Involvement of Rho kinase in endothelial barrier maintenance.

OBJECTIVE: Rho kinase mediates vascular leakage caused by many vasoactive agents including thrombin. Enhanced Rho kinase activity induces endothelial barrier dysfunction by a contractile mechanism via inactivation of Myosin Phosphatase (MP). Here, we investigated the contribution of basal Rho kinase activity to the regulation of endothelial barrier integrity. METHODS AND RESULTS: Using a phospho-specific antibody against the myosin phosphatase targeting subunit (Thr696-MYPT1) as a marker for Rho kinase activity, basal endothelial Rho kinase activity was observed at cell-cell contact sites, in vitro and in situ. Thrombin enhanced MYPT phosphorylation at F-actin stress fibers. Inhibition of basal Rho kinase activity for 24 hours or depletion of Rho kinase (ROCK-I and -II) by siRNA disrupted endothelial barrier integrity, opposite to the previously observed protection from the thrombin-enhanced endothelial permeability. This barrier dysfunction could not be explained by changes in RhoA, Rac1, eNOS, or apoptosis. Remarkably, basal Rho kinase activity was essential for proper expression of the adhesion molecule VE-cadherin. CONCLUSIONS: Rho kinase has opposing activities in regulation of endothelial barrier function: (1) an intrinsic barrier-protective activity at the cell margins, and (2) an induced barrier-disruptive activity at contractile F-actin stress fibers. These findings may have implications for long-term antivascular leak therapy.

[Synovial metastases from colonic cancer presenting as arthritis of the knee. Characterization by 18FDG PET scan]. / Métastase synoviale d'un cancer colique se présentant comme une arthrite du genou. Caractérisation par tomographie à émission de positons au 18FDG.

A case of carcinomatous monoarthritis involving the left knee due to colonic adenocarcinoma is described. Large recurrent synovial effusion, that will be later hematic, lytic lesion of the bones and chondrolysis were noted. Knee positron emitting tomography scan using FDG (FDG-PET) revealed a diffuse increased uptake in soft tissues assumed to be synovium, the hypertrophy of which was identified by ultrasonography. Whole body PET scan showed extensive lymph node, visceral and bone metastases, suggesting that the increase in the synovium could also be of metastatic origin. The final diagnosis of synovial carcinomatosis secondary to the known colonic adenocarcinoma was confirmed by histological analysis of biopsies obtained by arthroscopy. A review of the literature is realised. To our knowledge, this is the first synovial metastasis studied by FDG-PET.

[Horton's disease and extra-temporal vessel locations: role of 18FDG PET scan. Report of 3 cases and review of the literature]. / Maladie de Horton et atteintes artérielles extratemporales: utilité de la tomographie par émission de positons au 18FDG. A propos de trois observations et d'une revue de la littérature.

PURPOSE: We report three cases of Horton's disease, in which F18-Fluorine-2-Deoxy-D-Glucose (18FDG) positron emission tomography (PET) demonstrated a clinically unsuspected extra-cranial vessels hypermetabolism. METHODS: Fully corrected whole-body PET was performed in three patients (two women, one man) for exploring a marked inflammatory syndrome. Scanning was acquired 60 min after i.v. injection of 222 MBq of 18FDG in average. RESULTS: In two patients with histologically proven Horton's disease, PET alone showed increased glucose metabolism involving the carotid and sub-clavian arteries as well as the ascending aorta, aortic arch, thoracic and abdominal aorta, and the iliac and femoral arteries. In the third patient, by detecting cervical, thoracic and abdominal vessel hypermetabolism, PET non-invasively contributed to the diagnosis of giant cell arteritis. All patients had complete clinical and biological response to corticoids. PET controls performed 3- to 6-months post-treatment, confirmed the disappearance of the metabolic stigma. CONCLUSION: 18FDG PET may show an increased glucose metabolism in asymptomatic extracranial vessels locations of Horton's arterities. If these observations are confirmed on controlled trials, PET could be particularly useful for non-invasive diagnosing, staging and monitoring atypical clinical forms of Horton's disease. The metabolic imaging could also contribute to a better understanding of the pathogenesis of GCA.

