAEC and not seen with the gel substrate and was also demonstrated using human venous endothelium. When the adherence assay was repeated with varying ratios of elderly and young plasma, PMN adherence to BAEC correlated inversely with the proportion of elderly plasma in the assay. With greater than 70% elderly plasma, adherence was depressed below that observed in the absence of plasma. These data suggest the presence of a factor(s) in elderly plasma which may diminish adherence to endothelium. This factor(s) may be important in the increased risk of infection in a segment of the elderly population.

MEDIATION OF AUGMENTED MONOCYTE ADHESIVENESS BY THROMBOXANE

We examined the potential contribution of thromboxanes in human monocyte adherence to plastic. Monocyte adherence to plastic could be augmented by various stimuli including lipopolysaccharide, chemotactic peptide, and supernates of antigen-stimulated lymphocytes. Increments in monocyte adhesiveness were suppressed by inhibition of cyclooxygenase, thromboxane synthetase, or by antiserum to thromboxane B2. Neither prostaglandin E2 or F2α significantly affected baseline or lipopolysaccharide-stimulated monocyte adherence. Additional experiments confirmed incremental production of thromboxane B2 by monocytes after incubation with lipopolysaccharide. Thromboxane B2 itself did not stimulate monocyte adhesiveness. These data demonstrate that monocytes release thromboxane A2 following stimulation and suggest that thromboxane A2 may play a significant role in monocyte-substrate attachment.

Diethylcarbamazine citrate, an antifilarial drug, stimulates human granulocyte adherence.

Incubation with diethylcarbamazine citrate caused significant augmentation of human neutrophil and eosinophil adherence to tissue culture plastic. This effect was dose dependent and cell dependent, with eosinophils showing greater sensitivity and a greater adhesive response to the drug than did neutrophils. Eosinophils preincubated with diethylcarbamazine citrate demonstrated decreased adhesive responses to other adherence-augmenting stimuli. Use of Fc-treated plastic augmented diethylcarbamazine citrate-stimulated neutrophil (but not eosinophil) adherence. Direct stimulation of host effector cell adherence may explain, in part, the therapeutic action of diethylcarbamazine citrate in vivo.

Chemotaxis of human granulocytes toward microfilariae of Onchocerca volvulus

Summary Migration of human peripheral blood granulocytes in response to microfilariae of O. volvulus was demonstrated using modified Boyden chambers. Granulocyte migration was significantly enhanced when microfilariae were preincubated with heat‐inactivated immune serum (ΔIS), then added to a fresh serum source (P <0.025). This effect was not seen when microfilariae were incubated in medium alone, in ΔIS alone, in ΔIS plus C4‐deficient guinea‐pig serum, or in fresh serum alone. There was no significant difference between the response of cells from O. volvulus‐infected donors and that of cells from normal volunteers. Likewise, there was no significant difference between the migratory response seen toward nodule‐ versus skin‐derived microfilariae. These results suggest that the host inflammatory response to O. volvulus microfilariae is mediated in part by chemotactic factors generated by antibody and complement interaction with the organism and, furthermore, that these factors are product(s) of classical complement pathway activation.

Human neutrophil adhesion to bovine aortic endothelium. Evidence for endothelial lipoxygenase activity.

We examined the effect of phorbol myristate acetate on cultured bovine aortic endothelial cells to determine the role of endothelial cells in neutrophil‐endothelial cell adhesive interactions. Confluent endothelial cells were preincubated with phorbol myristate acetate and other inflammatory signals including JV‐formylmethionyl‐ leucyl‐phenylalanine (f‐Met‐Leu‐Phe), the ionophore A23187, and thrombin; washed extensively; and incubated with 51Cr‐labeled neutrophils. Preincubation of endothelium with A23187, phorbol ester, or thrombin increased adherence of neutrophils by 3.1‐, 5.7‐, and 3.7‐fold over baseline. In contrast, f‐Met‐Leu‐Phe preincubation failed to increase adhesion over baseline. Supernatants from endothelium preincubated with phorbol failed to augment adherence of untreated endothelial cells. Preincubation of endothelium with lipoxygenase inhibitors nordihydroguaiaretic acid (50μM), 5,8,11,14‐eicosatetraenoic acid (50 μM), and BW755C (50 μM) inhibited the effect of phorbol preincubation of endothelium significantly by 55, 27, and 22%, respectively. In contrast, inhibitors of cyclooxygenase and thromboxane synthase or thromboxane receptor antagonists had no effect on phorbol‐induced adhesion. Specific desensitization of neutrophil adhesion to phorbol‐treated endothelium could be demonstrated by prior exposure of neutrophils to low concentrations of leukotriene B4 (3.8 × 10−10 M). Endothelium preincubated with phorbol but not f‐Met‐Leu‐Phe or thrombin produced several fatty acid peaks at 280 nm, one of which comigrated with authentic leukotriene B4 (LTB4). This peak, isolated and purified, increased endothelial cell adherence in a temporal fashion in the same way as LTB4 and was demonstrated to be LTB4 by ultraviolet spectroscopy, high‐ performance liquid chromatography, and mass spectroscopy. These data demonstrate that endothelial cell‐derived lipoxygenase metabolites, in particular LTB4, are involved, in part, in the acute regulation of neutrophil adhesion to endothelium induced by inflammatory signals such as phorbol ester.

