Understanding the mechanisms underlying cognitive control in psychosis.

BACKGROUND: Cognitive control (CC) involves a top-down mechanism to flexibly respond to complex stimuli and is impaired in schizophrenia. METHODS: This study investigated the impact of increasing complexity of CC processing in 140 subjects with psychosis and 39 healthy adults, with assessments of behavioral performance, neural regions of interest and symptom severity. RESULTS: The lowest level of CC (Stroop task) was impaired in all patients; the intermediate level of CC (Faces task) with explicit emotional information was most impaired in patients with first episode psychosis. Patients showed activation of distinct neural CC and reward networks, but iterative learning based on the higher-order of CC during the trust game, was most impaired in chronic schizophrenia. Subjects with first episode psychosis, and patients with lower symptom load, demonstrate flexibility of the CC network to facilitate learning, which appeared compromised in the more chronic stages of schizophrenia. CONCLUSION: These data suggest optimal windows for opportunities to introduce therapeutic interventions to improve CC.

Sustained release of locally delivered celecoxib provides pain relief for osteoarthritis: a proof of concept in dog patients.

OBJECTIVE: Drug delivery platforms that allow for gradual drug release after intra-articular administration have become of much interest as a treatment strategy for osteoarthritis (OA). The aim of this study was to investigate the safety and efficacy of an intra-articular sustained release formulation containing celecoxib (CXB), a cyclooxygenase-2 (COX-2) selective inhibitor. METHODS: Amino acid-based polyesteramide microspheres (PEAMs), a biodegradable and non-toxic platform, were loaded with CXB and employed in two in vivo models of arthritis: an acute inflammatory arthritis model in rats (n = 12), and a randomized controlled study in chronic OA dog patients (n = 30). In parallel, the bioactivity of sustained release of CXB was evaluated in monolayer cultures of primary dog chondrocytes under inflammatory conditions. RESULTS: Sustained release of CXB did not alleviate acute arthritis signs in the rat arthritis model, based on pain measurements and synovitis severity. However, in OA dog patients, sustained release of CXB improved limb function as objective parameter of pain and quality of life based on gait analysis and owner questionnaires. It also decreased pain medication dependency over a 2-month period and caused no adverse effects. Prostaglandin E2 levels, a marker for inflammation, were lower in the synovial fluid of CXB-treated dog OA patients and in CXB-treated cultured dog chondrocytes. CONCLUSION: These results show that local sustained release of CXB is less suitable to treat acute inflammation in arthritic joints, while safe and effective in treating pain in chronic OA in dogs.

No added value for Computer-Assisted surgery to improve femoral component positioning and Patient Reported Outcomes in Hip Resurfacing Arthroplasty; a multi-center randomized controlled trial.

BACKGROUND: Computer Assisted Surgery (CAS) has proven to improve the accuracy in several orthopedic procedures. Therefore we used this technique to evaluate femoral component positioning in Hip Resurfacing Arthroplasty (HRA). The aim of this study was to evaluate imageless CAS compared to manually implanted femoral components and subsequently evaluates Patient Related Outcome Measures (PROMs). We hypothesized that the use of CAS optimizes the position of the femoral component and improves PROMs. METHODS: This is a multicenter, single-blinded, randomized, controlled trial of two groups. In the CAS group guiding of the femoral component was done with imageless navigation. In the Conventional (control) group the femoral component was placed manually according to the preplanned position. The primary outcome measure consists of a maximum of 3 degrees difference between the postoperative Stem Shaft Angle (SSA) and preplanned SSA. Secondary outcome measures consist of the Hip disability and Osteoarthritis Outcome Scale (HOOS), the Harris Hip Score (HHS) and Visual Analogue Scale (VAS) pain score. RESULTS: A total of 122 patients were randomized, 61 in the CAS group and 61 in the conventional group. There was no significant differences in accuracy of femoral implant position. The mean difference between the postoperative- and preplanned SSA was - 2.26 and - 1.75 degrees (more varus) respectively in the CAS and Conventional group. After surgery both groups show significant improvement in all PROMs compared to the baseline measurements, with no significant differences between the groups. CONCLUSION: Our cohort indicates no benefit for the use of CAS in accuracy of placement of the femoral component in HRA compared to manual implantation. There are no clinical differences in PROMs after 1 year follow up. This study showed no added value and no justification for the use of CAS in femoral component positioning in HRA. TRIAL REGISTRATION: This trial is registered at ClinicalTrails.gov ( https://clinicaltrials.gov/ ) on the 25th of October 2006: NCT00391937. LEVEL OF INCIDENCE: Level IIb, multicenter randomized controlled trial.

