Bridging the gap: heparan sulfate and Scube2 assemble Sonic hedgehog release complexes at the surface of producing cells.

Decision making in cellular ensembles requires the dynamic release of signaling molecules from the producing cells into the extracellular compartment. One important example of molecules that require regulated release in order to signal over several cell diameters is the Hedgehog (Hh) family, because all Hhs are synthesized as dual-lipidated proteins that firmly tether to the outer membrane leaflet of the cell that produces them. Factors for the release of the vertebrate Hh family member Sonic Hedgehog (Shh) include cell-surface sheddases that remove the lipidated terminal peptides, as well as the soluble glycoprotein Scube2 that cell-nonautonomously enhances this process. This raises the question of how soluble Scube2 is recruited to cell-bound Shh substrates to regulate their turnover. We hypothesized that heparan sulfate (HS) proteoglycans (HSPGs) on the producing cell surface may play this role. In this work, we confirm that HSPGs enrich Scube2 at the surface of Shh-producing cells and that Scube2-regulated proteolytic Shh processing and release depends on specific HS. This finding indicates that HSPGs act as cell-surface assembly and storage platforms for Shh substrates and for protein factors required for their release, making HSPGs critical decision makers for Scube2-dependent Shh signaling from the surface of producing cells.

Effect of decorin on overcoming the extracellular matrix barrier for oncolytic virotherapy.

The pressing challenge for contemporary gene therapy is to deliver enough therapeutic genes to enough cancer cells in vivo. With the aim of improving viral distribution and tumor penetration, we explored the use of decorin to enhance viral spreading and tumor tissue penetration. We generated decorin-expressing replication-incompetent (dl-LacZ-DCNG, dl-LacZ-DCNQ and dl-LacZ-DCNK) and replication-competent (Ad-DeltaE1B-DCNG, Ad-DeltaE1B-DCNQ and Ad-DeltaE1B-DCNK) adenoviruses (Ads). Point mutants of decorin gene (DCNG), DCNK and DCNQ, have a negative and moderate binding affinity to type-I collagen fibril, respectively. In both tumor spheroids and established solid tumors in vivo, tissue penetration potency of dl-LacZ-DCNG was greatly enhanced than those of dl-LacZ, dl-LacZ-DCNQ and dl-LacZ-DCNK, and this enhanced tissue penetration effect derived from decorin-expressing Ad was dependent on the binding affinity of decorin to collagen fibril. Expression of DCNG enhanced viral spread of replicating Ad, leading to improved tumor reduction and survival benefit. Moreover, the tumoricidal effects of Ad-DeltaE1B-DCNQ and Ad-DeltaE1B-DCNK were lessened, as the binding affinity to collagen was decreased, showing that the increased cancer cell cytotoxicity was driven by the action of decorin on extracellular matrix (ECM). Furthermore, Ad-DeltaE1B-DCNG substantially decreased ECM components within the tumor tissue. Finally, intratumoral injection of Ad-DeltaE1B-DCNG in primary tumor site greatly reduced the formation of B16BL6 melanoma cell pulmonary metastases in mice. Taken together, these data show the utility of decorin as a dispersion agent and highlight its utility and potential in improving the efficacy of replicating Ad-mediated cancer gene therapy.

TGF-beta1 generates a specific multicomponent extracellular matrix in human coronary SMC.

BACKGROUND: Transforming growth factor (TGF-beta(1)) is postulated to play an important role in maintaining the structure and function of arterial tissue and protection against development of arteriosclerosis. The TGF-beta(1)-induced production of a stable extra-cellular matrix-rich plaque phenotype is suggested to be part of the protection against a switch to an unstable rupture-prone arteriosclerotic plaque. MATERIALS AND METHODS: This study addresses the question of whether the expression profile and the type of extra-cellular matrix (ECM) generated by TGF-beta(1) stimulation have the structural feature of a fibril-rich stable matrix. Seventeen genes codings for ECM components of human coronary smooth muscle cells (SMCs) after a 24-h stimulation by TGF-beta(1) have been analyzed. RESULTS: Real-time RT-PCR was used to quantify the mRNA of genes under investigation. It was found that after TGF-beta(1) stimulation (a) the up-regulation of COL1A1-specific mRNA was associated with increased [(3)H]proline incorporation into the alpha-1 and -2 chains of collagen type I, (b) the up-regulation of biglycan- and syndecan-1-specific mRNA corresponded to an increased [(35)S]sulphate and [4,5-(3)H]leucine incorporation into the biglycan molecule and to an increase of syndecan-1 protein, (c) the up-regulated FGF-2 gene accounted predominantly for the ECM-bound subfraction of FGF-2-protein and (d) fibronectin and thrombospondin exhibited a significantly higher mRNA level. In contrast collagen XIV, a minor collagen type, and the proteoglycan decorin were down-regulated. The down-regulated decorin changed its structure by elongation and reduced GlcA to IdoA epimerization of the dermatan sulphate side-chain as judged by [(35)S]sulphate metabolic labelling experiments. No significant changes in response to TGF-beta(1) were observed for the collagen types III, VI and XVI, for versican, perlecan and the syndecans-2 and -4. CONCLUSIONS: It was concluded from the data that the TGF-beta(1)-induced formation of a highly specific multicomponent extra-cellular matrix on coronary arterial SMCs could provide in vivo mechanical strength to the neointima in arteriosclerotic lesions and to the fibrous cap overlying the lipid core.

