Increased endothelial cell apoptosis in symptomatic high-grade carotid artery stenosis: preliminary data.

OBJECTIVE: Endothelial cell (EC) apoptosis has been associated with thrombus formation on an eroded atherosclerotic plaque surface. Alongside plaque rupture, it may constitute another mechanism of plaque destabilisation. We investigated whether EC apoptosis also may be involved in plaque destabilisation in high-grade internal carotid artery (ICA) stenosis. METHODS: We compared the degree of EC apoptosis in carotid endarterectomy specimens from n=38 patients undergoing surgery for high-grade ICA stenosis (> or =70%; n=19 clinically asymptomatic; n=19 symptomatic). The total number of endothelial cells (ECs) and apoptotic cells were determined using CD31 immunohistochemistry and the TdT dUTP nick end-labeling (TUNEL) method respectively. RESULTS: Overall, EC apoptosis was a rare finding. The median percentage of apoptotic ECs was 0.0% (0.0-0.7%) in asymptomatic and 0.5% (0.0-7.3%) in symptomatic plaques (p=0.015, Mann-Whitney U test). No difference was observed between ruptured and unruptured plaque (0.0% [0.0-6.0%] vs 0.0% [0.0-5.7%]; p=0.446). CONCLUSIONS: Our results indicate that TUNEL-detected EC apoptosis is rare in carotid plaque from patients with >70% stenosis.

Fibrous cap thickness and smooth muscle cell apoptosis in high-grade carotid artery stenosis.

OBJECTIVE: There is growing evidence that, in high-grade internal carotid artery (ICA) stenosis, continuous fibrous cap thinning is not mandatory for plaque rupture and symptom development. The possibility that smooth muscle cell (SMC) apoptosis is involved in loss of fibrous cap volume has only been examined in a limited number of patients with high grade carotid artery stenosis. METHODS: Endarterectomy specimens from n = 38 consecutive patients undergoing surgery for high-grade ICA stenosis (> or = 70%) were transversely sectioned at 2 mm intervals. Plaque instability was defined clinically, by a history of recent ischemic symptoms (< 60 days before surgery; n = 19) attributable to the stenosis, or histopathologically by the presence of plaque rupture (n = 14). Detailed morphometric analyses of the fibrous cap was based on routine stains; for DNA in situ end labeling the TUNEL technique was used. SMCs were identified by immunostaining for SMC actin. RESULTS: We found no significant difference between symptomatic/asymptomatic or ruptured/unruptured plaque with respect to various morphometric measures of the fibrous cap (i.e. mean area, number of plaque sections with fibrous cap, necrotic core-to-lumen distance at its thinnest or thickest part). The mean (+/- SD) apoptotic SMCs per thousand within the fibrous cap was significantly higher in symptomatic vs. asymptomatic (64.53 +/- 77.3 vs. 6.71 +/- 11.9; P<0.001) but not in ruptured plaques (43.3 +/- 64.4 vs. 30.1 +/- 60.9; P=0.117). CONCLUSIONS: These data suggest that continuous thinning of the fibrous cap is not an essential prerequisite for plaque rupture in ICA stenosis. Symptomatic, but not ruptured plaque, were associated with the highest number of apoptotic SMC. Thus, it seems unlikely that SMC apoptosis promotes plaque rupture by fibrous cap thinning.

[The unstable carotid stenosis: definition and biological processes]. / Die instabile Karotisstenose: Definition und pathobiologische Mechanismen.

The occurrence of cerebral or retinal ischemic symptoms ipsilateral to high-grade internal carotid artery (ICA) stenosis indicates a status of instability with a substantial risk for future major stroke. Additionally, the detection of microembolic signals downstream of ICA stenosis is predictive for future cerebral ischemia in asymptomatic and symptomatic patients. There is substantial evidence that in unstable ICA stenosis plaque rupture and thrombus formation are the most frequent pathoanatomic findings. In contrast, in nearly the half of unstable carotid plaques the lumen surface appears to be intact. Within plaque tissue, the unstable plaque is mainly characterized by a substantial amount of inflammatory cell (i. e. macrophages, T-cells) infiltration. These cells are mainly localized in the fibrous cap near the necrotic core. Produced by macrophages, matrix degrading enzymes (e. g. MMP-9) are overexpressed in the unstable ICA stenosis. Thrombogenicity is mainly determined by the local concentration of activated tissue factor, also expressed by inflammatory cells. Furthermore, a significantly higher rate of apoptotic smooth muscle cells can be found within the fibrous cap of instable carotid stenoses. Whether infection with Chlamydia pneumoniae contribute to instability is unlikely, because a positive association to clinical instability has not been shown up to now. The exact and detailed characterization of the unstable ICA plaque and the correlation of different biological mechanisms to clinical instability may offer the possibility to use it as a human model of unstable atherosclerosis in general and to test the efficacy of new developed anti-atherosclerotic pharmaceutical agents.

Lower lipoteichoic and teichoic acid CSF concentrations during treatment of pneumococcal meningitis with non-bacteriolytic antibiotics than with ceftriaxone.