Scintigraphy using a technetium 99m-labelled anti-E-selectin Fab fragment in rheumatoid arthritis.

OBJECTIVE: We previously described a novel radiolabelled monoclonal antibody (1.2B6), which reacts with porcine E-selectin, for targeting activated endothelium as a means of imaging inflammatory disorders, and presented initial clinical work based on (111)In-labelled antibody. The aim of the present study was to evaluate a Fab fragment of 1.2B6 labelled with (99m)Tc in patients with rheumatoid arthritis (RA) by comparison with (i) (111)In-labelled 1.2B6 F(ab')(2) and (ii) conventional bone scanning. METHODS: (99m)Tc-1.2B6-Fab ( approximately 440 MBq) and (111)In-1.2B6-F(ab')(2) ( approximately 27 MBq) were compared in 10 patients using a double-isotope protocol. Images were obtained 4 and 20-24 h after injection. Two normal volunteers were also imaged. In a separate group of 16 patients, (99m)Tc-1.2B6-Fab and (99m)Tc-oxidronate ((99m)Tc-HDP) ( approximately 740 MBq) were compared on the basis of visual and semi-quantitative analysis of joint uptake (joint/soft tissue ratios) 4 h after injection. The respective biodistributions and blood clearances of the two 1.2B6 fragments were also compared. RESULTS: Image contrast was slightly better with (99m)Tc-Fab at 4 h but equal for the two tracers at 24 h. Diagnostic accuracy, taking joint tenderness or swelling as the clinical endpoint, was 76% for both fragments at 24 h. Plasma clearance of (99m)Tc-Fab was faster than that of (111)In-F(ab')(2) (t(1/2) 142 vs 421 min; P<0.0001). (99m)Tc-Fab appeared somewhat unstable in vivo, as shown by activity in the thyroid gland and bowel. The diagnostic accuracy of (99m)Tc-Fab was 88%, higher than that of (99m)Tc-HDP (57%) as a result of the low specificity of the latter in RA. Receiver operating characteristic (ROC) curve analysis using joint/soft tissue ratios as a variable cut-off showed that (99m)Tc-Fab discriminates better than (99m)Tc-HDP between actively inflamed and silent joints (Z=4.72; P<0.0001). No uptake of (99m)Tc-Fab was observed by inactive or normal joints, whereas (99m)Tc-HDP was taken up by all joints to a variable degree, making the decision as to whether a particular joint is actively involved or chronically damaged very difficult. CONCLUSION: (99m)Tc-anti-E-selectin-Fab scintigraphy can be used successfully to image synovitis with better specificity than (99m)Tc-HDP bone scanning. The advantages over (111)In-1.2B6-F(ab')(2) are easier availability of the radionuclide, improved physical properties and optimal imaging 4 h after injection.

The loss of cytoplasmic potassium upon host cell breakdown triggers egress of Toxoplasma gondii.

The ability of intracellular parasites to monitor the viability of their host cells is essential for their survival. The protozoan parasite Toxoplasma gondii actively invades nucleated animal cells and replicates in their cytoplasm. Two to 3 days after infection, the parasite-filled host cell breaks down and the parasites leave to initiate infection of a new cell. Parasite egress from the host cell is triggered by rupture of the host plasma membrane and the ensuing reduction in the concentration of cytoplasmic potassium. The many other changes in host cell composition do not appear be used as triggers. The reduction in the host cell [K(+)] appears to activate a phospholipase C activity in Toxoplasma that, in turn, causes an increase in cytoplasmic [Ca(2+)] in the parasite. The latter appears to be necessary and sufficient for inducing egress, as buffering of cytoplasmic Ca(2+) blocks egress and calcium ionophores circumvent the need for a reduction of host cell [K(+)] and parasite phospholipase C activation. The increase in [Ca(2+)](C) brings about egress by the activation of at least two signaling pathways: the protein kinase TgCDPK1 and the calmodulin-dependent protein phosphatase calcineurin.