Different usage of the glycosaminoglycan attachment sites of biglycan.

Biglycan is a member of the small leucine-rich proteoglycan family. Its core protein comprises two chondroitin/dermatan sulfate attachment sites on serine 42 and serine 47, respectively, which are the fifth and tenth amino acid residues, respectively, after removal of the prepro peptide. Because the regulation of glycosaminoglycan chain assembly is not fully understood and because of the in vivo existence of monoglycanated biglycan, mutant core proteins were stably expressed in human 293 and Chinese hamster ovary cells in which i) either one or both serine residues were converted into alanine or threonine residues, ii) the number of acidic amino acids N-terminal of the respective serine residues was altered, and iii) a hexapeptide was inserted between the mutated site 1 and the unaltered site 2. Labeling experiments with [(35)S]sulfate and [(35)S]methionine indicated that serine 42 was almost fully used as the glycosaminoglycan attachment site regardless of whether site 2 was available or not for chain assembly. In contrast, substitution of site 2 was greatly influenced by the presence or absence of serine 42, although additional mutations demonstrated a direct influence of the amino acid sequence between the two sites. When site 2 was not substituted with a glycosaminoglycan chain, there was also no assembly of the linkage region. These results indicate that xylosyltransferase is the rate-limiting enzyme in glycosaminoglycan chain assembly and implicate a cooperative effect on the xylosyl transfer to site 2 by xylosylation of site 1, which probably becomes manifest before the removal of the propeptide. It is shown additionally that biglycan expressed in 293 cells may still contain the propeptide sequence and may carry heparan sulfate chains as well as sulfated N-linked oligosaccharides.

Heroin and opiate emergencies in Vienna: analysis at the municipal ambulance service.

Vienna suffered an epidemic of heroin abuse in recent years, with drug-deaths due to opioids increasing from 62 in 1991 to 143 in 1993. The aim of this study was to make observations about illicit opioid-use with the ambulance service as a data source. From June 1994 to August 1995, the structured run records of the ambulance service were reviewed. Those with a presumptive diagnosis of "heroin or opiate" overdose were collected, characteristics of emergencies and patients were analyzed. The run records demonstrated a large number of non-fatal emergencies due to opioids, involving 528 men and 179 women in 1087 emergencies. These emergencies were on the average 6.8 times as prevalent as drug-fatalities. A group of 189 persons could be identified, who caused 52.2% of all emergencies and showed a threefold mortality rate during the observation period. In Vienna, the records of the municipal ambulance service provided valuable insights on opioid-abuse. We suggest local analysis of non-fatal emergencies due to opioids, as this might lead to a new source of information on illicit abuse of these drugs.

Expression of fibroblast growth factors 1 and 2 in a human retinal pigment epithelium cell line (K1034).

The primary source of fibroblast growth factors (FGFs) is the retinal pigment epithelium (RPE). Investigations on FGF secretion in RPE primary cultures are hampered by the rapid run-down of cell vitality after a few passages. Therefore, long-term experiments require an alternative to primary cultures. We detected FGF-1 and FGF-2 in the established human K1034 cell line by immunohistochemistry. In addition, mRNA for both FGFs was found by RT-PCR. By immunohistochemistry, the signal was more pronounced with FGF-2 than with FGF-1. K1034 is capable of expressing both FGF-1 and FGF-2. With respect to these features, this cell line can be used as an alternative to primary cultured human RPE cells.

Referral of outpatients with non-traumatic conditions for radiographic examinations in an emergency department.