A new semi-orthotopic bone defect model for cell and biomaterial testing in regenerative medicine.

In recent decades, an increasing number of tissue engineered bone grafts have been developed. However, expensive and laborious screenings in vivo are necessary to assess the safety and efficacy of their formulations. Rodents are the first choice for initial in vivo screens but their size limits the dimensions and number of the bone grafts that can be tested in orthotopic locations. Here, we report the development of a refined murine subcutaneous model for semi-orthotopic bone formation that allows the testing of up to four grafts per mouse one order of magnitude greater in volume than currently possible in mice. Crucially, these defects are also "critical size" and unable to heal within the timeframe of the study without intervention. The model is based on four bovine bone implants, ring-shaped, where the bone healing potential of distinct grafts can be evaluated in vivo. In this study we demonstrate that promotion and prevention of ossification can be assessed in our model. For this, we used a semi-automatic algorithm for longitudinal micro-CT image registration followed by histological analyses. Taken together, our data supports that this model is suitable as a platform for the real-time screening of bone formation, and provides the possibility to study bone resorption, osseointegration and vascularisation.

Synthesis of plasma membrane-associated and secretory immunoglobulin in diploid lymphocytes.

The half disappearance time for detectable plasma membrane-associated and cytoplasmic immunoglobulin after treatment of continuously growing diploid lymphocytes with inhibitors of protein and RNA synthesis was studied. Also, the amount of plasma membrane-associated and cytoplasmic immunoglobulin of synchronized cells in the G(1) phase of the cell cycle has been studied. Plasma membrane-associated immunoglobulin has a half disappearance time of 45 min after inhibition of protein synthesis. By contrast, after treatment of cells with actinomycin D for 24 hr, plasma membrane-associated immunoglobulin remains relatively unchanged whereas cytoplasmic immunoglobulin decreased by almost 90%. In the G(1) phase of the cell cycle, plasma membrane-associated immunoglobulin and cytoplasmic immunoglobulin were 70 and 10%, respectively, of that in logarithmically growing cells, and the half disappearance of M-Ig after treatment of cells with puromycin was again 45 min. In toto, these results suggest that perhaps secreted and plasma membrane-associated immunoglobulin may be separately controlled by the cells.

Strategies for managing multi-patient 3D mass spectrometry imaging data.

Mass spectrometry imaging (MSI) has emerged as a powerful tool in biomedical research to reveal the localization of a broad scale of compounds ranging from metabolites to proteins in diseased tissues, such as malignant tumors. MSI is most commonly used for the two-dimensional imaging of tissues from multiple patients or for the three-dimensional (3D) imaging of tissue from a single patient. These applications are potentially introducing a sampling bias on a sample or patient level, respectively. The aim of this study is therefore to investigate the consequences of sampling bias on sample representativeness and on the precision of biomarker discovery for histological grading of human bladder cancers by MSI. We therefore submitted formalin-fixed paraffin-embedded tissues from 14 bladder cancer patients with varying histological grades to 3D analysis by matrix-assisted laser desorption/ionization (MALDI) MSI. We found that, after removing 20% of the data based on novel outlier detection routines for 3D-MSI data based on the evaluation of digestion efficacy and z-directed regression, on average 33% of a sample has to be measured in order to obtain sufficient coverage of the existing biological variance within a tissue sample. SIGNIFICANCE: In this study, 3D MALDI-MSI is applied for the first time on a cohort of bladder cancer patients using formalin-fixed paraffin-embedded (FFPE) tissue of bladder cancer resections. This work portrays the reproducibility that can be achieved when employing an optimized sample preparation and subsequent data evaluation approach. Our data shows the influence of sampling bias on the variability of the results, especially for a small patient cohort. Furthermore, the presented data analysis workflow can be used by others as a 3D FFPE data-analysis pipeline working on multi-patient 3D-MSI studies.

