Phosphorylation of human pro-urokinase on Ser138/303 impairs its receptor-dependent ability to promote myelomonocytic adherence and motility.

Serine phosphorylation of human pro-urokinase (pro-uPA) by A431 human carcinoma cells results in a catalytically active molecule with reduced sensitivity to plasminogen activator inhibitor type 1. We mapped the phosphorylated seryl residues by analyzing the in vivo phosphorylation state of engineered pro-uPA variants carrying a COOH-terminal poly-histidine tag. Stably transfected A431 cells do not incorporate radioactive phosphate into tagged pro-uPA in which the serines 138 and 303 have been replaced with glutamic residues, although endogenous nontagged pro-uPA is 32P-labeled on A and B chains. Moreover, the catalytic-independent ability of the mono- and di-substituted "phosphorylation-like" variants to bind to the GPI-anchored urokinase receptor (uPAR) and promote adherence of differentiating U937, HL-60, and THP-1 myelomonocytic cells was examined. We found that glutamic residues as well as the naturally occurring phosphoserines at positions 138 and 303 abolish proadhesive ability, although they do not interfere with receptor binding. In addition, pro-uPA carrying Glu138/303 lacks the capability to induce a chemotactic response of THP-1 cells. The exclusive presence of Glu138 reduces pro-uPA proadhesive and chemotactic ability by 70-80%, indicating that a phosphoserine residue at the same position plays a major inhibitory role of myeloid cell response to pro-urokinase. The di-substitution does not affect pro-uPA ability to interact with vitronectin or to enhance binding of urea-denatured vitronectin to uPAR. However, unlike wild-type tagged pro-uPA, the di-substituted variant does not induce receptor polarization in pre-adherent U937 cells. Taken together, the data support the possibility that pro-uPA phosphorylation on Ser138/303 can modulate uPAR transducing ability.

A high throughput assay for inhibitors of HIV-1 protease. Screening of microbial metabolites.

A novel method for discovery of HIV-1 protease inhibitors in complex biological samples has been developed. The assay is based on two specific reagents: a recombinant protein constituted by a portion of the HIV-1 Gag polyprotein comprising the p17-p24 cleavage site, fused to E. coli beta-galactosidase, and a monoclonal antibody which binds the fusion protein in the Gag region. Binding occurs only if the fusion protein has not been cleaved by the HIV-1 protease. The assay has been adapted for the screening of large numbers of samples in standard 96-well microtiter plates. Using this method about 12000 microbial fermentation broths have been tested and several HIV-1 protease inhibitory activities have been detected. One of these has been studied in detail.

Differential detection of single-chain and two-chain urokinase-type plasminogen activator by a new immunoadsorbent-amidolytic assay (IAA).

A new immunoadsorbent-amidolytic assay (IAA) for the specific differential detection of two-chain urokinase-type plasminogen activator (tcu-PA) and its single-chain precursor (scu-PA) in cell culture supernatants has been developed. The assay combines the selectivity of immunoassays with the specificity of enzyme activity assays exploiting both the antigenic and enzymatic properties of the two proteins. tcu-PA and scu-PA are selectively immunoadsorbed on the wells of a microtiterplate coated with the monoclonal antibody 5B4 and tested for enzymatic activity before and after activation by plasmin treatment. Both proteins are determined with similar efficiency since overlapping dose-response curves were obtained in the range between 12.5-200 ng/ml. The assay has been used to determine tcu-PA and scu-PA in A431 human epidermoid carcinoma cell supernatants. The analytical recoveries for tcu-PA and scu-PA added to A431 cell supernatants were 95.2% and 96.9% respectively. The intra- and inter-assay variations (CV) were 5.5% and 9.0% for tcu-PA and 9.7% and 9.8% for scu-PA respectively.

A monoclonal antibody that recognizes the receptor binding region of human urokinase plasminogen activator.

An anti-urokinase monoclonal antibody 5B4 (MAB 5B4) was obtained by fusing the murine myeloma cell line X63-Ag8.653 with the spleen cells from a female BALB/c mouse immunized with high-molecular-weight urokinase (HMW-uPA). MAB 5B4 is an IgG1 that binds selectively to the single-chain form of uPA (sc-uPA), to HMW-uPA and to the 17,000 Mr aminoterminal fragment of the A-chain (ATF) but not to the low-molecular-weight urokinase (LMW-uPA) nor to the reduced form of HMW-uPA. This strongly suggests that MAB 5B4 recognizes a conformational determinant on the A-chain. The antibody has an affinity constant for uPA-Sepharose of 1.42 X 10(7) M-1, calculated from equilibrium binding data, and can be used for one step purification of HMW-uPA by immunoaffinity chromatography. MAB 5B4 and the previously obtained antibody 105IF10 recognize the A-chain: the epitopes, however, are distinct as shown by double-antibody-sandwich enzyme immunoassay. Finally MAB 5B4 inhibits the binding of ATF to the uPA receptor of different human cells, whereas 105IF10 does not. Thus this antibody represents a potentially, useful tool for the study of uPA receptor physiology.