Characterization of the subpellicular network, a filamentous membrane skeletal component in the parasite Toxoplasma gondii.

Electron microscopic examination of detergent-extracted Toxoplasma tachyzoites reveals the presence of a mechanically stable cytoskeletal structure associated with the pellicle of this parasite. This structure, composed of interwoven 8-10 nm filaments, is associated with the cytoplasmic face of the pellicle and surrounds the microtubule-based cytoskeleton. Two protein components of this network, TgIMC1 and TgIMC2, were identified. Both are novel proteins, but have a resemblance to mammalian filament proteins in that they are predicted to have extended, coiled-coil domains. TgIMC1 is also homologous to articulins, the major components of the membrane skeleton of algae and free-living protists. A homologue of TgIMC1 in the related malaria parasite Plasmodium falciparum was also identified suggesting the presence of structurally similar membrane skeletons in all apicomplexan parasites. We suggest that the subpellicular network, formed by TgIMC1 and 2 in Toxoplasma gondii and related parasites, plays a role in the determination of cell shape and is a source of mechanical strength.

Toxoplasma gondii attachment to host cells is regulated by a calmodulin-like domain protein kinase.

The role of calcium-dependent protein kinases in the invasion of Toxoplasma gondii into its animal host cells was analyzed. KT5926, an inhibitor of calcium-dependent protein kinases in other systems, is known to block the motility of Toxoplasma tachyzoites and their attachment to host cells. In vivo, KT5926 blocks the phosphorylation of only three parasite proteins, and in parasite extracts only a single KT5926-sensitive protein kinase activity was detected. This activity was calcium-dependent but did not require calmodulin. In a search for calcium-dependent protein kinases in Toxoplasma, two members of the class of calmodulin-like domain protein kinases (CDPKs) were detected. TgCDPK2 was only expressed at the mRNA level in tachyzoites, but no protein was detected. TgCDPK1 protein was expressed in Toxoplasma tachyzoites and cofractionated precisely with the peak of KT5926-sensitive protein kinase activity. TgCDPK1 kinase activity was calcium-dependent but did not require calmodulin or phospholipids. TgCDPK1 was found to be inhibited effectively by KT5926 at concentrations that block parasite attachment to host cells. In vitro, TgCDPK1 phosphorylated three parasite proteins that migrated identical to the three KT5926-sensitive phosphoproteins detected in vivo. Based on these observations, a central role is suggested for TgCDPK1 in regulating Toxoplasma motility and host cell invasion.

Efficacy of Artichoke dry extract in patients with hyperlipoproteinemia.

Efficacy and tolerability of artichoke dry extract (drug/extract ratio 25-35:1, aquous extract, CY450) as coated tablets containing 450 mg extract (tradename: Valverde Artischocke bei Verdauungsbeschwerden) was investigated in the treatment of hyperlipoproteinemia and compared with placebo. 143 adult patients with initial total cholesterol of > 7.3 mmol/l (> 280 mg/dl) were included in a double blind, randomized, placebo controlled, multi-center clinical trial. Patients received 1,800 mg artichoke dry extract per day or placebo over 6 weeks. Changes of total cholesterol and LDL-cholesterol from baseline to the end of treatment showed a statistically significant superiority (p = 0.0001) of artichoke dry extract over placebo. The decrease of total cholesterol in the CY450 group was 18.5% compared to 8.6% in the placebo group. LDL-cholesterol decrease in the CY450 group was 22.9% and 6.3% for placebo. LDL/HDL ratio showed a decrease of 20.2% in the CY450 group and 7.2% in the placebo group. There were no drug related adverse events during this study indicating an excellent tolerability of artichoke dry extract. This prospective study could contribute clear evidence to recommend artichoke dry extract CY450 for treating hyperlipoproteinemia and, thus, prevention of atherosclerosis and coronary heart disease.

Efficacy and tolerability of intranasally applied dimetindene maleate solution versus placebo in the treatment of seasonal allergic rhinitis.