High prevalence of hypogonadotropic hypogonadism in men referred for obesity treatment.

BACKGROUND: Obesity can be associated with biochemical evidence of isolated hypogonadotropic hypogonadism (IHH) in men. Prevalence and severity of IHH in obese men are not exactly known. OBJECTIVE: To assess the prevalence of IHH in obese men. DESIGN AND SUBJECTS: Cross-sectional study of 160 obese men, BMI >30 kg/m2, who applied for medical or surgical treatment of obesity in a general teaching hospital. MAIN OUTCOME MEASURES: Total and calculated free testosterone (TT and FT) in relation to body mass index (BMI). RESULTS: Mean age of the study population was 43.3 +/- 0.8 years (mean +/- SEM), BMI ranged from 30.0 to 65.7 kg/m2. TT and FT levels were inversely related to BMI (-0.48, p<0.0001). Total testosterone was subnormal in 57.5% and free testosterone in 35.6% of the subjects. The group of men with IHH was more obese, had higher Hba IC levels and had a 2.6 higher risk for cardiovascular disease. Decreased libido and erectile dysfunction were 7.1 and 6.7 times as common in IHH than in eugonadal obese men. CONCLUSION: Reduced T levels, well into the hypogonadal range, are common in male obesity. Assessment of its clinical implications, and a search for the best mode of treatment are warranted.

Value of Quantitative Collateral Scoring on CT Angiography in Patients with Acute Ischemic Stroke.

BACKGROUND AND PURPOSE: Many studies have emphasized the relevance of collateral flow in patients presenting with acute ischemic stroke. Our aim was to evaluate the relationship of the quantitative collateral score on baseline CTA with the outcome of patients with acute ischemic stroke and test whether the timing of the CTA acquisition influences this relationship. MATERIALS AND METHODS: From the Multicenter Randomized Clinical Trial of Endovascular Treatment of Acute Ischemic Stroke in the Netherlands (MR CLEAN) data base, all baseline thin-slice CTA images of patients with acute ischemic stroke with intracranial large-vessel occlusion were retrospectively collected. The quantitative collateral score was calculated as the ratio of the vascular appearance of both hemispheres and was compared with the visual collateral score. Primary outcomes were 90-day mRS score and follow-up infarct volume. The relation with outcome and the association with treatment effect were estimated. The influence of the CTA acquisition phase on the relation of collateral scores with outcome was determined. RESULTS: A total of 442 patients were included. The quantitative collateral score strongly correlated with the visual collateral score (ρ = 0.75) and was an independent predictor of mRS (adjusted odds ratio = 0.81; 95% CI, .77-.86) and follow-up infarct volume (exponent ß = 0.88; P < .001) per 10% increase. The quantitative collateral score showed areas under the curve of 0.71 and 0.69 for predicting functional independence (mRS 0-2) and follow-up infarct volume of >90 mL, respectively. We found significant interaction of the quantitative collateral score with the endovascular therapy effect in unadjusted analysis on the full ordinal mRS scale (P = .048) and on functional independence (P = .049). Modification of the quantitative collateral score by acquisition phase on outcome was significant (mRS: P = .004; follow-up infarct volume: P < .001) in adjusted analysis. CONCLUSIONS: Automated quantitative collateral scoring in patients with acute ischemic stroke is a reliable and user-independent measure of the collateral capacity on baseline CTA and has the potential to augment the triage of patients with acute stroke for endovascular therapy.

Intradiscal delivery of celecoxib-loaded microspheres restores intervertebral disc integrity in a preclinical canine model.