In the rabbit model of Streptococcus pneumoniae meningitis, treatment with rifabutin, quinupristin-dalfopristin, moxifloxacin and trovafloxacin led to smaller increases of the CSF concentrations of the pro-inflammatory cell wall components lipoteichoic and teichoic acids (LTA and TA) than did treatment with ceftriaxone. Low doses of moxifloxacin were associated with higher LTA and TA concentrations in CSF than were high doses.

Reduction of meningeal macrophages does not decrease migration of granulocytes into the CSF and brain parenchyma in experimental pneumococcal meningitis.

Leukocyte infiltration of the CSF and brain parenchyma and other parameters of inflammation during pneumococcal meningitis were investigated after reduction of meningeal macrophages in rabbits by intracisternal injection of dichloromethylene-diphosphonate (Cl2MDP)-containing liposomes. Macrophages in the meninges were reduced, in median, by approximately 77% after three intrathecal injections of 100 microl of liposomes containing Cl2MDP at 12 h intervals. Production of the cytokines interleukin-1 and tumor necrosis factor-alpha as well as infiltration of the CSF and nervous tissue by leukocytes was not significantly altered in infected animals after treatment with Cl2MDP-containing liposomes. The median CSF concentration of neuron specific enolase (NSE) as a parameter of neuronal damage was higher in infected Cl2MDP-treated animals (median [median (25th/75th percentiles): 44.7 (33.2/54.3) microg/l vs. 13.9 (10.4/23.9) microg/l; P = 0.01]). Therefore, the reduction of meningeal macrophages does not appear to attenuate inflammation in the subarachnoid space in experimental pneumococcal meningitis. Meningeal macrophages seem, however, to be important for the protection of neuronal tissue in bacterial meningitis.

Quinupristin/dalfopristin attenuates the inflammatory response and reduces the concentration of neuron-specific enolase in the cerebrospinal fluid of rabbits with experimental Streptococcus pneumoniae meningitis.

The inflammatory response following initiation of antibiotic therapy and parameters of neuronal damage were compared during intravenous treatment with quinupristin/dalfopristin (100 mg/kg as either a short or a continuous infusion) and ceftriaxone (10 mg/kg/h) in a rabbit model of Streptococcus pneumoniae meningitis. With both modes of administration, quinupristin/dalfopristin was less bactericidal than ceftriaxone. However, the concentration of proinflammatory cell wall components (lipoteichoic acid (LTA) and teichoic acid (TA)) and the activity of tumour necrosis factor (TNF) in cerebrospinal fluid (CSF) were significantly lower in the two quinupristin/dalfopristin groups than in ceftriaxone-treated rabbits. The median LTA/TA concentrations (25th/75th percentiles) were as follows: (i) 14 h after infection: 133 (72/155) ng/mL for continuous infusion of quinupristin/dalfopristin and 193 (91/308) ng/mL for short duration infusion, compared with 455 (274/2042) ng/mL for ceftriaxone (P = 0.002 and 0.02 respectively); (ii) 17 h after infection: 116 (60/368) ng/mL for continuous infusion of quinupristin/dalfopristin and 117 (41/247) ng/mL for short duration infusion, compared with 694 (156/2173) ng/mL for ceftriaxone (P = 0.04 and 0.03 respectively). Fourteen hours after infection the median TNF activity (25th/75th percentiles) was 0.2 (0.1/1.9) U/mL for continuous infusion of quinupristin/dalfopristin and 0.1 (0.01/3.5) U/mL for short duration infusion, compared with 30 (4.6/180) U/mL for ceftriaxone (P = 0.02 for each comparison); 17 h after infection the TNF activity was 2.8 (0.2/11) U/mL (continuous infusion of quinupristin/dalfopristin) and 0.1 (0.04/6.1) U/mL (short duration infusion), compared with 48.6 (18/169) U/mL for ceftriaxone (P = 0.002 and 0.001). The concentration of neuron-specific enolase (NSE) 24 h after infection was significantly lower in animals treated with quinupristin/dalfopristin: 4.6 (3.3/5.7) microg/L (continuous infusion) and 3.6 (2.9/4.7) microg/L (short duration infusion) than in those treated with ceftriaxone (17.7 (8.8/78.2) microg/L) (P = 0.03 and 0.009 respectively). In conclusion, antibiotic treatment with quinupristin/dalfopristin attenuated the inflammatory response within the subarachnoid space after initiation of antibiotic therapy. The concentration of NSE in the CSF, taken as a measure of neuronal damage, was lower in quinupristin/dalfopristin-treated rabbits than in ceftriaxone-treated rabbits.

Streptococcal meningitis: effect of CSF filtration on inflammation and neuronal damage.