The aim of the study was to evaluate the referral of outpatients with non-traumatic conditions for radiographic examinations and to assess the impact of the radiological report on the patient's management in an emergency department. In a prospectively designed study, 1223 X-ray examinations of 1116 non-trauma outpatients (640 males, 476 females; mean age: 44 +/- 18 years) requested over a 10-month period were evaluated. Patients were classified into four groups according to the presenting complaints (respiratory, abdominal, neurological or non specific symptoms). Analysis of data included the influence of age and presenting symptoms on the likelihood of abnormal radiological findings and the impact of the radiological result on the further management of the patient. In 455 (40.8%) patients an abnormal radiological result was observed. The likelihood of an abnormal radiological findings increased with age (age < 40 years: 33%; age > 40 years; 47%; p < 0.05). Whereas the rate of abnormal radiological findings was high in patients with specific symptoms (respiratory: 69%; abdominal: 37%; neurological: 38%), in patients with non-specific symptoms only 25% of all radiological examinations revealed an abnormality. The radiological result had an impact on the further management in 948 (85%) patients. As 45% of all radiographic examinations revealing a normal radiological result had a clinical impact, normal radiological reports are just as helpful as abnormal radiological findings in the management of non-trauma outpatients in an emergency department. Thus, we assume that the radiological result has a major impact on the management of non-trauma outpatients in the emergency department.

Accuracy and reliability of noninvasive continuous finger blood pressure measurement in critically ill patients.

OBJECTIVE: To evaluate the accuracy and reliability of noninvasive continuous finger blood pressure measurement in critically ill patients. DESIGN: Prospective data collection. SETTING: Emergency department in a 2,000-bed hospital. PATIENTS: Thirty-nine patients admitted to the emergency department requiring invasive arterial blood pressure monitoring were enrolled to the study protocol. INTERVENTIONS: Continuous noninvasive blood pressure measurement was performed on the middle phalanx of the second and third finger, using a test instrument which provides continuous arterial waveform display with the use of a finger cuff. Invasive mean arterial blood pressure measurement was done by cannulation of the radial artery and direct transduction of the systemic arterial pressure waveform. MEASUREMENTS AND MAIN RESULTS: Three thousand one hundred eighteen pairs of simultaneous finger cuff and intra-arterial blood pressure measurements were collected in 1-min intervals from 39 patients over a total of 51.8 hrs. The overall discrepancy between both measurements was 0.10 mm Hg. The standard deviation of the differences was +/- 5.02 mm Hg. The mean bias in patients treated with catecholamines was 0.01 mm Hg and was not different from the bias observed in patients without catecholamines (mean bias: 0.23 mm Hg; p > .22). Whereas 95% of all comparisons between finger cuff and intra-arterial measurement had a discrepancy < or = +/- 10 mm Hg, 4.7% had a discrepancy between +/- 10.1 to 15 mm Hg and 0.3% exhibited a discrepancy > +/- 15 mm Hg. In 29 (74%) patients, the duration of errors > 10 mm Hg was < or = 1 min. In seven (18%) patients, the duration of errors > 10 mm Hg was between 2 to 3 mins and in three (8%) patients, the errors lasted for > 3 mins. CONCLUSIONS: Our data provide a guide to the accuracy and reliability of noninvasive finger blood pressure measurements in critically ill patients. Although most test instrument measurements were reliable, in 8% of all patients large discrepancies (> 10 mm Hg) between both measurements with a duration of > 3 mins were noted. Concerning the considerable risk for arterial cannulation, our preliminary data demonstrate that the test instrument (PORTAPRES, TNO Biomedical Instrumentation Research Unit; The Netherlands) is an advance in noninvasive monitoring of critically ill patients and may be useful in most emergency clinical settings.

After antagonization of acute opiate overdose: a survey at hospitals in Vienna.

This study describes the clinical management and characteristics of people who, following acute opioid overdose, are taken to hospital after efficient antagonization by the pre-hospital emergency service. In addition, it defines areas of interest for further research. Over a 4-month period (September-December 1993) we collected data by a structured protocol sheet on patients' characteristics, anamnestic data on abuse and emergencies, clinical presentation, treatment by specific antidote and routine laboratory investigations. Outcome was verified by retrospective review of prehospital and forensic data. We studied 77 subjects, predominantly young males, who were involved in 83 emergencies, mostly occurring at weekends. In more than 60% of cases a single administration of specific antidote sufficed to stabilize the patients; 64% of patients left hospital against medical advice after an average stay of less than 6 hours; 46% denied daily opioid abuse and half the subjects, especially younger drug-users, seemed interested in counselling. This hospital-based study did not provide reliable data on the epidemiology of opioid overdose. Clinical management is determined by experience, pragmatism and beliefs. Efforts towards secondary prevention of drug problems at emergency departments might be warranted, and further research on pattern and management of opioid overdose is needed.

