Michele Fuortes

Protection from Alzheimer's-like disease in the mouse by genetic ablation of inducible nitric oxide synthase

Brains from subjects who have Alzheimers disease (AD) express inducible nitric oxide synthase (iNOS). We tested the hypothesis that iNOS contributes to AD pathogenesis. Immunoreactive iNOS was detected in brains of mice with AD-like disease resulting from transgenic expression of mutant human β-amyloid precursor protein (hAPP) and presenilin-1 (hPS1). We bred hAPP-, hPS1-double transgenic mice to be iNOS+/+ or iNOS−/−, and compared them with a congenic WT strain. Deficiency of iNOS substantially protected the AD-like mice from premature mortality, cerebral plaque formation, increased β-amyloid levels, protein tyrosine nitration, astrocytosis, and microgliosis. Thus, iNOS seems to be a major instigator of β-amyloid deposition and disease progression. Inhibition of iNOS may be a therapeutic option in AD.

MyD88 Primes Macrophages for Full-Scale Activation by Interferon-γ yet Mediates Few Responses to Mycobacterium tuberculosis

Macrophages are activated from a resting state by a combination of cytokines and microbial products. Microbes are often sensed through Toll-like receptors signaling through MyD88. We used large-scale microarrays in multiple replicate experiments followed by stringent statistical analysis to compare gene expression in wild-type (WT) and MyD88−/− macrophages. We confirmed key results by quantitative reverse transcription polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay. Surprisingly, many genes, such as inducible nitric oxide synthase, IRG-1, IP-10, MIG, RANTES, and interleukin 6 were induced by interferon (IFN)-γ from 5- to 100-fold less extensively in MyD88−/− macrophages than in WT macrophages. Thus, widespread, full-scale activation of macrophages by IFN-γ requires MyD88. Analysis of the mechanism revealed that MyD88 mediates a process of self-priming by which resting macrophages produce a low level of tumor necrosis factor. This and other factors lead to basal activation of nuclear factor κB, which synergizes with IFN-γ for gene induction. In contrast, infection by live, virulent Mycobacterium tuberculosis (Mtb) activated macrophages largely through MyD88-independent pathways, and macrophages did not need MyD88 to kill Mtb in vitro. Thus, MyD88 plays a dynamic role in resting macrophages that supports IFN-γ–dependent activation, whereas macrophages can respond to a complex microbial stimulus, the tubercle bacillus, chiefly by other routes.

Central venous oxygen saturation: a useful clinical tool in trauma patients.

An accurate method of estimating acute blood loss is essential in the evaluation of injured patients. Central venous oxygen (CVO2) saturation has been shown to be a sensitive and reliable correlate of blood loss in an animal model but its clinical validity is unproven. We evaluated 26 consecutive patients with an injury mechanism suggesting blood loss but who were deemed stable after initial evaluation. Vital signs (pulse, blood pressure, pulse pressure, urine output, CVP) and CVO2 saturation were serially measured. Blood loss was estimated by direct intracavitary collection or serial hematocrits and acute transfusion requirements. Despite stable vital signs, ten patients (39%) had CVO2 saturations under 65%. These patients had more serious injuries, significantly larger estimated blood losses, and required more transfusions than those patients with CVO2 saturation greater than 65%. Linear regression analysis demonstrated the superiority of CVO2 saturation to predict blood loss with a p value less than 0.005 relative to any of the normally followed parameters. CVO2 saturation is a reliable and sensitive method for detecting blood loss. It is a useful tool in the evaluation of acutely injured patients.

Central venous blood oxygen saturation: an early, accurate measurement of volume during hemorrhage.

Accurate and relatively simple monitoring is essential in managing patients with multiple injuries, and becomes particularly important when there is substantial occult blood loss. Tachycardia, said to occur following a 15% blood loss, is generally regarded as the first reliable sign of hemorrhage. However, heart rate is a nonspecific parameter which is affected by factors other than changing intravascular volume. The purpose of this study was to evaluate available means of monitoring volume status and to identify the parameter which is the earliest and most reliable indication of blood loss. Sixteen mongrel dogs were anesthetized and bled by increments of 3% of their total blood volume until the onset of sustained hypotension or a 25% blood loss. All dogs were monitored with a Swan-Ganz catheter and an arterial line. Vital signs, full hemodynamic parameters, and arterial and mixed venous blood gases were measured after each 3% blood loss. Statistical analysis of the data demonstrated that only Cardiac Index and Mixed Venous Oxygen Saturation showed linearity as function of measure blood loss. Linear regression analysis generated r values that ranged from 0.85-0.99 with a mean of 0.95 for Mixed Venous Oxygen Saturation; r values for Cardiac Index ranged from 0.39-0.98 with a mean of 0.85. Furthermore, all dogs had increased tissue oxygen extraction after 3-6% blood loss. Because Central Venous Blood Oxygen Saturation mirrors Mixed Venous Oxygen Saturation and is easily and rapidly measured, we extended our study by repeating all of the previously measured parameters, with the addition of CVP blood gases in an unanesthetized animal model.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular Analysis of the Prokaryotic Ubiquitin-Like Protein (Pup) Conjugation Pathway in Mycobacterium tuberculosis

