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Biomedical subjects

D Neri

Publications and source records attributed to D Neri.

At least 73 records · Page 4Linked to original sources
PubMed1996

Ultrasound-guided fine needle biopsy and laparoscopy in the study of pancreatic masses: report on 136 cases.

The authors report their experience from 136 fine needle ultrasound (FN-US)-guided biopsies and laparoscopies. The pancreatic diseases considered by these methods were as follows: 9 cases of Pancreatitis, 11 cases of Pancreatic cysts, 5 cases of endocrine cancer, 109 cases of Exocrine cancer and 2 not conclusive cases. Diagnostic accuracy of FNB and laparoscopy was evaluated for each group and, in particular, for cancer patients. In the latter group, FNB helped to detect abdominal diffusion in 25 cases (33%) while laparoscopy, including laparoscopic washing, revealed a micro-diffusion in 31 cases (55%), the latter not shown previously by CT, RNM and US. The combination of these methods allows us to confirm the advanced stage of the majority of pancreatic cancers at onset. Furthermore, this seems to be a very reliable method to select resectable patients, thus avoiding useless, sometimes hazardous and expensive further investigation.

Biopsy, Needle↗
PubMed1996

Multipurpose high sensitivity luminescence analyzer (LUANA): use in gel electrophoresis.

Many applications in molecular biology require the rapid and high-sensitivity detection of biological macromolecules. Here we describe a luminescence analyzer (LUANA), featuring xenon lamp-based illumination and a cooled CCD camera as detector. Luminescent samples (gels, membranes or microplates) are placed in a light-tight chamber, and computer software is used to control the camera and to display and analyze the images. LUANA can be used with a broad spectrum of excitation and emission wavelengths (200-1000 nm), allowing a choice of many fluorescent dyes and facilitating multicolor imaging. We show that LUANA is readily applicable to gel electrophoresis and highly sensitive; on gels we could detect < 100 fg of Cy5TM-labeled protein and 0.1 ng of plasmid DNA labeled with a fluorescent intercalator. We observed a linear response for sample concentrations differing by two orders of magnitude using a single acquisition time. This range could be further extended by using different acquisition times. We have also used LUANA in two-color imaging of DNA and for screening of antigen-antibody and peptide-protein complexes by gel bandshift.

Cloning, Molecular↗
PubMed1995

High-affinity antigen binding by chelating recombinant antibodies (CRAbs).

We have developed a strategy for making antibody fragments with high binding affinities by harnessing the chelate effect. We create a bispecific antibody fragment (Chelating Recombinant Antibody or CRAb) that recognizes adjacent and non-overlapping epitopes of the target antigen, and is flexible enough to bind to both epitopes simultaneously. Here the strategy is illustrated with two antibodies that form complexes of known three-dimensional structure against different epitopes of lysozyme. Computer graphic modelling indicated that two single-chain antibody fragments (scFv) derived from antibodies D1.3 (Ka = 10(8) M-1) and mutant HyHEL-10 (Ka = 10(6) M-1) could be linked together on the surface of lysozyme by a flexible and hydrophilic polypeptide between the C terminus of one fragment and the N terminus of the other. The CRAb gene was assembled and the CRAb expressed by secretion from bacteria. The purified CRAb was shown to have a much higher affinity than either of the scFv fragments, as shown by competition ELISA (Kd > 10(9) M-1), bandshift on gels (Ka > 2 x 10(9) M-1) and fluorescence quench (Ka > 1.3 x 10(10) M-1).

Amino Acid Sequence↗
PubMed1995

Efficient one-step direct labelling of recombinant antibodies with technetium-99m.

High-affinity bacterially expressed antibody fragments can nowadays be cloned from established hybridomas or, more conveniently, isolated directly from antibody libraries displayed on filamentous phage. Such antibodies can be tagged with C-terminal peptide tags containing one cysteine residue, which represents a convenient functionalisation site for a number of applications, including technetium-99m labelling. Here we describe a simple one-step method for 99mTc labelling of cysteine-tagged recombinant antibodies with more than 50% radionuclide incorporation. The labelled antibodies displayed full retention of immuoreactivity and good stability.

Antibodies, Bacterial↗
PubMed1995

Engineering recombinant antibodies for immunotherapy.

Recombinant antibody fragments binding with high affinity to their target can be obtained either from hybridomas or directly from antibody libraries on filamentous phage. These fragments are devoid of any activity other than antigen binding, and have to be processed and functionalized in order to be suitable for clinical applications. This article presents the authors' view on the procedures and the features that are important for effective transformation of recombinant antibodies into useful immunotherapeutic agents. The topics presented include phage display methodologies, engineering of high-affinity binding, purification, and functionalization strategies of recombinant antibodies.

