Epithelial lining fluid neutrophil-gelatinase-associated lipocalin levels in premature newborns with bronchopulmonary dysplasia and patency of ductus arteriosus.

Patency of the ductus arteriosus (PDA) and bronchopulmonary dysplasia (BPD) development represent severe affections for premature newborns, therefore the research of early markers for these two conditions is really important. The aim of this study is to analyze epithelial lining fluid (ELF) Neutrophil-gelatinase-associated lipocalin (NGAL) levels for prediction of lung injury or possible involvement of this molecule in PDA. Only scarce and contrasting results have previously been published in this field. In contrast, this molecule, included in a large macromolecular complex together with matrix metalloproteinase-9 (MMP-9), is considered an acceptable marker of infectious/inflammatory processes, cancer monitoring and induction of apoptotic pathway. NGAL was detected in 28 pre-term newborns by means of a commercially available kit in bronchoalveolar lavage fluid (BALF). The results have been corrected to ELF levels, by the urea method, to eliminate bias due to BALF collection. ELF NGAL levels were found significantly increased both in infants developing BPD or in those affected by PDA. By means of multivariate logistic regression analysis the significances were confirmed after adjusting for possible interfering variables such as gestational age and concomitant presence of both PDA and BPD. Our results stress the involvement of NGAL in the mechanisms leading to BPD and also suggest a possible association with PDA, which is often linked to prematurity and BPD development, probably due to the involvement of inflammatory and angiogenetic processes in both pathologies.

A dominant negative RAS-specific guanine nucleotide exchange factor reverses neoplastic phenotype in K-ras transformed mouse fibroblasts.

Ras proteins are small GTPases playing a pivotal role in cell proliferation and differentiation. Their activation state depends on the competing action of GTPase Activating Proteins (GAP) and Guanine nucleotide Exchange Factors (GEF). A tryptophan residue (Trp1056 in CDC25Mm-GEF), conserved in all ras-specific GEFs identified so far has been previously shown to be essential for GEF activity. Its substitution with glutamic acid results in a catalytically inactive mutant, which is able to efficiently displace wild-type GEF from p21ras and to originate a stable ras/GEF binary complex due to the reduced affinity of the nucleotide-free ras/GEF complex for the incoming nucleotide. We show here that this 'ras-sequestering property' can be utilized to attenuate ras signal transduction pathways in mouse fibroblasts transformed by oncogenic ras. In fact overexpression of the dominant negative GEFW1056E in stable transfected cells strongly reduces intracellular ras-GTP levels in k-ras transformed fibroblasts. Accordingly, the transfected fibroblasts revert to wild-type phenotype on the basis of morphology, cell cycle and anchorage independent growth. The reversion of the transformed phenotype is accompanied by DNA endoreduplication. The possible use of dominant negative ras-specific GEFs as a tool to down-regulate tumor growth is discussed.

Tyrosine protein kinase in boar spermatozoa: identification and partial characterization.

A protein-tyrosine kinase has been isolated from a detergent-soluble extract of boar spermatozoa, using poly(Glu, Tyr)4:1 as a substrate. The purification procedure involves sequential column chromatographies on phosphocellulose, polyamino acid affinity and Sephadex G-100 molecular sieving, and results in more than a 1200-fold enrichment. Analysis of the most purified preparation by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a major Coomassie blue-stained band of molecular mass 42 kDa. The Tyr-protein kinase does not seem to be autophosphorylable. The Km value for poly(Glu, Tyr)4:1 is relatively low, 2.3 microM, and the tyrosine-polymer phosphorylating activity is apparently inhibited by tyrphostin. The characteristics shown by this new tyrosine kinase--the first to be described in mature male germ cells--support the hypothesis that it belongs to the group of non-receptor-associated tyrosine kinases.

Evidence for a calsequestrin-like calcium-binding protein in human spermatozoa.