Low back pain, related to degeneration of the intervertebral disc (IVD), affects millions of people worldwide. Clinical studies using oral cyclooxygenase-2 (COX-2) inhibitors have shown beneficial effects, although side-effects were reported. Therefore, intradiscal delivery of nonsteroidal anti-inflammatory drugs can be an alternative treatment strategy to halt degeneration and address IVD-related pain. In the present study, the controlled release and biologic potency of celecoxib, a selective COX-2 inhibitor, from polyesteramide microspheres was investigated in vitro. In addition, safety and efficacy of injection of celecoxib-loaded microspheres were evaluated in vivo in a canine IVD degeneration model. In vitro, a sustained release of celecoxib was noted for over 28 days resulting in sustained inhibition of inflammation, as indicated by decreased prostaglandin E2 (PGE2) production, and anti-catabolic effects in nucleus pulposus (NP) cells from degenerated IVDs on qPCR. In vivo, there was no evidence of adverse effects on computed tomography and magnetic resonance imaging or macroscopic evaluation of IVDs. Local and sustained delivery of celecoxib prevented progression of IVD degeneration corroborated by MRI, histology, and measurement of NP proteoglycan content. Furthermore, it seemed to harness inflammation as indicated by decreased PGE2 tissue levels and decreased neuronal growth factor immunopositivity, providing indirect evidence that local delivery of a COX-2 inhibitor could also address pain related to IVD degeneration. In conclusion, intradiscal controlled release of celecoxib from polyesteramide microspheres prevented progression of IVD degeneration both in vitro and in vivo. Follow-up studies are warranted to determine the clinical efficacy of celecoxib-loaded PEAMs in chronic back pain.

Controlled release of celecoxib inhibits inflammation, bone cysts and osteophyte formation in a preclinical model of osteoarthritis.

Major hallmarks of osteoarthritis (OA) are cartilage degeneration, inflammation and osteophyte formation. COX-2 inhibitors counteract inflammation-related pain, but their prolonged oral use entails the risk for side effects. Local and prolonged administration in biocompatible and degradable drug delivery biomaterials could offer an efficient and safe treatment for the long-term management of OA symptoms. Therefore, we evaluated the disease-modifying effects and the optimal dose of polyesteramide microspheres delivering the COX-2 inhibitor celecoxib in a rat OA model. Four weeks after OA induction by anterior cruciate ligament transection and partial medial meniscectomy, 8-week-old female rats (n = 6/group) were injected intra-articular with celecoxib-loaded microspheres at three dosages (0.03, 0.23 or 0.39 mg). Unloaded microspheres served as control. During the 16-week follow-up, static weight bearing and plasma celecoxib concentrations were monitored. Post-mortem, micro-computed tomography and knee joint histology determined progression of synovitis, osteophyte formation, subchondral bone changes, and cartilage integrity. Systemic celecoxib levels were below the detection limit 6 days upon delivery. Systemic and local adverse effects were absent. Local delivery of celecoxib reduced the formation of osteophytes, subchondral sclerosis, bone cysts and calcified loose bodies, and reduced synovial inflammation, while cartilage histology was unaffected. Even though the effects on pain could not be evualated directly in the current model, our results suggest the application of celecoxib-loaded microspheres holds promise as novel, safe and effective treatment for inflammation and pain in OA.

[Fatal intoxication with nicotine for e-cigarette]. / Fatale intoxicatie met nicotine voor e-sigaret.

BACKGROUND: Since the introduction of the electronic e-cigarette a few years ago, its use has greatly increased. The liquid formulations used in these e-cigarettes contain nicotine in high concentrations; ingestion of these liquids can be fatal. CASE DESCRIPTION: A 42-year-old male was admitted to the Intensive Care ward due to cardiac arrest. The patient had ingested highly concentrated liquid nicotine, originating from a vial with liquid for e-cigarettes. When the ambulance personnel found the patient he did not have a pulse; following CPR and administration of adrenaline his pulse returned. Upon admission, the plasma nicotine level was high at 3.0 mg/l (reference values for a smoker are 0.01-0.05 mg/l) and the patient's neurological function was poor. The patient was treated symptomatically, but eventually died of a postanoxic encephalopathy. CONCLUSION: Nicotine e-liquids are highly concentrated. Intentional ingestion can lead to toxic levels of nicotine which are associated with cardiac arrhythmias or arrest. Because even a few millilitres can be lethal, nicotine intoxication due to e-liquid ingestion should be considered potentially life-threatening.

Comparison of CTA- and DSA-Based Collateral Flow Assessment in Patients with Anterior Circulation Stroke.