Antibodies↗
PubMed1995

Calmodulin as a versatile tag for antibody fragments.

Calmodulin is a highly acidic protein (net charge -24 at pH 8.0 in the absence of calcium) that binds to peptide and organic ligands with high affinity (Ka > 10(9) M-1) in a calcium-dependent manner. We have exploited these properties to develop calmodulin as a versatile tag for antibody fragments. Fusions of calmodulin with single chain Fv fragments (scFv) could be expressed by secretion from bacteria in good yield (5-15 mg/l in shaker flasks), and purified from periplasmic lysates or broth to homogeneity in a single step, either by binding to anion-exchange resin (DEAE-Sephadex), or to an organic ligand of calmodulin (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide-agarose). The antibody fusions could be detected by binding of fluorescently labeled peptide ligands, as illustrated by their use in confocal microscopy, fluorescent activated cell sorting and "band shift" gel electrophoresis. Moreover, the interaction between calmodulin and peptide ligands could provide a means of heterodimerization of proteins, as illustrated by the assembly of an antibody-calmodulin fusion with maltose binding protein tagged with a peptide ligand of calmodulin.

Amino Acid Sequence↗
PubMed1994

Recombinant proteins L and LG. Two new tools for purification of murine antibody fragments.

Several bacterial cell wall proteins, like proteins A and G, with valuable affinity for immunoglobulins have been discovered and are currently employed in immunological techniques. In 1988, protein L, a bacterial cell wall protein with Ig-binding capacity, was isolated from the anaerobic bacterial species Peptostreptococcus magnus. Binding data with immunoglobulin fragments suggested that protein L could selectively bind the variable region of human kappa light chains. More recently a recombinant LG fusion protein was expressed in E. coli containing four repeated Ig-binding domains of protein L (fragment B1-4) and two IgG Fc-binding protein G domains (fragment CDC). Recombinant L and LG proteins were tested in the purification of murine monoclonal IgG and their fragments. After affinity-constant evaluation in different buffer systems, high-pressure affinity-chromatography columns were prepared by coupling the proteins to Affi-prep 10 resin and tested with eight different murine monoclonal antibodies and their fragments of various isotypes. Affinity-chromatography experiments confirming radioimmunoassay results showed 75% fragment-binding capacity of protein L and 100% of protein LG. These results evidenced protein LG as the most powerful Ig-binding tool so far described. Therefore, application of these proteins may be suggested in the purification of murine immunoglobulins and their fragments, including the engineered ones.

Animals↗
PubMed1994

[The palliative treatment of hepatocarcinoma: chemoembolization vs. the combination of tamoxifen plus beta-interferon].

Clinical and experimental data show that beta-IFN enhances the effect of tamoxifen on advanced breast cancer. There is a similarity between breast and liver as far as the proliferating effect on normal and neoplastic tissue of estrogen and progestin receptors is concerned. The authors tested this pharmacological association in unresectable liver neoplasms. They considered 76 (not randomized) patients affected with HCC; 38 were treated by trans-arterial chemoembolization (TACE) and 38 to beta-INF and tamoxifen (the 2 groups were comparable according to age, sex, Child-Pugh score, Okuda and TNM stages, cirrhosis etiology). The treatment response (positive when a tumor diameter decreased or stabilization was observed) was similar in the two groups; in the TACE group, the presence of a peritumoral capsula had a significant influence on survival (p < 0.02); on the other hand, in the patients treated with beta-INF and tamoxifen important factors for a better prognosis were the TNM stage (I and II, p < 0.02) and a symptom-free condition (p < 0.04). The authors believe the beta-INF and tamoxifen treatment could represent an effective alternative in the management of unresectable HCC. A better knowledge of the presence and meaning of estrogen and progestin receptors in the neoplastic tissue may allow a better selection of patients.

Aged↗
PubMed1994

Pancreatic cancer and retroperitoneal neural tissue invasion. Its implication for survival following radical surgery.

To analyze the implication of retroperitoneal perineural lymphatic infiltration (rpli) for the survival of patients affected by exocrine pancreatic cancer, we considered 17 cases which underwent radical resection at our Institute from 1980 to 1993. Histology of specimens showed an rpli in 15 cases, without any correlation between this anatomopathologic aspect and the neoplasm size or the presence of lymph node metastases. The 2 patients without rpli are both alive and have been free from disease during a follow-up period of more than 5 years. In the group of 15 patients with positive rpli, 3 (21.4%) have remained free from disease (2 alive and 1 dead for other reason). Eleven remained with disease (1 is alive and 10 have died). One died in the postoperative period and could not be included in the evaluation. These observations have led the surgeon to consider the necessity, during resection, of an accurate dissection not only of the regional lymph nodes but also of the retropancreatic tissue and the neural structures present in this area. This approach may lead to a more correct staging of the neoplasia, a better radical surgery and perhaps a longer survival. Moreover it may represent an important marker for an adjuvant protocol of chemotherapy and/or radiotherapy.