Human spermatozoa were investigated for the presence of protein(s) recognized by antibodies against calsequestrin, the high capacity, moderate affinity Ca2(+)-binding protein, originally described in striated muscle fibers. Western immunoblots of detergent-soluble sperm extracts probed with polyclonal antibodies raised against human skeletal muscle calsequestrin identified a strongly cross-reactive protein. This protein resembles muscle calsequestrin in many respects. In fact, its migration in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is pH dependent, its apparent molecular mass being 64 kDa in alkaline SDS-PAGE and 44 kDa in neutral SDS-PAGE; its isoelectric point is acidic (4.6); it is metachromatically stained blue by the carboxycyanine dye, Stains-All; it is a Ca2(+)-binding protein (45Ca blot overlay). Indirect immunofluorescence experiments showed that the immunoreactive protein has an intracellular localization confined to the tail mid-piece. From these findings we conclude that human sperm cells express a protein structurally and antigenically related to skeletal muscle calsequestrin; a basis for a novel interpretation of Ca2(+)-mediated events in spermatozoa is thus provided.

Penicillamine-related neurologic syndrome in a child affected by Wilson disease with hepatic presentation.

OBJECTIVES: To describe a case of penicillamine-related neurologic symptoms in a 9-year-old boy affected by asymptomatic Wilson disease with hepatic presentation; to compare this case with similar cases in adults; and to discuss the role of zinc therapy as an alternative treatment for patients who have an adverse reaction to penicillamine therapy. SETTING: Referral hospital. MAIN OUTCOME MEASURE: The occurrence of a neurologic syndrome that severely impaired a child's usual daily activities and his health-related quality of life after the institution of penicillamine therapy. RESULTS: Initial penicillamine therapy was chronologically related to the development of progressive neurologic deterioration in the absence of other causes of neurologic syndrome. The discontinuation of penicillamine therapy and the initiation of zinc therapy were followed by a prompt disappearance of neurologic symptoms and a return to neurologic baseline status. CONCLUSIONS: Penicillamine therapy, even in children affected by Wilson disease with hepatic presentation alone and without neurologic disease at the beginning of treatment, may trigger neurologic symptoms. Zinc therapy may be a satisfactory alternative.

A simple, automatic method for morphometric analysis of the left ventricle in rats with myocardial infarction.

Induction of acute myocardial infarction in the rat is an established model for studying effects of therapeutic interventions. Images of sections of the rat left ventricle, stained with nitroblue tetrazolium, were digitized and several parameters estimated by dedicated software on an image analyzer (IBAS 2.0). The method was tested on 7 rats with 48-hr-old myocardial infarction and 4 sham-operated controls. Infarct size can be evaluated by two largely used methods, based on area or on angular extension of the lesion. Results of the two methods are linearly correlated, but area calculations give values half of those obtained from angular extension. Five minutes were needed for a complete evaluation of a section of the left ventricle. Estimates of the parameters showed a relatively low between- and within-operator variability and a good correlation with a classic, but time-consuming, planimetric method. The method simultaneously measures infarct size and left ventricular geometry in the rat. The advantages over previous nonautomatic methods are simplicity, good reproducibility, and speed of execution, which make it particularly useful in the evaluation of drug effects.

In vitro permeation screening of a new formulation of thiocolchicoside containing various enhancers.

Thiocolchicoside, a muscle relaxant agent with anti-inflammatory and analgesic actions, also is used topically for the treatment of muscular spasms and for rheumatologic, orthopedic, and traumatologic disorders. In this study, thiocolchicoside was formulated to use as foam to avoid contact with the afflicted area during the spreading phase. To enhance drug penetration, various enhancers were added to the base formulation. The tested enhancers were ethoxyethylendiglycol (Transcutol), highly purified phosphatidylcholine (Lipoid S20), capsaicin, propylene glycol dipelargonate (DPPG), and glycolysed ethoxylated glycerides (Labrafil M1944 CS). The transdermal absorption of the tested formulations containing enhancers, in comparison with base formulation, was evaluated in vitro through rat skin using standard Franz diffusion cells. Base formulation was found to have a higher permeation profile than the simple aqueous and hydroalcoholic solutions of the drug, meaning that the base formulation by itself enhances the drug permeation. Among the tested formulations, only the formulation containing DPPG/ethanol was found to be statistically different, showing an enhancement factor of 3.58. In the same experimental session, Muscoril ointment, the commercially available pharmaceutical product containing the same thiocolchicoside concentration (0.25%), also was tested. The formulation containing DPPG/ethanol showed a 4.86 times increase of permeability constant in comparison with Muscoril ointment. The formulation containing DPPG/ethanol as an enhancer could be a good candidate for a new topical foam, considering its good characteristics of permeability and compliance.