BACKGROUND AND PURPOSE: Collateral flow is associated with clinical outcome after acute ischemic stroke and may serve as a parameter for patient selection for intra-arterial therapy. In clinical trials, DSA and CTA are 2 imaging modalities commonly used to assess collateral flow. We aimed to determine the agreement between collateral flow assessment on CTA and DSA and their respective associations with clinical outcome. MATERIALS AND METHODS: Patients randomized in MR CLEAN with middle cerebral artery occlusion and both baseline CTA images and complete DSA runs were included. Collateral flow on CTA and DSA was graded 0 (absent) to 3 (good). Quadratic weighted κ statistics determined agreement between both methods. The association of both modalities with mRS at 90 days was assessed. Also, association between the dichotomized collateral score and mRS 0-2 (functional independence) was ascertained. RESULTS: Of 45 patients with evaluable imaging data, collateral flow was graded on CTA as 0, 1, 2, 3 for 3, 10, 20, and 12 patients, respectively, and on DSA for 12, 17, 10, and 6 patients, respectively. The κ-value was 0.24 (95% CI, 0.16-0.32). The overall proportion of agreement was 24% (95% CI, 0.12-0.38). The adjusted odds ratio for favorable outcome on mRS was 2.27 and 1.29 for CTA and DSA, respectively. The relationship between the dichotomized collateral score and mRS 0-2 was significant for CTA (P = .01), but not for DSA (P = .77). CONCLUSIONS: Commonly applied collateral flow assessment on CTA and DSA showed large differences, indicating that these techniques are not interchangeable. CTA was significantly associated with mRS at 90 days, whereas DSA was not.

Alterations of inosinate branchpoint enzymes in cultured human lymphoblasts.

The specific activities of the three enzymes of the inosinate branchpoint are independently regulated when lymphoblasts are grown under various tissue culture conditions. In comparison to rapidly dividing cells, lymphoblasts at high cell density with no cellular division have decreased activity of the enzymes which commit inosinate to adenylate or guanylate, while cytoplasmic 5'-nucleotidase is relatively preserved. A linear relationship between inosinate dehydrogenase activity and growth rate (r = 0.92) exists in lymphoblasts with slowed growth rates. In contrast, in dividing cells adenylosuccinate synthetase and 5'-nucleotidase do not vary with growth rate. Adenylosuccinate synthetase and inosinate dehydrogenase activities appear to be related to the presence or rate of cellular division, as opposed to the presence or degree of neoplastic transformation. Lymphoblast lines with alterations of specific purine metabolic enzymes have characteristic alteration of the inosinate utilizing enzymes. Deficiencies of purine nucleoside phosphorylase or hypoxanthine phosphoribosyltransferase, abnormalities which render the cell unable to salvage purine effectively, are associated with depressed inosinate dehydrogenase activity. Insertion of the hypoxanthine phosphoribosyltransferase gene into hypoxanthine phosphoribosyltransferase-deficient cells normalizes inosinate dehydrogenase activity, while a hypoxanthine phosphoribosyltransferase-deficient mutant selected from a hypoxanthine phosphoribosyltransferase-containing line has depressed inosinate dehydrogenase activity. In contrast, overactivity of phosphoribosylpyrophosphate synthetase, with enhanced excretion of purines due to excessive production, is associated with elevated inosinate dehydrogenase activity. Inosinate dehydrogenase appears to be regulated according to the availability of purine nucleotides. Patients who overproduce uric acid and potentially have undescribed purine metabolic defects are now being screened for abnormalities in the inosinate branchpoint enzymes.

Clinical phenotype of Gaucher disease in relation to properties of mutant glucocerebrosidase in cultured fibroblasts.