The aim of this study was to investigate the efficacy and tolerability of a 0.1% dimetindene maleate spray (Fenistil Nasal Dosierspray) compared to placebo when applied intranasally. Dimetindene (dimetindene maleate, CAS 3614-69-5, DMM) is a very potent and well established H1-receptor antagonist. A total of 36 asymptomatic patients (17 female, 19 male), suffering from seasonal allergic rhinitis from grass pollen, were randomly assigned to treatment with matching topicalnasal sprays with either dimetindene maleate 0.1% or placebo as control in a double-blind, randomised, cross-over-design, with 2 weeks wash-out periods between. The trial period was chosen in a pollen-free time from 20th October to 5th November 1998 to guarantee asymptomatic patients. The patients being allergic to grass pollen, verified by positive case history, positive skin prick test and positive nasal provocation test, were challenged under controlled conditions with purified airborne grass pollen in the Vienna Challenge Chamber. The nasal spray were applied as single doses (1 puff = 0.14 ml of the respective solution with or without 0.14 mg dimetindene maleate) in the evening before at 7.30 p.m. and in the morning at 7.30 a.m. to each nostril exactly 15 min before the onset of allergen provocation. The dosage scheme relates to a daily dose of 0.56 mg DMM in the active treatment group. Subjective nasal and ocular symptoms were measured on-line in time intervals of 30 min during the 4 h allergen provocation. The statistical analysis was a priori sequentially ordered to account for multiple testing and keep the 5% level of significance. All measured primary criteria, Total Nasal Symptom Score (p < 0.0001) calculated from the three single symptoms running of the nose (p = 0.0032) sneezing stimulus (p < 0.0001) and nasal itching (p < 0.0001), as well nasal secretion (p = 0.0031), resulted consistently in a statistically significant and clinically relevant superiority of 0.1% DMM compared to placebo. The same superior treatment effect was observed for all the other criteria, despite the nasal flow, but including the ocular variables. This can be interpreted as a positive efficacy also in secondary allergic conjunctivitis. No systemic or topical adverse events were reported. The results of the study demonstrate that 0.1% DMM as nasal spray is an efficient and safe application form for patients suffering from seasonal allergic rhinitis.

The microneme protein MIC3 of Toxoplasma gondii is a secretory adhesin that binds to both the surface of the host cells and the surface of the parasite.

Assay of the adhesion of cultured cells on Toxoplasma gondii tachyzoite protein Western blots identified a major adhesive protein, that migrated at 90 kDa in non-reducing gels. This band comigrated with the previously described microneme protein MIC3. Cellular binding on Western blots was abolished by MIC3-specific monoclonal and polyclonal antibodies. The MIC3 protein affinity purified from tachyzoite lysates bound to the surface of putative host cells. In addition, T. gondii tachyzoites also bound to immobilized MIC3. Immunofluorescence analysis of T. gondii tachyzoite invasion showed that MIC3 was exocytosed and relocalized to the surface of the parasite during invasion. The cDNA encoding MIC3 and the corresponding gene have been cloned, allowing the determination of the complete coding sequence. The MIC3 sequence has been confirmed by affinity purification of the native protein and N-terminal sequencing. The deduced protein sequence contains five partially overlapping EGF-like domains and a chitin binding-like domain, which can be involved in protein-protein or protein-carbohydrate interactions. Taken together, these results suggest that MIC3 is a new microneme adhesin of T. gondii.

Hormone synthesis and storage in the thyroid of human preterm and term newborns: effect of thyroxine treatment.