Pharmacokinetics of diltiazem and a new analogue, LR-A/113, in the conscious rat.

Pharmacokinetics and metabolism of diltiazem and a new analogue, LR-A/113, have been studied in the rat. Conscious rats, with the jugular vein cannulated, received the compounds by intravenous (3 mg/kg body weight) or oral (50 mg/kg body weight) route. Parent compounds and their N-demethyl and N-deacetyl metabolites were assayed at serial times in blood. Half-life of elimination of diltiazem was significantly shorter than that of LR-A/113, both after oral (37 +/- 9 vs 59 +/- 26 min) and intravenous (29 +/- 12 vs 57 +/- 16 min) administration. N-deacetyl-diltiazem concentrations after oral administration were higher than the parent compound and N-demethyldiltiazem; LR-A/113 blood concentrations were higher than those of its two metabolites. Metabolites were measurable only in traces after intravenous administration. Oral bioavailability was very low, 3.5% for diltiazem and 4.2% for LR-A/113. In conclusion, the substitution of a methyl by an isopropyl group appears to slow in vivo elimination of the analogue of diltiazem, LR-A/113.

[Diverticulosis of the gallbladder. A case report and review of the literature]. / Diverticolosi della colecisti. Considerazioni su un caso clinico e revisione della letteratura.

We report a case of diffuse diverticulosis of the gallbladder in a 32 year old patient, who presents epigastric pains and decayed general conditions. With the suspicion of a primitive tumor of the gallbladder, we make a cholecystectomy and the intraoperative histological examination shows a "diffuse diverticulosis of the gallbladder wall"; it displays micro-stones inside diverticulums, too. The patient, after the surgical treatment, has no problem and he gets back to his normal weight. The literature shows the rarity of this disease; its etiopathogenesis is connected with congenital factors. In our opinion, the many terms used (cholecystitis glandularis proliferans, hypertrophic, adenomyomatosis) to define those conditions are not appropriate for our case but rather for the gallbladder's chronic inflammation characterized by the Rokitansky-Ashoff sinuses.

[Surgical treatment of complicated diverticular disease of the colon]. / Il trattamento chirurgico della malattia diverticolare complicata del colon.

The present study analyzes the results obtained by the AA with the different types of surgery adopted in the treatment of the complicated diverticulosis of the colon, highlighting, on the basis of data available in literature, the possible treatments in the different clinical settings. A retrospective study analyzing type of complication, the surgical technique adopted, Hinchey stage, mortality and morbidity rates and average hospital stay correlated with the kind of intervention has been carried out on 83 surgical interventions performed between 1984 and 1988. The results show that 43 R.A.P. (R.A.P. = primitive anastomosis resection) (32 cases at the I-II stage and 11 cases at the III-IV stage), 27 Hartmann (11 at the I-II and 16 at the III-IV), 9 colostomies (2 at the I-II and 7 at the III-IV), 2 esteriorizations and 2 simple drains have been carried out on a total of 44 intestinal perforations, 16 recurrent diverticulitis, 13 intestinal occlusions, 2 fistulae, 5 abscesses and 3 hemorrhages. The total mortality rate amounts to 10.6%; the morbidity rate of the R.A.P. interventions to 14.4 (I-II stage-related morbidity = 15.6%, III-IV stage = 63.6%), Hartmann's to 9.6% and that of the colostomies to 3.6%. Furthermore, in this work, we have considered the cases of riconversation after Hartmann interventions (9 cases): in the second operations the mortality and morbility rate amounts to 0 and the hospital stay to 9 days. The AA analyze on the surgical technique adopted in the different cases and the of choice criteria. According to the data obtained and to current literature, it results that the primitive anastomosis resection represents the first choice intervention at the I-II stage, although, in selected cases, it can be carried out also at the III-IV stage. Hartmann surgery confirms its effectiveness while simple colostomy is no longer accepted in literature.

[Endothoracic goitre: anatomoclinical and therapeutic considerations]. / Gozzo endotoracico: considerazioni anatomocliniche e terapeutiche.