Purification and characterization of single-chain urokinase-type plasminogen activator (pro-urokinase) from human A431 cells.

A single-chain urokinase-type plasminogen activator (A431sc-uPA) was purified approximately 18,000-fold from A431 human epidermoid carcinoma cell supernatants by monoclonal antibody immunoaffinity chromatography on 5B4-agarose and ion-exchange FPLC (overall yield 63%). More than 100 micrograms of A431sc-uPA can be recovered per liter of supernatant. The product is homogeneous by SDS-PAGE and reverse phase FPLC analysis while two main isoelectric forms of pI 9.05 and pI 9.20 were observed by IEF. SDS-PAGE in reducing and non-reducing conditions, Western blot analysis and zymography showed that A431sc-uPA is a single-chain protein of about 50,000 Mr immunologically related to urokinase (uPA) and distinct from tissue plasminogen activator (tPA). The N-terminal aminoacid sequence of A431sc-uPA (27 residues) is identical to that of human kidney single-chain uPA. A431sc-uPA does not incorporate 3H-diisopropylfluorophosphate and is virtually inactive on the synthetic substrate S-2444. Plasmin treatment converts A431sc-uPA into a two-chain active form with a fibrinolytic specific activity of 123,000 I. U./mg.

The differential glycosylation of human pro-urokinase from various recombinant mammalian cell lines does not affect activity and binding to PAI-1.

Human chromosomal DNA encoding single-chain urokinase-type Plasminogen Activator (scu-PA, or pro-urokinase) was inserted in an expression plasmid and transfected in human A431, mouse LB6 and CHO cells. LB6 cells were also transfected with a Bovine Papilloma Virus derivative containing the scu-PA gene. Human scu-PA was purified from cell supernatants of recombinant clones and characterized for structure and function. All recombinant scu-PAs are undistinguishable from human urine-derived scu-PA for peptide backbone, but possess a higher sugar content, as revealed by SDS-PAGE analysis after digestion with glycopeptidase F. This difference is partly due to an increased sialic acid content, as shown by analysis of neuraminidase-treated scu-PAs. No difference was found, however, among recombinant and natural scu-PAs in the kinetics of conversion into two-chain active forms (tcu-PAs) by human plasmin, and in the KM and kcat values of tcu-PA activity on the chromogenic substrate S-2444 and on human plasminogen. Also, recombinant and non-recombinant tcu-PAs displayed similar dose-response curves for binding to the endothelial inhibitor PAI-1. In conclusion, the glycosylation pattern of u-PA does not affect its interaction with the plasma proteins directly involved in its fibrinolytic function.

Epitope mapping of the anti-urokinase monoclonal antibody 5B4 by isolated domains of urokinase.

The amino terminal fragment (ATF) of urokinase-type plasminogen activator (uPA) is a degradation product comprising the entire growth factor-like and kringle domains. It has been previously shown that ATF is able to bind to the u-PA receptor through the growth factor-like domain and that the anti u-PA monoclonal antibody 5B4 (Mab 5B4) binds to ATF preventing u-PA receptor binding. To localize more precisely the epitope recognized by Mab 5B4, ATF was subfragmented by controlled enzymatic proteolysis with V8 protease. Three subfragments of 4,000 Mr (F-4k), 11,000 Mr (F-11k) and 12,000 Mr (F-12k) were purified from the reaction mixture and characterized. SDS-PAGE under reducing and non-reducing conditions, N-terminal aminoacid sequence analysis and C-terminal aminoacid analysis of each fragment indicate that F-4k and F-11k correspond to intact growth factor-like domain and kringle domain (residues 4-43 and 44-135 respectively) while F-12k corresponds to the kringle domain cleaved in the first loop at the glu52-gly53 bond. By Western blot and competitive binding experiments we show that Mab 5B4 recognizes an epitope located on the kringle domain of u-PA and that the binding is strongly reduced when the kringle contains an additional cleavage in its first loop. Since the receptor binding site of u-PA has been previously shown to be located on the growth factor-like domain, Mab 5B4 inhibits the binding of uPA to its cellular receptor likely by steric hindrance. Besides the proven utility in epitope localization of anti u-PA monoclonal antibodies, these u-PA fragments may represent powerful tools for studies of structure-function relationship of u-PA.