Proteins targeted for degradation by the Mycobacterium proteasome are post‐translationally tagged with prokaryotic ubiquitin‐like protein (Pup), an intrinsically disordered protein of 64 residues. In a process termed ‘pupylation’, Pup is synthesized with a terminal glutamine, which is deamidated to glutamate by Dop (deamidase of Pup) prior to attachment to substrate lysines by proteasome accessory factor A (PafA). Importantly, PafA was previously shown to be essential to cause lethal infections by Mycobacterium tuberculosis (Mtb) in mice. In this study we show that Dop, like PafA, is required for the full virulence of Mtb. Additionally, we show that Dop is not only involved in the deamidation of Pup, but also needed to maintain wild‐type steady state levels of pupylated proteins in Mtb. Finally, using structural models and site‐directed mutagenesis our data suggest that Dop and PafA are members of the glutamine synthetase fold family of proteins.

Secretory leukocyte protease inhibitor, an inhibitor of neutrophil activation, is elevated in serum in human sepsis and experimental endotoxemia.

Objectives: To document changes in serum secretory leukocyte protease inhibitor (SLPI) in human sepsis and in experimental endotoxemia in vivo. To compare changes in serum SLPI in human sepsis with changes in interleukin (IL)‐6, IL‐10, and tumor necrosis factor (TNF)‐α. To determine whether or not changes in SLPI correlate with the severity of multiple organ dysfunction syndrome as measured by the maximal multiple organ dysfunction score. Finally, because neutrophils have been implicated in tissue injury associated with organ dysfunction, to determine whether recombinant human SLPI blocks activation of isolated human neutrophils. Design: Case‐control study and ex‐vivo cellular assay. Setting: Surgical intensive care unit and clinical research center of university hospitals; laboratory of a medical school. Interventions: None. Measurements and Main Results: There was a significant dose‐dependent elevation (50.2 ± 4.0 ng/mL, p = .01) in plasma SLPI 12 hrs after administration of lipopolysaccharide to seven healthy adults (36.4 ± 2.3 ng/mL). Further, serum concentrations of SLPI (132 ± 15 ng/mL) were elevated in septic surgical patients compared with healthy controls (43 ± 2 ng/mL, p < .01) and nonseptic surgical controls (69 ± 10 ng/mL, p = .01). Serum SLPI concentrations correlated (r2 = .71, p < .01) better with organ dysfunction as measured by maximal multiple organ dysfunction score than did serum IL‐6 (r2 = .49, p < .01), IL‐10 (r2 = .05, p = .22), or TNF‐α (r2 = .02, p = .44). We found that recombinant human SLPI in vitro inhibits TNF‐α‐induced hydrogen peroxide production by human neutrophils (ID50 = 1‐2 μg/mL). Conclusions: Serum SLPI is elevated in human sepsis and experimental endotoxemia. Maximal concentrations of serum SLPI correlate significantly with maximal multiple organ dysfunction scores in patients with sepsis. Secretory leukocyte protease inhibitor may function to limit ongoing neutrophil‐mediated tissue injury associated with organ dysfunction.

Critical Role of the Carboxyl Terminus of Proline-rich Tyrosine Kinase (Pyk2) in the Activation of Human Neutrophils by Tumor Necrosis Factor: Separation of Signals for the Respiratory Burst and Degranulation

Transduction of Tat-tagged fusion proteins confirmed a hypothesis based on pharmacologic inhibitors (Fuortes, M., M. Melchior, H. Han, G.J. Lyon, and C. Nathan. 1999. J. Clin. Invest. 104:327–335) that proline-rich tyrosine kinase (Pyk2) plays a critical role in the activation of adherent human neutrophils, and allowed an analysis of individual Pyk2 domains not possible with chemical inhibitors. Acting as a dominant negative, the COOH terminus of Pyk2 fused to a Tat peptide (Tat-CT), but not other regions of Pyk2, specifically inhibited the respiratory burst of cells responding to tumor necrosis factor (TNF), Salmonella, or Listeria, while sparing responses induced by phorbol ester. Tat-CT suppressed TNF-triggered cell spreading and the phosphorylation of endogenous Pyk2 and the associated tyrosine kinase Syk without blocking the ability of neutrophils to degranulate and kill bacteria. Thus, separate signals control the respiratory burst and degranulation, and a normal rate of killing of some bacteria can be sustained by granule products in conjunction with a minimal residual respiratory burst. Inhibition of select inflammatory functions without impairment of antibacterial activity may commend the Pyk2 pathway as a potential target for antiinflammatory therapy.