Iodine and thyroglobulin concentrations, as well as iodine, T3, T4 and sialic acid contents of thyroglobulin, were measured in thyroid glands collected postmortem from 42 human premature or term newborns and infants. Three groups were considered: very preterm newborns (24-32 postmenstrual weeks, < 5 days postnatal life), preterm and term newborns (34-41 postmenstrual weeks, < 5 days postnatal life) and infants (born at term, postnatal age 1-8 months). Five very preterm and seven preterm newborns received a daily dose of 10 microg/kg L-T4 for at least 3 days. Thyroid weight and sialic acid content of thyroglobulin progressed with maturation. Intrathyroidal concentrations of iodine and thyroglobulin did not increase significantly before the 42nd week of postmenstrual age. The level of thyroglobulin iodination increased during the postnatal life, except in the very preterm neonates. T4 and T3 content of thyroglobulin was directly proportional to its degree of iodination and positively related to its sialic acid content. L-T4 treatment of preterm newborns increased thyroglobulin iodination and T4-T3 content, without increasing thyroglobulin concentration in the thyroid. It was concluded that the storage of thyroglobulin and iodine in the thyroid develops around term birth. This, associated with the resulting rapid theoretical turnover of the intrathyroidal pool of T4 in Tg, could be an important factor of increased risk of neonatal hypothyroxinemia in the premature infants. The L-T4 treatment of preterm newborns does not accelerate the maturational process of the thyroid gland.

Dimethindene maleate in the treatment of sunburn. A double-blind, placebo-controlled pilot study.

The efficacy of topical dimethindene maleate (DMM, CAS 31614-69-5, Fenistil Gel) in the treatment of sunburn was evaluated in a placebo-controlled, 1-period crossover trial in 24 healthy volunteers. An UV-erythema (sunburn) of a well-defined intensity and extent was experimentally induced on three different skin test-areas by means of UV-A/B irradiation with three times the minimal erythema dose (MED). About 24 h after irradiation, one skin test-area was subjected to a 1-h occlusive treatment with DMM gel, the second test area was subjected to treatment with a placebo gel and the third one remained untreated. As objective-quantitative indicators of tenderness, a key symptom of sunburn, sensory and pain thresholds to CO2-Laser stimulation and laser somatosensory evoked potentials (SEPs) in Vertex-EEG were assessed about 1.5 h postdose. The reaction times (RTs) to painless and painful CO2-laser stimulation (sensory and pain threshold level, respectively) on the DMM-treated area were significantly longer than RTs to stimulation on the placebo-treated area. Thresholds in terms of laser energy showed no differences between the treatments. The SEP N1-amplitude on the DMM-area was markedly decreased in comparison to placebo. With regard to subjective sensations of pain, itching and tenderness assessed by means of visual analogue scales (VAS), no clinically relevant differences between treatments were observed after sole UV-irradiation. After additional laser stimulation tenderness was--objectively but not subjectively--decreased on the DMM-area versus placebo. Both gel preparations were well tolerated.

Recommended restrictions after 131I therapy: measured doses in family members.

Absorbed doses to family members of patients treated with (131)I were measured using thermoluminescent dosimeters worn on the chest. Twenty-two patients with thyroid cancer were hospitalized for 2 d for treatment with 3,700-7,400 MBq, and 18 hyperthyroid patients were treated on an outpatient basis with 200-600 MBq. Doses were measured over periods of 15-21 d following the administration of radioiodine in 35 partners and 38 children, aged 4 mo to 25 y. These results were correlated with dose rate measurements performed with an ionization chamber, and residual thyroid uptake was assessed by scintigraphy over the same period. In the cancer group, the residual activity in thyroid remnants was less than 50 MBq in all cases at day 4 following treatment and decayed with a mean half-life of 2.2 (SD: 0.8) d. The dose measured with thermoluminescent dosimeters was lower than 0.5 mSv in all partners and children. In the hyperthyroid group, the effective half-life averaged 6.2 (SD: 1.2) d. The median of the doses measured in partners and children were 1.04 mSv (range: 0.05-5.2) and 0.13 mSv (range: 0.04-3.1), respectively. Fifteen children (88%) received less than the dose constraint of 0.5 mSv. The ICRP recommend an annual limit of 1 mSv for the members of the public. In addition, dose constraints (for example: 0.5 mSv) should be complied with whenever possible. The recommended dose limits are generally well met among family members of patients treated with 1311 for cancer. The higher doses measured in hyperthyroid patients, compared to thyroid cancer patients, relate to a higher (131)I retention by the gland and justify more extended and stringent restriction periods, based on residual thyroid activity.

131I therapy of toxic and non-toxic goiters.