Bacteria and bacterial cell wall constituents induce the production of regulatory cytokines in dendritic cell clones.

The primary function of dendritic cells (DC) is the uptake, processing, and presentation of antigens to unprimed T cells, but the regulation of these functions is largely unknown. The study of the signals that maintain DC in a resting state or that drive their activation has been hampered by the difficulties in obtaining pure DC populations. The availability of immortalized DC clones from different tissues (spleen and skin) allowed us to investigate the regulation of cytokine production in response to physiological signals in the absence of contaminating cells. The DC clones exhibited the phenotypical and functional features of DC precursors and could phagocytose, albeit at a low rate, whole bacteria. Heat-inactivated bacteria and bacterial cell wall products were tested for cytokine induction. Lipopolysaccharide, lipoteichoic acid, and gram-negative bacteria were potent inducers of tumor necrosis factor alpha and interleukin 6 release, whereas gram-positive bacteria were less efficient. The results suggest that microbial infections can directly promote cytokine DC release of relevant inflammatory responses as well as in the autocrine activation of DC.

Pain language in fibromyalgia, rheumatoid arthritis and osteoarthritis.

Using the Italian version of the McGill Pain Questionnaire (MGPQ), the language of pain was assessed in three chronic rheumatic diseases, namely Fibromyalgia syndrome (FS), Rheumatoid Arthritis (RA) and Osteoarthritis (OA). The Pain Rating Index (PRI) of the MGPQ clearly allowed OA to be distinguished from RA and FS. Words of the affective and sensory subclasses were chosen more frequently in FS, RA and OA respectively. By analyzing words chosen by at least 33% of the patients, a thermal sensory descriptor arose in FS. Data from this study suggest that the MGPQ-PRI might be useful for the assessment of fibromyalgic pain in a clinical setting and during follow-up of the disease.

Modifications of plasma concentrations of hormonal and tissue factors during mechanical ventilation with positive end-expiratory pressure.

AIM: The aim of this study is to analyse if the decrease of cardiac performance due to positive end-expiratory pressure (PEEP) application, within low values applied in clinical practice (5 cm H(2)O) is able to trigger a response of the main endogenous factors which control and maintain the mean arterial pressure (MAP). METHODS: This study was applied to 18 patients, admitted to the Intensive Care Unit (ICU) of the University Hospital of Modena, who underwent oro-tracheal intubation and mechanical ventilation. On admission, patients did not suffer from cardiac or lung disease. This study analyses plasma concentrations of epinephrine, norepinephrine, ET-1, NO metabolites, renin, aldosterone at 4 different times: before PEEP application, 60 minutes after the beginning of mechanical ventilation with PEEP, and respectively 30 and 60 minutes after withdrawal of PEEP. At the same time, MAP values and heart rate (HR) have been observed. RESULTS: Results show an increase of epinephrine and norepinephrine after PEEP application and a decrease to basal values at PEEP withdrawal. All variations are statistically significant. After PEEP introduction, ET-1 showed an increased concentration, although it was not statistically significant, while a significant decreasing trend was observed after PEEP withdrawal. A significant increase of NO metabolite values has been observed together with the increase of ET-1, followed by a decrease to basal values after the withdrawal of PEEP. Concentrations of renin increased when PEEP was applied even though they were not significant and decreased significantly when PEEP was withdrawn. A similar trend was revealed by aldosterone even though it underwent constant significant variations. CONCLUSION: The administration of PEEP produces an effective response of endogenous substances whose function is to maintain a proper tissue perfusion.

Use of a Caco-2 cell culture model for the characterization of intestinal absorption of antibiotics.

The use of cell culture models, based on human cell lines derived from the intestinal epithelium, is a promising new tool for the in vitro study of oral absorption of drugs. An assay has been developed using the Caco-2 cell line with the aim of studying the in vitro permeability of antibiotics. The reproducibility of the assay conditions have been assessed by means of the transport of two different marker molecules: 3H-mannitol and fluorescein, and transepithelial electrical resistance (TEER) value for cells monolayers. The results show that cells after 21 days of culture give significantly tighter monolayers than those after 15 days with higher reproducibility. Apparent permeability coefficients (Papp) have been measured for 13 antibiotics, known to be absorbed at different rates in humans. Papp values span from 0.18 x 10(-6) cm/s for cephaloridine to 5.79 x 10(-6) cm/s for rifampicin where the corresponding bioavailability values, known from literature, span from < 3 to 98%. A Caco-2 in vitro model appears to be suitable to investigate the transport of drugs across the intestinal epithelium. This model gives no information about the metabolic phase that follows the absorption of a drug but could provide information to investigate its pharmacokinetical behavior.