Efficacy of different antihypertensive drugs in the emergency department.

Hypertensive crises are a commonly observed problem in an emergency department. The aim of the study was to evaluate the efficacy and safety of different antihypertensive agents in the treatment of patients with hypertensive crises. 168 patients (mean age: 52 +/- 12 years) admitted to the emergency department with a hypertensive urgency (systolic (SBP) blood pressure > 210 mm Hg and/or diastolic (DBP) blood pressure > 110 mm Hg) or a hypertensive emergency (DBP > 100 mm Hg and evidence of end-organ damage) were included into the study protocol. Blood pressure (BP) was measured every 5 min automatically using a noninvasive BP measurement unit. After a resting period of 30 min the patients received the following drugs: 5 mg enalaprilat intravenous (n = 43) or 25 mg urapidil intravenous (n = 48) or 10 mg nifedipine-capsule sublingual (n = 47) or 2 x 5 mg nifedipine-spray sublingual (n = 30). The aim of treatment was to reduce SBP below 180 mm Hg and DBP below 95 mm Hg within 45 min after start of treatment. When evaluating the response rates the highest rate was observed in the urapidil group (96%). The response rate of enalaprilat and both preparations of nifedipine were similar (70-72%). The rate of major side effects was higher in the urapidil compared to the other drugs (4% vs 2% in the nifedipine-group or 0% in the enalaprilat-group). All four drugs are suitable in the treatment of patients with hypertensive crisis in the emergency department. Urapidil should be used as a first choice drug in critically ill patients with hypertensive crisis due to its higher response rate.

Prehospital opiate emergencies in Vienna.

To establish baseline data on prehospital emergencies caused by opiates during a 4-month period, a retrospective analysis of run records of the Emergency Medical System in Vienna, the capital of Austria, was conducted. During the study period, there were 308 opioid emergencies involving 240 persons, an average of 2.5 overdoses per day. Severely compromised patients were treated in 67.8% of the 308 emergencies, and 79.3% of emergencies were transported to hospital; 52.5% of the involved persons were younger than 22 years of age. Sex distribution and periodicity and frequency of emergencies differed among age groups. A subgroup of individuals involved repeatedly in emergencies was identified, partly showing temporal clustering of fatal and nonfatal overdoses. Persons involved in opiate emergencies belong to heterogenous subgroups. At a local level, research should be initiated to clarify the pattern and impact of these emergencies on overall drug abuse prevention.

[Evaluation system in assessing radiological examinations of clinical significance for the patient]. / Bewertungssystem zur Erfassung des Stellenwertes radiologischer Leistungen für die klinische Arbeit am Patienten.

PURPOSE: The purpose of this study was to test a scoring system and its utility for the assessment of the clinical significance of radiological examinations. MATERIAL AND METHODS: The reports of all diagnostic studies obtained in patients with a clinical suspicion of pancreatitis, urinary tract obstruction, biliary or gallbladder diseases, ileus and gastrointestinal perforation were scored prospectively in 4 categories from 0 to 3. These categories were related to the ability to establish a definite diagnosis, to exclude suspected disease, to influence further diagnostic work-up and therapeutic decision-making. RESULTS: This scoring system showed significant differences of the efficacy of different imaging modalities in these 5 clinical settings. CONCLUSION: This scoring system was helpful to determine the clinical significance of radiological examinations.

Pulmonary embolism. II. Diagnosis and treatment.

Pulmonary embolism is an often underestimated, underdiagnosed, and undertreated disease. As symptoms and signs of pulmonary embolism are nonspecific, the diagnosis still remains a challenge to the attending physician. Diagnostic and therapeutic procedures depend on the clinical presentation of the patient. First we must suspect pulmonary embolism and consider its likelihood in the presence of a number of clinical signs and symptoms. Once pulmonary embolism is suspected, heparin should be administered. Additional basic support is mandatory if required. If the patient's hemodynamic situation is stable, available preferable noninvasive diagnostic options should be considered to confirm or rule out the diagnosis of venous thromboembolism before further administration of anticoagulant or thrombolytic agents. If the patient's status has deteriorated, bedside diagnostic techniques should be applied to reinforce the suspicion or establish the diagnosis. To restore pulmonary perfusion more rapidly than conventional anticoagulation is suspected to do, several dosing regimens of thrombolytic agents are proposed, with recent interest in short-term thrombolysis and bolus lysis with urokinase or recombinant tissue plasminogen activator. If thrombolysis fails or is contraindicated, catheter embolectomy or surgical embolectomy is indicated. The main therapy is prevention. In this article, clinical assessment, imaging techniques, and therapeutic options described in the published literature are discussed and clinical experiences of an emergency department with a noninvasive diagnostic approach are described.