We have investigated several parameters of glucocerebrosidase in cultured skin fibroblasts from patients with various clinical phenotypes of Gaucher disease. In this study no strict correlation was found between the clinical manifestations of Gaucher disease and the parameters investigated in fibroblasts. These parameters included the specific activity of the enzyme in extracts towards natural lipid and artificial substrate in the presence of different activators; the enzymic activity per unit of glucocerebrosidase protein; the rate of synthesis of the enzyme and its stability; and the post-translational processing of the enzyme. In addition, the activity in situ of glucocerebrosidase in fibroblasts was investigated using a novel method by analysis of the catabolism of NBD-glucosylceramide in cells that were loaded with bovine serum albumin-lipid complexes. Again, no complete correlation with the clinical phenotype of patients was detectable. Glucocerebrosidase in fibroblasts from most non-neuronopathic (type 1) Gaucher disease patients differs in some aspects from enzyme in cells from patients with neurological forms (types 2 and 3). The stimulation by activator protein and phospholipid is clearly more pronounced in type 1 than in types 2 and 3; the enzymic activity per unit of glucocerebrosidase protein in type 1 is severely reduced in the presence of taurocholate and the amount of glucocerebrosidase appears (near) normal in contrast to the situation in types 2 and 3 Gaucher fibroblasts. However, this distinction was not always consistent; glucocerebrosidase in fibroblasts from some type 1 Gaucher patients, particularly some South African cases, was comparable in properties to enzyme in type 2 and 3 patients.

Tuberculin skin test: estimation of positive and negative predictive values from routine data.

OBJECTIVE: To estimate, using routinely available data, the predictive values of tuberculin testing with 2TU RT23 for detection of latent tuberculosis infection (LTBI) in the Dutch population as a basis for recommendations on cut-off values at various levels of infection prevalence. DESIGN: Smoothed distributions of TST reactions among 312 tuberculosis patients and 2848 healthy non-BCG-vaccinated persons were used to estimate the sensitivity and specificity at various cut-off values. RESULTS: Sensitivity was 98.9% at 5 mm, 95.4% at 10 mm and 79.8% at 15 mm cut-off. Specificity with the corresponding cut-offs was 95.3%, 96.3% and 97.1% before and 98.0%, 98.8% and 99.6% after adjustment for presumed LTBI. At 10 mm, the positive predictive value (PPV) was > 75% if the infection prevalence in the tested population was at least 10%, but strongly declined with lower prevalences. For lower prevalences a cut-off of 15 mm was proposed, as this results in a higher PPV without greatly affecting the negative predictive value. CONCLUSION: Estimation of the predictive values of the TST from routine data can be useful for establishing cut-off values for detection of LTBI in different populations with different tuberculin preparations.

Type VI collagen is phagocytosed by fibroblasts and digested in the lysosomal apparatus: involvement of collagenase, serine proteinases and lysosomal enzymes.

Type VI collagen is present in most connective tissues, where it is considered to play a crucial role in the attachment of cells to the extracellular matrix and/or in the three-dimensional organization of the collagen meshwork. Although some information is available on its formation, the mechanisms involved in its degradation are not understood. Here, we present evidence for lysosomal digestion of type VI collagen by fibroblasts of periosteal explants. In the lysosomal apparatus of these cells, broad-banded filamentous aggregates characterized by 100-nm periodicity were found, which proved to consist of type VI collagen as indicated by their stainability with anti-type VI collagen antibodies. By interfering with synthesis (ascorbate or alpha, alpha-dipyridyl), intracellular translocation of collagen-containing vesicles (colchicine) as well as phagocytosis (cytochalasin B), it was shown that the intracellular broad-banded type VI collagen represented phagocytosed material. In the presence of acidotropic agents (NH4Cl and methylamine) the amount of intracellular type VI collagen increased significantly (5- to 10-fold), suggesting that a rise of pH in the endosomal/lysosomal apparatus causes inhibition of its degradation. By using a variety of proteinase inhibitors, it was found that inhibition of collagenase (when used in combination with NH4Cl), or inhibition of cysteine proteinases (both with and without NH4Cl), resulted in an increased amount of intracellular type VI collagen, whereas inhibition of serine proteinases significantly lowered the level of intracellular type VI collagen. The data presented are the first to indicate a pathway by which type VI collagen degradation may occur: fibroblasts phagocytose type VI collagen and subsequently digest this collagen in their lysosomal apparatus. Degradation depends on the activity of several enzymes, among them collagenase and serine proteinases, probably exerting their activity in the extracellular space just before the actual internalization. After uptake, digestion involves pH-sensitive lysosomal enzymes, including those belonging to the class of cysteine proteinases.

Gelatinase A (MMP-2) and cysteine proteinases are essential for the degradation of collagen in soft connective tissue.