Antibodies against the antibiotics: an overview.

Antibiotics are small molecules used in the treatment of diseases due to pathogenic microorganisms such as bacteria, viruses, fungi. They have different mechanisms of actions and by exploiting their selective toxicity on the infecting microorganism they do not necessarily damage the host cell. Assay for antibiotic concentrations in blood, urine and other body fluids is motivated primarily in conjunction with antibiotic pharmacology and pharmacokinetics to: a) establish the therapeutic levels of the drug; b) monitor antibiotics with narrow toxic/therapeutic ratios; c) detect accumulation of the antibiotic metabolites. The assay used to measure a wide variety of antibiotics is based on the intrinsic capability of these molecules to inhibit the growth of a suitable microorganism. In the last few years other types of tests, e.g., enzyme immunoassays, have been developed based on polyclonal and monoclonal antibodies. Antibodies against non antigenic molecules like antibiotics can be raised by using as antigen the antibiotic (hapten), conjugated with a carrier protein. Polyclonal antibodies containing mixed populations of antibodies (against different antigenic sites of the hapten, the carrier and the site of conjunction between the hapten and the carrier) are largely used to set up enzyme immunoassays for antibiotics. Monoclonal antibodies by recognizing one antigenic site of the hapten have a better specificity but sometimes less affinity than polyclonals. In the case of antibiotics, to raise monoclonal antibodies with good affinity it is important to use targeted immunization and screening strategies that utilize the antibiotic conjugated in different ways to different carriers.(ABSTRACT TRUNCATED AT 250 WORDS)

[Prosthetic bilateral laparoscopic hernioplasty. Extra-peritoneal approach]. / Ernioplastica laparoscopica bilaterale protesica. Accesso pre-peritoneale.

The use of prosthetic mesh in inguinal hernia repairs is becoming increasingly popular. In recent years different laparoscopic procedures for prosthetic repair of inguinal hernias have been developed. The authors describe their initial experience with a totally extra-peritoneal prosthetic approach in laparoscopic repair of bilateral inguinal hernias. From November 1993 to May 1994, ten consecutive patients with bilateral primary inguinal hernias underwent laparoscopic repair under general anesthesia. A totally extra-peritoneal approach has been performed beginning through a 2 centimeter vertical midline sub-umbilical incision. Two additional trocars have been inserted on the midline: a 10/12 mm one halfway between the umbilicus and the pubis and 5 mm one 2 cm above the pubis. Average operative time was 141 minutes. Two cases were converted to traditional open Stoppa procedure because of holes made in the peritoneum during blunt dissection of the hernia sac. In the remaining 8 cases a polypropylene mesh of about 8 cm in height and 13 cm in length have been placed on each hernia site. No major complications have been observed and recovery was quick in all cases. In conclusion we think that laparoscopic hernia repair through a totally extra-peritoneal approach is technically feasible for general surgeons trained in laparoscopic surgery. Nevertheless the operation in costly and the patient's benefit in terms of rapid recovery, complications and recurrences has not yet been demonstrated in controlled prospective trials.

Wernicke's encephalopathy in a malnourished surgical patient: clinical features and magnetic resonance imaging.

We report a clinical and neuroradiological description of a severe case of Wernicke's encephalopathy in a surgical patient. After colonic surgery for neoplasm, he was treated for a long time with high glucose concentration total parenteral nutrition. In the early post-operative period, the patient showed severe encephalopathy with ataxia, ophthalmoplegia and consciousness disorders. We used magnetic resonance imaging (MRI) to confirm the clinical suspicion of Wernicke's encephalopathy. The radiological feature showed hyperintense lesions which were symmetrically distributed along the bulbo-pontine tegmentum, the tectum of the mid-brain, the periacqueductal grey substance, the hypothalamus and the medial periventricular parts of the thalamus. This progressed to typical Wernicke-Korsakoff syndrome with ataxia and memory and cognitive defects. Thiamine deficiency is a re-emerging problem in non-alcoholic patients and it may develop in surgical patients with risk factors such as malnutrition, prolonged vomiting and long-term high glucose concentration parenteral nutrition.