The effect of CSF filtration on inflammation and neuronal damage was studied in experimental Streptococcus pneumoniae meningitis. New Zealand white rabbits received either antibiotic therapy alone (ceftriaxone i.v., 20 mg/kg bolus, 10 mg/kg maintenance dose; n = 10) or ceftriaxone plus CSF filtration (n = 11) 12 h after intracisternal infection. Immediately after the onset of antibiotic therapy 300 microliters cisternal CSF was removed, passed through a miniaturized CSF-1 filter at a constant flow of 20 microliters/min, and then reinjected. This procedure was repeated six times at intervals of 20 min. Antibiosis plus CSF filtration caused a transient reduction in CSF bacterial titers and leukocyte counts compared with antibiosis alone (P = 0.04 and 0.02 5 h after initiation of therapy). CSF lipoteichoic acid concentrations were not reduced. The concentration of neuron-specific enolase in CSF and the density of apoptotic neurons in the dentate gyrus were almost equal 12 h after the onset of treatment. Adjuvant CSF filtration accelerated the elimination of viable bacteria from CSF in comparison to antibiotic treatment alone. Parameters of neuronal destruction, however, were not reduced.

Granulocytes in the subarachnoid space of humans and rabbits with bacterial meningitis undergo apoptosis and are eliminated by macrophages.

The contribution of leukocyte apoptosis to the resolution of meningeal inflammation in bacterial meningitis was studied in the cerebrospinal fluid (CSF) and in meningeal infiltrates of humans and rabbits by in situ tailing, flow cytometry, agarose gel electrophoresis, and electron microscopy. In humans, the rate of apoptotic granulocytes was 21.0+/-20.8% (n=11) in cytocentrifuge preparations and 3.3+/-3.4 (n=14) in putrid infiltrates of autopsy cases (P=0.02). In rabbits, CSF pleocytosis peaked 8 h after the initiation of antibiotic treatment (5311+/-3122/microl). At this time, the rate of apoptotic granulocytes was 15.2+/-7.3% in CSF and 1.8+/-1.4% in the meningeal infiltrates (each group n=6, P=0.007). Thereafter, the rate of apoptotic granulocytes in CSF declined below 10%. In humans and rabbits, bands representing internucleosomal fragments of approximately 180 base pairs and multiples thereof were documented on agarose gels. Phagocytosis of apoptotic granulocytes by macrophages was visualized by light and electron microscopy. In conclusion, during resolution of subarachnoid space inflammation in bacterial meningitis, a substantial fraction of granulocytes undergoes apoptosis. These granulocytes are removed by phagocytosis by macrophages. Apoptosis is more frequent in granulocytes floating in the CSF than in adherent cells.

Moxifloxacin in the therapy of experimental pneumococcal meningitis.

The activity of moxifloxacin (BAY 12-8039) against a Streptococcus pneumoniae type 3 strain (MIC and minimum bactericidal concentration [MBC] of moxifloxacin, 0.06 and 0.25 microgram/ml, respectively; MIC and MBC of ceftriaxone, 0.03 and 0.06 microgram/ml, respectively) was determined in vitro and in a rabbit model of meningitis. Despite comparable bactericidal activity, 10 micrograms of moxifloxacin per ml released lipoteichoic and teichoic acids less rapidly than 10 micrograms of ceftriaxone per ml in vitro. Against experimental meningitis, 10 mg of moxifloxacin per kg of body weight per ml reduced the bacterial titers in cerebrospinal fluid (CSF) almost as rapidly as ceftriaxone did (mean +/- standard deviation, -0.32 +/- 0.14 versus -0.39 +/- 0.11 delta log CFU/ml/h). The activity of moxifloxacin could be described by a sigmoid dose-response curve with a maximum effect of -0.33 delta log CFU/ml/h and with a dosage of 1.4 mg/kg/h producing a half-maximal effect. Maximum tumor necrosis factor activity in CSF was observed later with moxifloxacin than with ceftriaxone (5 versus 2 h after the initiation of treatment). At 10 mg/kg/h, the concentrations of moxifloxacin in CSF were 3.8 +/- 1.2 micrograms/ml. Adjunctive treatment with dexamethasone at 1 mg/kg prior to the initiation of antibiotic treatment only marginally reduced the concentrations of moxifloxacin in CSF (3.3 +/- 0.6 micrograms/ml). In conclusion, moxifloxacin may qualify for use in the treatment of S. pneumoniae meningitis.

Central nervous system TNFalpha-mRNA expression during rabbit experimental pneumococcal meningitis.

In pneumococcal meningitis inflammatory mediators such as tumor necrosis factor alpha (TNFalpha) are produced in large quantities and play a major role in pathogenesis. It is not known exactly which cells produce these mediators during infection. We investigated the localisation of TNFalpha-mRNA in the central nervous system (CNS) by in situ hybridisation during experimental Streptococcus pneumoniae meningitis. TNF-positive cells were detected only in inflammatory infiltrates within the meninges. Cells within the brain parenchyma and the choroid plexus were completely negative. After monocyte depletion, no TNFalpha-mRNA positive cells were detected in the CNS. These findings suggest that TNFalpha in pneumococcal meningitis is produced in the CNS mainly by blood-derived, infiltrating monocytes.