Substernal goitre is a clinical condition in which the masin bulk of the enlarged gland is firmly located in the chest. The incidence of this pathology ranges in literature from 1.7% and 30%. This study examines 230 cases of substernal goitre out of a total 5.362 operations performed from 1965 to 2000, for thyroid gland pathologies (4.36%). According to their experience the Authors propose a classification based on the anatomical location of the goitre: right, left, anterior and posterior goitre are therefore identified. The surgical procedures performed include 136 subtotal thyroidectomies (59.1%), 59 emithyroidectomies (25.7%) and 23 total thyroidectomies (10%). In 12 cases the operation was confined to removal of the mediastinal mass (5.2%). The cervical approach was the only surgical access route used in all the patients, regardless of the different anatomical variants. Appreciable venous stasis, due to the mediastinal mechanical obstruction exerted by the goitre, was always evident at the operation. In order to limit the risk of bleeding during operation, careful hemostasis of the major vascular pedicles must be performed. Any attempt to legate the smallest vessel, should be avoided since it is a difficult, useless and time-consuming procedure. Minor bleeds promptly stop as soon as the pathological mass is removed. Ligation of the vascular pedicles can be easily achieved; in this way, the goitre is freed from its anatomical connections and the surgeon can safely manage the substernal portion of the mass. The mortality reported in this study was 0.43% (one patient died 30 days postoperatively due to respiratory complications), whereas the morbidity rate was 2.6%.

Lipopolysaccharide-induced lung injury in mice. I. Concomitant evaluation of inflammatory cells and haemorrhagic lung damage.

Intratracheal instillation of lipopolysaccharide (LPS) induces an inflammatory response characterized by infiltration of polymorphonuclear neutrophils (PMNs) into the extracellular matrix and by the release of mediators that play a fundamental role in lung damage. In the present study, we developed a mouse model which allows correlation of the inflammatory response and haemorrhagic tissue injury in the same animal. In particular, the different steps of the inflammatory response and tissue damage were evaluated by the analysis of three parameters: myeloperoxidase (MPO) activity in the parenchyma, reflecting PMNs accumulation into the lung, inflammatory cells count in the bronchoalveolar lavage fluid (BALF), reflecting their extravasation, and total haemoglobin estimation in BALF, a marker of haemorrhagic tissue damage consequent to PMNs degranulation. In our experimental conditions, intra-tracheal administration of 10 microg/mouse of LPS evoked an increase of MPO activity in the lung at 4 h (131%) and 6 h (147%) from endotoxin challenge. A significant increase of PMNs in the BALF was noticed at these times with a plateau between the 12nd and 24th h. PMN accumulation produced a time-dependent haemorrhagic lung damage until 24 h after LPS injection (4 h: +38%; 6 h: +23%; 12 h: +44%; 24 h: +129% increase of haemoglobin concentration in the BALF vs. control). Lung injury was also assessed histopathologically. Twenty-four hours after the challenge, diffuse alveolar haemorrhage, as well as PMN recruitment in the interstitium and alveolus were observed in the LPS group. This model was pharmacologically characterized by pretreatment of LPS-treated mice with antiinflammatory drugs acting on different steps of the <>. We demonstrated that: a) betamethasone (1, 3, 10, 30 mg/kg p.o.) reduced in a dose-dependent manner the MPO activity, the number of inflammatory cells and, at the same time, lung injury; b) pentoxifylline, a TNFalpha production inhibitor (200 mg/kg i.p.), inhibited PMN extravasation and lung haemorrhage but it was not able to reduce MPO activity in the lung; c) L-680,833, an anti-elastase compound (30 mg/kg po), decreased significantly only the haemorrhagic lung damage; d) indomethacin, a non steroidal antiinflammatory drug (5 mg/kg p.o.), did not show any effect on any of the parameters considered. In conclusion, our in vivo mouse model is a practical alternative to animal models of ARDS (Adult Respiratory Distress Syndrome) recently described and it permits to dissect and to characterize the different steps of PMNs infiltration and, at the same time, the damage caused by their activation.

Effects of an early treatment with lisinopril and isosorbide-5-mononitrate on hemodynamics and late ventricular remodelling in rats with 9-week myocardial infarction.