[Postoperative pain management. Aims and organization of a strategy for postoperative acute pain therapy]. / La gestione del dolore postoperatorio. Obiettivi, identificazione e organizzazione delle procedure di sviluppo di un programma di terapia del dolore acuto postoperatorio.

The Health Services, not only the Italian one, is under pressure because of request for improving treatment quality and the financial need for reorganization and cost-saving. It's required a rationalization of intervention, together with a careful choice of the best and cheapest techniques and the demonstration of their efficacy. The anaesthesia service activity, in a period of cost rationalization and funds restriction should be aimed to appropriate outcome measures corrected by both patient's risk factors and surgical-anaesthesiological case-mix. The development of a complete strategy for surgical pain management might run into two phases. The first phase, internal and mono-specialistic, should develop like the creation of an Acute Pain Team. The main processes are: focusing the problem (charge of the care), training, information, teaching methodology (timing, methods, drugs, techniques, etc.) and the audit (before and after changes). The main aims are the evaluation of the level of analgesia and pain relief or patient's satisfaction which are partial endpoints useful to demonstrate the improvement and the efficacy of the new pain management strategies. The second phase, multidisciplinary, is directed toward the creation of a Postoperative Evaluation Team. The main objective is to set up a collaborative clinical group able to identify the criteria for quality, efficacy and safety. The major purpose is the evaluation of major outcome measures: surgical outcome, morbidity, mortality and length of hospitalization. The improvement in the quality of postoperative pain treatment goes through a better organization and a progressive increase of the already available therapy. The achievement of the result and the quality projects depend on the interaction among staff members with different behaviours and settings. Internal teaching and training, continuous education for doctors and nurses, and external information, marketing and improvement of attractive capability of Institution, are the procedures of a growing integrated program for postoperative pain treatment. The organizational processes should interact effectively with a plan of education, updating, revision and information in a definite development timing. It should be emphasized a collaborative, interdisciplinary approach to pain control, assessment and treatment, including all the members of the health care team, with an input from the patient and a protective collaboration from the Institution. The development of a postoperative care team must be considered as part of the largest project for "a Painfree Hospital". It represents a keystone of a Institutional Quality Assurance Plan for health care providers, patients (customs) and Institution.

Postoperative analgesia in Italy. National survey on the anaesthetist's beliefs, opinions, behaviour and techniques in postoperative pain control in Italy.

BACKGROUND: Using a personal, anonymous questionnaire developed ad hoc, we tried to document the role, the problems and the activities of Italian anaesthetists in postoperative pain control. METHODS: A random selection-stratified by region-was made from a list of 971 hospitals. The final sample was made up of 395 departments of anaesthesia and intensive care from 369 hospitals. The completeness and consistency of each questionnaire were assessed. RESULTS: The response rate achieved was 78.9% (312 centres in 293 hospitals) with 2435 evaluable questionnaires corresponding to 30% of all Italian anaesthesia departments and 25% of Italian anaesthetists. Only 15.2% of anaesthetists are completely responsible for the postoperative analgesia (POA) (prescriptions and evaluations) and an established attitude for POA (protocols/guidelines) exists in 18 departments only. 60% of anaesthetists use routinely the intramuscular route, 55.2% the intravenous route (infusion plus bolus), and 23.7% the spinal/epidural route. Only 6.2% of the anaesthetists use the patient-controlled analgesia system. Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most commonly used agents (47% NSAID vs 37.9% opioids; NSAID-opioid association 14.7%). A local anesthetics-opioids association is preferred for the spinal/epidural route (15.1%) only (local anaesthetic 4.9% only; opioids alone 2.7%). Lack of time, work organization, technique knowledge/training, trust in nursing-ward staff, difficulty in monitoring the patient on the ward, concern about opioid use leading to ventilatory depression, and administration errors are reported to be the main factors that limit an appropriate approach to postoperative pain (POP), the correct use of analgesics and a free choice of POA technique. CONCLUSION: This survey shows that Italian anaesthetists do not consider the postoperative period to be their own personal work area and that POA is to be considered as a matter of individual choice.

[Intraoperative anaphylaxis from latex. A case report]. / Anafilassi intraoperatoria al lattice. A proposito di un caso.

The authors report a case of anaphylactic reaction in a 58-year-old woman during total gastrectomy under general anesthesia. Before induction a peridural catheter had been inserted for the purpose of postoperative antalgia. Anaphylaxis occurred fifteen minutes after the start of surgical resection. The tests performed later led the authors to regard latex as the triggering agent.