Duodenum↗
PubMed1993

Effect of ursodeoxycholic acid administration on bile duct proliferation and cholestasis in bile duct ligated rat.

The origin, mechanism, and significance of the bile duct proliferation (BDP) associated with cholestasis remain unexplained. This study examined the effect of oral administration of ursodeoxycholic acid (UDCA) on both BDP and cholestasis in the rat. After bile duct ligation, male Sprague-Dawley rats were treated for 30 days with either UDCA (5 mg/day) (group A) or saline solution (group B). Animals were sacrificed at day 30. The serum activity of aminotransferase (ALT, AST), alkaline phosphatase, and gamma-glutamyltransferase (GGT) was significantly lower (P < 0.01) in the UDCA-treated rats. Total serum bilirubin and total serum bile acids were lower (P < 0.001) in group A. Moreover, the control of BA in bile was reduced also (P < 0.02). Conversely, serum cholesterol levels were not different between the two groups. Histological examination showed that the number of ductular cells in the portal areas was significantly (P < 0.001) reduced in UDCA-treated as compared to saline-treated rats. The replication activity, assessed as the number of bromodeoxyuridine-positive cells, was also significantly lower in treated animals (33 +/- 11 vs 64 +/- 22 per 1000 cells; P < 0.001). Lobular bile ductules were three times larger in group B, and extrahepatic duct measurements confirmed this increase in size of the larger biliary ducts (P < 0.001). These findings demonstrate that UDCA reduces BDP in response to BD ligation. Although the mechanism(s) of this effect is still hypothetical, UDCA may reduce the level of irritating bile salts such as chenodeoxycholic acid and lithocolate and increase periductular bile acid recirculation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Biological activities of terthiophenes and polyynes from the Asteraceae.

We evaluated a number of terthiophenes and polyynes, from the Asteraceae, for biological activities against microorganisms, viruses, and tumor cells, with and without the aid of UVA (long wavelength ultraviolet) radiation. The terthiophenes, which represented the basic alpha-terthienyl nucleus with simple side chains, showed impressive UVA-dependent activities, some of which were superior to alpha-terthienyl itself. In contrast, the polyynes had no significant biological activity, with or without UVA. We believe that these terthiophenes would be worthwhile evaluating in animal models of infectious diseases.

Alkynes↗
PubMed1993

Sex hormones and trace elements in rat CCL4-induced cirrhosis and hepatocellular carcinoma.

Several biochemical events accompany and mediate the development of chronic liver disease and its evolution into cancer. Low plasma zinc and high copper levels have been observed in various liver diseases, such as liver cirrhosis and viral hepatitis, while increased oestradiol levels have been documented in chronic liver damage and hepatocellular carcinoma. We administered CCL4 intragastrically to 10 female Sprague Dawley rats for 30 weeks. All animals developed cirrhosis and four also developed hepatocellular carcinoma. Plasma levels of zinc, copper and oestradiol were significantly higher in the latter group than in animals with simple cirrhosis. Progesterone, AST and bilirubin showed a trend toward significant differences whereas testosterone and ALP levels were unchanged. These findings add to the evidence that sex hormones and trace elements are involved in the process of the development of chronic liver damage and carcinogenesis.

Alkaline Phosphatase↗
PubMed1993

Palliative treatment of hepatocellular carcinoma with transcatheter arterial embolization.

Thirty-three consecutive patients with previously untreated hepatocellular carcinoma (HCC) and 6 patients with recurrent HCC were treated with transcatheter arterial embolization (TAE). The patients were not eligible for surgical resection or percutaneous ethanol injection. TAE was performed with Lipiodol Ultra-Fluid, epidoxorubicin and Gelfoam, with a mean of 1.7 treatments per patient. CT was performed 15 days after TAE. The mean cumulative survival was 14.2 months in patients with previously untreated HCC. The survival of patients stages Okuda I and II did not differ significantly (p > 0.05); tumor size did not affect survival (p > 0.05). Two patients with recurrent HCC died 7.0 and 9.3 months after the diagnosis of tumor recurrence; the remaining 4 patients are still alive with a maximum follow-up of 22.5 months from the diagnosis of HCC recurrence. Ten complications occurred in 8 patients, and were controlled by medical therapy. Eleven patients died during the study; no death was related to TAE. The series was not randomized, but comparison with the natural history of HCC suggests that TAE is effective as palliative treatment of advanced or recurrent HCC.