This study was undertaken to assess whether the converting enzyme inhibitor lisinopril, and the long-acting nitrate, isosorbide-5-mononitrate, affect left ventricle dysfunction and anatomical remodelling in rats with myocardial infarction. Lisinopril, isosorbide-5-mononitrate or vehicle were given to rats (n = 10-14 per group) immediately after coronary artery occlusion (by an intravenous bolus) and then for nine weeks (in drinking water). At the end of the study, left ventricular pressures were measured, the heart arrested in diastole, and infarct size, left ventricular chamber volume and wall thicknesses measured. Lisinopril significantly lowered systemic blood pressure and left ventricular systolic pressure in rats with small (< 15% scarred tissue of the left ventricle) and large (> 15%) infarcts; the weight of the left ventricle (including the septum) was reduced by 24% and 28% in animals with small and large infarcts, respectively. Lisinopril lowered left ventricular end-diastolic pressure (by 33% and 39%) and chamber volume (by 4% and 34%) in rats with small and large infarcts, respectively, compared with controls (NS). The combined anatomical and hemodynamic changes led to a reduction of the circumferential wall stress by 20% and 44% in lisinopril-treated rats with small and large infarcts, respectively (NS). No significant changes were seen in the nitrate-treated hearts compared with controls. Lisinopril, given early after myocardial infarction and continued for nine weeks, significantly affected cardiac hemodynamics and ventricular weights in rats with infarcts of different sizes.(ABSTRACT TRUNCATED AT 250 WORDS)

Lymphoblastoid interferon alfa treatment in chronic hepatitis C.

Interferon is becoming the standard treatment in adults for chronic hepatitis C. Twenty one children with histologically proved chronic hepatitis C (10 boys, range 2.5-13 years), who were otherwise healthy, were enrolled in a randomised controlled study to test their response to interferon alfa. Eleven children were treated with lymphoblastoid interferon alfa (3 million units/m2) for 12 months; 10 children received no treatment. All had raised transaminases and positive antihepatitis C virus (HCV) antibodies and HCV-RNA. Alanine aminotransferase (ALT) serum levels became normal in five (45%) treated patients after a mean of three weeks (range 1-6 weeks) and no relapse had occurred by the end of follow up (30th month). Only one (10%) untreated patient had normal ALT serum levels from the 11th until the 30th month. Disappearance of serum HCV-RNA, persisting throughout the follow up period, was observed in the six children (five treated) whose ALT became normal. Biopsy specimens in treated patients showed a significant improvement in Knodell's score (median (SD) basal 9.0 (2.2); final 2.0 (0.4)). Interferon treatment was well tolerated in all. This study confirms the efficacy of interferon in children with chronic hepatitis C, not only by restoring normal ALT serum levels, but also viral clearance and histological amelioration of liver inflammation. Contrary to reports in adults no biochemical and virological relapses occurred in responder children.

Chronic non-A, non-B hepatitis: role of hepatitis C virus.

Thirty three consecutive children with chronic non-A, non-B hepatitis (NANBH) were studied during a four year period to evaluate clinical and histological features and the role of hepatitis C virus (HCV). All patients were asymptomatic. Thirteen (39%) of them were anti-HCV positive. A history of parenteral exposure was significantly more frequent among anti-HCV positive (69%) than anti-HCV negative patients (15%). Aminotransferase serum values were not statistically different between anti-HCV positive and anti-HCV negative patients. Unlike adults, cirrhosis was never found in the children studied. Our results suggest that chronic NANBH is, during childhood, an asymptomatic disease and that the prevalence of HCV infection is lower than in adults. As the majority of the children with chronic NANBH showed no evidence of HCV infection, it seems unwarranted to identify NANBH with HCV infection in children. The lack of cirrhosis in paediatric patients is probably related to a shorter duration of liver disease.

Effects of a new angiotensin-converting enzyme inhibitor (idrapril) in rats with left ventricular dysfunction after myocardial infarction.