The degradation of soft connective tissue collagen is considered to depend on the activity of various proteolytic enzymes, particularly those belonging to the group of matrix metalloproteinases and cysteine proteinases. In the present study, we investigated the contribution of these enzymes to this process. Using a general inhibitor of MMPs (SC44463), collagen degradation was strongly inhibited, by about 40% after 24 h and up to 80% after 72 h of culturing. Blockage of cysteine proteinase activity (with leupeptin or E-64) reduced breakdown at these time intervals by 50% and 20%, respectively. Given the abundant presence of gelatinases--in particular gelatinase A (MMP-2)--in the tissue, the effect of an inhibitor selective for gelatinases (CT1166) was studied. Gelatinase inhibition resulted in a dose-dependent decrease of collagen breakdown up to 90% after 48 h. The ability of gelatinase A to degrade collagens was demonstrated by the induction of breakdown in devitalized explants by addition of activated gelatinase A, or by activation of endogenous enzyme with 4-aminophenylmercuric acetate. This latter effect was not found with plasmin, an activator of MMPs other than gelatinase A. Finally, the relevance of gelatinase A to the in vivo degradation of soft connective tissue collagen was implicated by the significant correlation found between its activity and the collagen turnover rates of four soft connective tissues (tooth pulp, periodontal ligament, molar gingiva and skin). We conclude that collagen degradation in soft connective tissue is mediated by MMPs and to a lesser extent by cysteine proteinases. Our data are the first to attach a key role to gelatinase A in this process.

Mechanisms of action of endothelin on isolated feline cerebral arteries: in vitro pharmacology and electrophysiology.

Vascular endothelium has been found to produce a strong and potent vasoconstrictor peptide, endothelin. In this study, we have examined basic mechanisms underlying the contractile response of cerebral vessels to endothelin using in vitro pharmacology and electrophysiology. It was found that endothelin produced strong concentration-dependent contractions of circular segments of the feline middle cerebral artery. The response was slow in onset and long lasting. The vessels showed a remarkably strong tachyphylactic reaction upon repeated exposure to endothelin. The contractile effect of endothelin was not modified by the alpha-adrenoceptor antagonist phentolamine (10(-6) M) or the 5-hydroxytryptamine antagonist ketanserin (10(-6) M). Mechanical removal of the endothelium decreased potassium contractions while the maximum response to endothelin was only slightly reduced. There was no change in sensitivity of the cerebral artery to endothelin. The addition of a calcium antagonist (10(-6) M diltiazem or 3 X 10(-8) M nimodipine) or removal of extracellular calcium from the buffer solution did not change the sensitivity of the artery to endothelin but the maximum response to endothelin was reduced by between 40 and 60% by these procedures. The resting membrane potential of the cat middle cerebral artery was -62.8 +/- 3.5 mV. There was no significant depolarization in conjunction with cumulative administration of endothelin in concentrations below 1 X 10(-9) M. However, bursts of excitatory junction potentials were occasionally seen in response to high concentrations of endothelin (5 X 10(-9) M).(ABSTRACT TRUNCATED AT 250 WORDS)

PACAP, a VIP-like peptide: immunohistochemical localization and effect upon cat pial arteries and cerebral blood flow.

Pituitary adenylate cyclase activating peptide (PACAP) is a vasoactive intestinal polypeptide (VIP)-like peptide recently isolated from ovine hypothalami. Nerve fibers containing PACAP immunoreactivity were present in the adventitia and the adventitia-media border of cat cerebral arteries. Double immunostaining revealed that PACAP-immunoreactive nerve fibers constituted a subpopulation of the VIP-containing fibers. PACAP effected a concentration-dependent relaxation of feline middle cerebral arteries that had been precontracted with prostaglandin F2 alpha. The maximum relaxation, 24 and 34% of precontraction, was achieved with PACAP-38 and PACAP-27, respectively, at a concentration of 10(-6) M. In cats anesthetized with alpha-chloralose, intracerebral microinjection of PACAP effected a moderate increase in cerebral blood flow. The maximal increase (18.6 +/- 6%) was observed following the injection of 5 micrograms PACAP.