For more than 50 years, 131I has been recognized as an effective tool for controlling thyroid hyperplasia and hyperactivity. The fixed dose administration is the simplest method with doses of 111-370 MBq (3 to 10 mCi) 131I being administered. More sophisticated methods aiming to deliver a well-defined amount of 131I per gram of thyroid tissue are handicapped by problems related to the evaluation of the goiter size and the prediction of the sensitivity of thyroid cells to radiation. The use of 131I in nontoxic multinodular goiter is to be reserved for specific situations.

MFR, a putative receptor mediating the fusion of macrophages.

We had previously identified a macrophage surface protein whose expression is highly induced, transient, and specific, as it is restricted to actively fusing macrophages in vitro and in vivo. This protein is recognized by monoclonal antibodies that block macrophage fusion. We have now purified this protein and cloned its corresponding cDNA. This protein belongs to the superfamily of immunoglobulins and is similar to immune antigen receptors such as the T-cell receptor, B-cell receptor, and viral receptors such as CD4. We have therefore named this protein macrophage fusion receptor (MFR). We show that the extracellular domain of MFR prevents fusion of macrophages in vitro and therefore propose that MFR belongs to the fusion machinery of macrophages. MFR is identical to SHPS-1 and BIT and is a homologue of P84, SIRPalpha, and MyD-1, all of which have been recently cloned and implicated in cell signaling and cell-cell interaction events.

Iodine-131-MIBG scintigraphy in adults: interpretation revisited?

UNLABELLED: Iodine-131-metaiodobenzylguanidine (MIBG) scintigraphy is a reliable method used to diagnose pheochromocytoma. Although the adrenal medulla usually is not visualized, faint uptake can be observed in 16% of the patients 48-72 hr after injection of 18.5-37 MBq 131I-MIBG. We recently observed an increase in the frequency of visualization of the adrenal medulla in patients injected with 74 MBq 131I-MIBG. Therefore, we retrospectively evaluated the pattern of uptake and potential changes between 1984 and 1994. METHODS: Scintigraphic data from 103 patients referred for suspected pheochromocytoma were reviewed randomly. Data from 19 patients with medullary thyroid carcinoma were analyzed separately. Patients were injected with 74 MBq 131I-MIBG and imaged at 24 hr postinjection, 48 hr postinjection, or both. Adrenal uptake was scored visually as 0 (no visible uptake) and 1 (uptake just visible) to 4 (most intense activity in the picture). Semiquantitative indicies were evaluated for discriminating between normal adrenal medullae and pheochromocytomas. Twenty-seven pheochromocytomas were surgically proven in 25 patients. RESULTS: A visual score > or =3 was noted in 81% and 90% of the pheochromocytomas at 24 hr and 48 hr postinjection, respectively. From 1984 to 1988, 16% and 31% of adrenal medullae were seen at 24 and 48 hr postinjection, respectively, whereas from 1989 to 1994, 56% and 73% were visualized at 24 and 48 hr postinjection, respectively. Before 1989, the best cutoff criterion to identify a pheochromocytoma, determined from receiver operating characteristic curve analysis, was a score > or =1 at 24 hr and > or =3 at 48 hr postinjection, with a sensitivity and specificity of 92% and 84% at 24 hr and 92% and 99% at 48 hr postinjection. From 1989, the best cutoff was a score > or =3 at both imaging sessions, with a sensitivity and specificity of 82% and 100% at 24 hr and 100% and 97% at 48 hr postinjection. Among the semiquantitative indicies, the adrenal-to-liver and adrenal-to-heart ratios were the best discriminators between normal and pathological adrenals. They were, however, of little use because of the overlap between normal adrenal medullae and pheochromocytomas. CONCLUSION: The high rate of visualization of the normal adrenal medulla in this study was related to the larger-than-usual injected dose (74 MBq). Over recent years, however, this rate has been increasing, possibly because of the increased specific activity of 31I-MIBG. Adequate interpretation should take into account that a faint or definite uptake may be visible in more than 50% of normal adrenal medullae.