The effect of verapamil on cellular uptake, organ distribution, and pharmacology of cyclosporine

Verapamil has been shown to potentiate cyclosporines effect in inhibiting lectin-stimulated proliferation of murine and human lymphocytes, and in prolonging graft survival in experimental heterotopic cardiac transplantation in rats. A series of experiments were designed to determine whether verapamils effect occurred by increasing cyclosporine uptake or decreasing cyclosporines clearance by lymphocytes utilizing human peripheral blood lymphocytes and radiolabeled cyclosporine. Verapamil had no effect. The distribution of radiolabeled cyclosporine was also studied in mice that had been given verapamil (10 mg/kg) 1 hr prior to cyclosporine injection. No significant changes in organ distribution occurred. Lectin-stimulated release of intracellular ionized calcium was studied using a flurometric technique (Quin-2 and Fura-2). Neither cyclosporine nor verapamil had any effect on either lectin-stimulated or phorbol ester-stimulated release of intracellular ionized calcium. Phorbol ester and subproliferative doses of lectin were used to determine the effect of cyclosporine and verapamil on protein kinase C-mediated lymphocyte activation. Cyclosporine inhibited phorbol ester stimulated proliferation and verapamil potentiated this inhibition. Verapamil does not change cell or organ uptake of cyclosporine, and it does not affect the initial increase in intracellular ionized calcium that occurs with lymphocyte activation. Verapamil potentiates cyclosporine in inhibiting protein kinase C-mediated events in lymphocyte activation.

Effect of endothelin-1 and vasoactive intestinal contractor on blood flow and output of vasoactive intestinal polypeptide in the feline colon

Injection of the structurally related peptides, endothelin-1 and vasoactive intestinal contractor (VIC), into a branch of the superior mesenteric artery in anesthetized cats caused dose-dependent reductions in blood flow in the portal vein and inferior mesenteric artery. The maximum effect occurred after 1 minute and was more prolonged in the portal vein. The effects of the two peptides were not significantly different. The colonic output of vasoactive intestinal polypeptide (VIP) into portal venous blood was decreased significantly by endothelin-1 and VIC, returning to baseline more rapidly than blood flow. When norepinephrine was injected to produce comparable reductions in blood flow, the output of VIP into portal venous blood was not altered significantly. These results suggest that inhibition of output of the vasodilator VIP contributes to the vasoconstrictor effects of endothelin-1 and VIC in the feline colonic vascular bed.

Cyclosporine effect on anti-CD3 monoclonal antibody-stimulated mitogenesis, phorbol ester comitogenesis, and PGE2 production☆

Human peripheral blood mononuclear cells (H-PBMC) from 10 healthy donors were stimulated to proliferate with phytohemagglutinin lectin (PHA), anti-CD3 monoclonal antibody (mAb), and anti-CD3 mAb plus phorbol 12, myristate 13 acetate (TPA), a protein kinase C (PKC) agonist. Anti-CD3 mAb-mediated mitogenesis was 35-75% of that observed with PHA. When TPA was added to a dose of mAb that by itself did not cause mitogenesis, proliferation equal to 50-90% of the maximally mitogenic dose occurred. TPA did not enhance proliferation with maximally mitogenic doses of antibody. Dimethyl-prostaglandin E2, dibutyryl cyclic AMP, and forskolin (an adenyl cyclase agonist) inhibited PHA, anti-CD3, and anti-CD3/PMA-mediated mitogenesis. Cyclosporine (CSA) inhibited anti-CD3 and anti-CD3/TPA mitogenesis in a dose-dependent fashion. While CSA inhibited anti-CD3 and anti-CD3/TPA mitogenic signals, it did not affect PGE2 production by anti-CD3 mAb-stimulated H-PBMC. In the presence of CSA, PGE2 production in PHA-stimulated H-PBMC was increased. PGE2 inhibits lymphocyte proliferation via a cyclic AMP-mediated mechanism and may enhance maturation of suppressor cells. CSA inhibits anti-CD3 mAb and anti-CD3/TPA proliferative signals in H-PBMC yet has no effect or may even enhance production of suppressive PGE2. The maturation of antigen-specific suppressor cells elicited by CSA may involve active down-regulation of CD3 receptor and PKC-dependent events while PGE2 production continues.