Hormonal derangements in patients with severe alcohol intoxication.

Controversial results of fluid and electrolyte derangements in patients with moderate alcohol intoxication have been described. However, no information is available about severe alcohol intoxication. We investigated differences of hormonal disorders between alcohol-habituated and alcohol-naive subjects with severe ethanol intoxication. The hormonal derangements and recommendations on therapy of these patients are discussed. Thirty-three patients [10 alcohol-naive (group A) and 23 alcohol-habituated (group B) subjects] with severe alcohol intoxication (blood ethanol > 200 mg/dl) were selected for the study. Electrolytes and osmolarity of serum and urine, blood ethanol, vasopressin, renin, and aldosterone were determined on admission 2, 4, and 6 hr later. Fluid balance was calculated for each hour. All patients received isotonic saline solution according to urine production. Group A: On admission, serum osmolarity was increased (308 mOsmol/kg). Concomitantly, vasopressin level was elevated on admission (9.12 pg/ml). Increased serum osmolarity was correlated with elevated vasopressin levels (r = 0.8211; p < 0.005). Serum electrolytes, renin, and aldosterone values were within normal ranges. Group B: On admission, vasopressin level was significantly decreased (0.9 pg/ml), despite an elevated serum osmolarity (309 mOsmol/kg). Serum osmolarity remained high despite a sufficient fluid substitution. In addition, vasopressin level remained suppressed over the observation period. Aldosterone level was significantly increased on admission (319 ng/ml). Accordingly, serum sodium was increased from 142 to 148 mM/liter, and serum potassium was decreased from 3.9 to 3.4 mM/liter. Response to hyperosmolarity due to severe alcohol intoxication is different in alcohol-naive and alcohol-habituated subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of low dose sedation with diazepam on ventilatory response at moderate altitude.

UNLABELLED: Benzodiazepines are in wide use in out of hospital emergency medicine in at sea level, as well as in alpine rescue services. Ventilatory depression is a common side effect of high dose intravenous benzodiazepines. The objective of our study was to evaluate the effect of low dose benzodiazepines on ventilation at moderate altitude. DESIGN: Randomized, double blind, placebo controlled crossover trial. Blood gas analysis of 8 healthy subjects was performed before and one hour after 5 mg of oral diazepam or placebo at 171 m and at 3,000 m altitude. PaO2 and PaCO2 before and after diazepam or placebo medication at 171 m did not change significantly. At 3,000 m altitude. PaO2 decreased and PaCO2 increased significantly one hour after 5 mg of oral diazepam (PaO2 64.7 +/- 3.4 vs. 60.0 +/- 1.5 mmHg, PaCO2 28.3 +/- 2.3 vs. 33.3 +/- 1.8 mmHg, p < 0.05). Placebo did not influence PaO2 and PaCO2. CONCLUSION: Low dose diazepam inhibits ventilatory adaptation to mild hypoxia at moderate altitude. Caution in benzodiazepine use in moderate altitude is therefore necessary.

[Local complications after poisonous snake bite]. / Lokale Komplikationen nach einem Giftschlangenbiss.

The case of a zoo keeper who was bitten on the left finger by a venomous snake (Vipera xanthina) is reported. The administration of antivenom prevented the development of systemic poisoning but had no effect on the extent of the local complications. A compartment syndrome with a concomitant severe reaction at the bite site required fasciotomy of the upper and lower arm. The extensor tendon of the involved finger ruptured spontaneously, many weeks after wound healing was completed. Therefore, delayed local complications following snake bites may occur, even if signs of systemic poisoning are missing.

[Carbamazepine poisoning: protracted course with development of intestinal atony and hepatic toxicity]. / Intoxikation mit Carbamazepin: Protrahierter Verlauf unter Entwicklung von Darmatonie und Hepatotoxizität.

A 43 year-old female patient with a history of manic-depressive illness and prophylactic carbamazepine (CBZ) medication ingested a potentially lethal overdose of 20 g of the substance. Neurotoxic symptoms reached full intensity after about 24 hours. Subsequently, the patient developed a gastrointestinal atony, which proved to be refractory to treatment for several days. Moreover, there was an increase of bilirubin. Parallel to this we observed the persistence-and even intermittent re-increase--of toxic serum CBZ concentrations for one week with corresponding protracted clinical symptomatology.