Platelet activating factor amplifies human neutrophil adherence to bovine endothelial cells: Evidence for a lipoxygenase dependent mechanism

Platelet activating factor (PAF) is a potent lipid mediator that induces the release of leukotrienes and prostaglandins from various cells and tissues. We examined the capacity of PAF alone and in combination with soluble stimuli to enhance eicosanoid synthesis and adherence of human neutrophils. Neutrophils were preincubated with PAF and washed before exposure to the soluble stimuli F-Met-Leu-Phe (FMLP), calcium ionophore A23187, and phorbol myristate acetate. Preincubation of neutrophils with 1μM PAF enhanced the release of both LTB4 and LTC4 in response to each of the three agonists, in contrast with the unprimed neutrophils. Priming was specific for PAF since lyso-PAF was inactive. Priming concentrations of PAF also augmented the adherence of neutrophils to endothelium in the presence of the soluble agonists A23187, phorbol myristate acetate, and FMLP. The priming effect of PAF on eicosanoid release and neutrophil adherence was shown to have similar time- and dose-dependent effects. Further, the priming effects of PAF on adherence could be reversed by preincubation of neutrophils with the lipoxygenase inhibitors nordihydroguiaretic acid and 5,8,11,14-ETYA but not by preincubation with the cycloxygenas e inhibitor indomethacin. These data demonstrate that PAF amplifies neutrophil adherence to endothelium through a lipoxygenase dependent mechanism.

Effects of antiviral treatment on influenza-related complications over four influenza seasons: 2006–2010

Abstract Purpose: The objective of the study is to evaluate the effect of antiviral treatment, pre-existing diseases, and sociodemographic factors on the risk of influenza-related complications and healthcare utilization. Methods: Case data was obtained from U.S. MarketScan Research Databases. Cases had a clinical diagnosis of influenza between 2006 and 2010 and continuous healthcare insurance from 90 days before to 30 days after diagnosis. Logistic regression models were applied to explore the impact of antiviral treatment on complications and healthcare utilization. Modified generalized estimating equation regression models in propensity score matched samples were used to address the robustness of the study. Results: Analyses included 1,557,437 cases from four influenza seasons. In each season, 34.82%–43.42% of patients received antiviral treatment, mostly oseltamivir. On average, 1.86% of patients were hospitalized, 9.56% visited the emergency room and 41.14% made ≥2 outpatient visits. The incidence of complications ranged from 17.62 to 19.67 per 100 patient-months. The relative risk of complications was increased in patients aged 0–4 years and those with pre-existing diseases, including asthma, Parkinson’s disease, and cystic fibrosis. Overall, patients receiving antiviral treatment had an 11% reduction in the risk of complications. Among oseltamivir-treated patients, the risk of complications was significantly reduced by 81% in those treated ≤2 days after diagnosis compared with later. Antiviral treatment significantly reduced the risk of hospitalization, emergency room visits and need for ≥2 outpatient visits by 29%, 24% and 11%, respectively. The propensity score matching method improved the strength of the study. Conclusions: Early treatment with antivirals, and specifically oseltamivir, significantly reduced the risk of influenza-related complications and healthcare utilization. However, lacking information about disease severity and the time from onset of symptoms to fulfillment of a prescription may bias the outcomes.