Aged↗
PubMed1993

[Gastric localization of sarcoidosis].

Sarcoidosis is a systemic granulomatous disease of unknown etiology, characterized by an immunological disorder with accumulation of activated lymphocytes and macrophages in all the organs and apparatus. The intrathoracic lymphnodes and the lung remain the most common sites of such disease. The gastrointestinal sarcoidosis, particularly of the stomach, is very rare. The stomach may be the primitive or the secondary (systemic sarcoidosis) site of sarcoid granuloma. The endoscopic aspects of the gastric mucosa are variable: localized or diffused hyperemia, single or multiple ulcers, aspects of atrophic gastritis with easy bleeding during contact, rigid mucosa and so on. Generally asymptomatic, the disease may show symptoms as pain in the epigastrium, nausea, vomiting, haematemesis and so on. The wide range of gastric pathologies resembling sarcoidosis both on a histological level and on a clinic-endoscopical one (syphilis, histoplasmosis, Crohn's disease, stomach cancer) require an extremely accurate diagnosis above all for the setting out of the therapy with steroids which are the most appropriate drugs (prednisone). Three out of thirty-two patients observed for respiratory problems, already affected by cutaneous and pulmonary sarcoidosis, started suffering from gastric symptoms of different kind: pain in the epigastrium, haematemesis, weight loss, nausea and post-prandial vomiting. Gastroscopy and biopsy, with histopathologic examination of gastric mucosal specimens taken from the most suspicious sites, confirmed the diagnosis of sarcoidosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1992

NMR determination of residual structure in a urea-denatured protein, the 434-repressor.

A nuclear magnetic resonance (NMR) structure determination is reported for the polypeptide chain of a globular protein in strongly denaturing solution. Nuclear Overhauser effect (NOE) measurements with a 7 molar urea solution of the amino-terminal 63-residue domain of the 434-repressor and distance geometry calculations showed that the polypeptide segment 54 to 59 forms a hydrophobic cluster containing the side chains of Val54, Val56, Trp58, and Leu59. This residual structure in the urea-unfolded protein is related to the corresponding region of the native, folded protein by simple rearrangements of the residues 58 to 60. Based on these observations a model for the early phase of refolding of the 434-repressor(1-63) is proposed.

Amino Acid Sequence↗
PubMed1992

1H, 15N and 13C NMR assignments of the 434 repressor fragments 1-63 and 44-63 unfolded in 7 M urea.

An E. coli overexpression system for the N-terminal domain of the 434 repressor with residues 1-63 (434 repressor(1-63)) was constructed and used to produce this polypeptide with uniform 15N-labeling, and with 13C-labeling of the methyl groups of valine and leucine. Using these protein preparations almost complete sequence-specific resonance assignments were obtained for the urea-unfolded form of the 434 repressor(1-63). In addition, the isotope-labeled tryptic peptide, 44-63, was produced by enzymatic cleavage of the recombinant 434 repressor(1-63), and its NMR spectrum was assigned. Corresponding residues in 434 repressor(1-63) and 434 repressor(44-63) in 7 M urea were found to have nearly identical chemical shifts, and in both species similar deviations from 1H random coil shifts were found as previously in 434 repressor(1-69). These indicate the presence of residual non-random structure in the polypeptide segment 50-60. The present NMR assignments, which include stereospecific assignments for the diastereotopic methyl groups of Val and Leu, are the basis for detailed studies of this residual structure in the urea-unfolded form of the 434 repressor.

Amino Acid Sequence↗
PubMed1992

Complete 15N and 1H NMR assignments for the amino-terminal domain of the phage 434 repressor in the urea-unfolded form.

The amino-terminal domain of the phage 434 repressor consisting of residues 1-69 forms a globular structure of five tightly packed helices, with nearly identical molecular architectures in crystals and in solution. Upon addition of urea to an aqueous solution of this protein, the NMR spectrum of a second form of the protein appears in addition to the native form, and at a urea concentration of 7 M, this urea-unfolded form is the only species observed. At intermediate urea concentrations, the two forms of the protein inter-convert at a rate that allows the observation of the exchange process by NMR. Starting from the previous assignments for the native protein, we obtained nearly complete sequence-specific (1)H and (15)N NMR assignments for the unfolded form of the protein. For most amino acid residues, the (1)H chemical shifts of the urea-unfolded protein are very similar to the random coil values, but some discrete regions of the polypeptide chain were identified that are likely to retain residual nonrandom spatial structure as evidenced by deviations of (1)H chemical shifts and amide proton exchange rates from the expected random coil values.

Amino Acid Sequence↗