We evaluated the effects of a new angiotensin-converting enzyme (ACE) inhibitor (idrapril) in terms of hemodynamics and ventricular remodeling after myocardial infarction in rats. The animals were randomly assigned to four experimental groups. Myocardial infarction was induced by left coronary artery ligation in the first two groups treated with either idrapril (300 mg kg-1 day-1) or vehicle for 4 weeks after myocardial infarction. Two groups of sham-operated rats were treated accordingly. Hemodynamics were measured, and the diastole-arrested hearts were analyzed morphometrically to quantify left ventricular (LV) remodeling and infarct size. In infarcted rats, idrapril reduced the arterial systolic blood pressure (SBP) from 128 +/- 10 to 97 +/- 6 mm Hg (p < 0.05) and LV end-diastolic pressure (LVEDP) from 19 +/- 3 to 13 +/- 3 mm Hg (p < 0.01). The decrease in diastolic wall stress conferred by idrapril to infarcted rats (from 499 +/- 99 to 269 +/- 68 dynes mm-2, p < 0.05) was mainly due to a reduction in LVEDP and, to a lesser extent, in LV volume. Idrapril also reduced body and heart weights as compared with those of vehicle-treated animals. Four-week treatment with idrapril initiated immediately after myocardial infarction reduced LVEDP and limited LV wall stress, a major prognostic factor for the progression toward chronic ventricular failure.

Differential contribution of R and S isomers in ketoprofen anti-inflammatory activity: role of cytokine modulation.

Among nonsteroidal anti-inflammatory drugs (NSAIDs), 2-arylpropionic acids exist as a racemic mixture of its enantiomeric forms, with S-enantiomers primarily responsible for inhibition of prostaglandin synthesis and of inflammatory events. The aim of this study was to compare the anti-inflammatory effects of R- and S-ketoprofen in vitro and in vivo. S-Ketoprofen efficiently inhibited carrageenan-induced edema formation, but it could also amplify the LPS-induced production of the inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1), in close correlation with its ability to inhibit prostaglandin synthesis. Because these inflammatory cytokines are among the factors involved in carrageenan-induced inflammation and also are possibly involved in gastric damage, enhanced cytokine production could partially mask the analgesic effect of S-ketoprofen, and it can be associated with the clinical evidence of its gastric toxicity. On the other hand, R-ketoprofen contributes to the overall activity of the racemate, by playing the main role in ketoprofen-induced analgesia. Unlike the S-isomer, R-ketoprofen did not induce a significant increase of cytokine production even at cyclooxygenase-blocking concentrations. It is concluded that the R-isomer directly contributes to the anti-inflammatory effects of ketoprofen, being more analgesic, and because it does not amplify inflammatory cytokine production.

Efficacy of a new topical gel-spray formulation of ketoprofen lysine salt in the rat: percutaneous permeation in vitro and in vivo and pharmacological activity.

The aim of this study was to evaluate the percutaneous permeation of a new topical Gel-Spray formulation, containing 15% of ketoprofen lysine salt (KLS), both in vitro, using the Franz-type diffusion cells and in vivo, by evaluating urinary recovery after topical administration and to correlate the absorption data with KLS pharmacological activity in the rat. Concentrations of ketoprofen free acid (KFA) were determined by HPLC in the receptor compartment (in vitro), or in urine (in vivo). The permeation of ketoprofen evaluated in vitro after the application of KLS Gel-Spray was higher than that observed with the marketed formulation Profénid gel (containing KFA at 2.5%). The same evidence was found in vivo, except when the ratio between the administered dose and the area treated was higher than 1 mg cm-2. Thus, the difference between the two formulations seems to be the resultant of two opposing components: a positive gradient of concentration that favours the absorption of ketoprofen from KLS Gel-Spray and the presence of the enhancer ethanol that could favour the efficacy of Profénid gel. Under our conditions the former prevailed. As for the efficacy, evaluated in the carrageenan-induced oedema and hyperalgesia model, KLS Gel-Spray confirmed the data obtained for in vivo absorption, being more efficient than the reference standard Profénid gel. The observed inhibitory effects were due only to dermal absorption, oral absorption was excluded by an Elizabethan collar applied around the neck of the rat. In these experimental conditions, no significant damage of the rat stomach mucosa was observed. These results indicate that KLS Gel-Spray, due to its high KLS concentration, allows a very high efficiency in delivering ketoprofen to the inflamed area using a minimal amount of formulation, even in the absence of permeation enhancers.