Biomedical subjects
G Motta
Publications and source records attributed to G Motta.
Production of a functional anti-scorpion hemocyanin scFv in Escherichia coli.
A58 is a murine monoclonal antibody raised against the internal dimeric subunit A alpha 1 of the hemocyanin of the scorpion Androctonus australis. When A58 is tested against a range of related hemocyanins, it exhibits cross-reactivity with a limited number of scorpion species. All of them belong to a single Scorpion Family: the Buthidae. We have utilized a bacterial expression-export system in which the variable regions of A58 are expressed as a single-chain variable fragment (scFv). Polymerase chain reaction (PCR) was used for the cloning and modification of the heavy and light variable regions which were assembled into a scFv. After insertion into a vector which contains the pectate lyase signal sequence from Erwinia caratovora for periplasmic expression, a scFv protein was produced in high yield as a soluble and functional protein. The bacterial produced A58 scFv has binding properties similar to those of the original murine antibody. It binds specifically the heterodimeric subunit of Androctonus australis hemocyanin, cross-reacts with only one subunit of each Buthidae investigated, and blocks the recognition of the hemocyanin by antibody A58 in competitive enzyme-linked immunosorbant assay. Previous reports have demonstrated the value of monoclonal antibodies in taxonomical and molecular evolution studies. A58scFv is the first of a new generation of antigen-binding proteins which should prove useful both in taxonomical studies and in the analysis of VH--VL structure-function relationships of antibodies.
Auditory brainstem responses in thyroid diseases before and after therapy.
The aim of this study was to evaluate through the auditory brainstem responses (ABRs) the electrical events generated along the auditory pathway in 56 adult patients affected with hyper- and hypothyroidism. Twenty-four normal-hearing hyperthyroid patients affected with Graves' disease and 32 normal-hearing hypothyroid patients (9 with subclinical and 23 with overt hypothyroidism) were subjected to standard (clicks at 21 pps) and sensitized ABR with masking wide-band (masking noise). In addition, thyroid scintiscan and ultrasonography, free T3 and T4, total T3 and T4, TSH, antimicrosomal and antithyroglobulin antibodies, audiogram and impedance tests were performed in all the patients. This study was repeated after 6-12 months of treatment in conditions of euthyroidism. The results showed changes of ABRs both in the standard procedure as well as in the sensitized test in 6 hyperthyroid (25%) and 8 hypothyroid patients (25%). All the patients with abnormal ABRs had overt hypothyroidism (8/23; 34.7%). The ABRs became normal in 5 out of 6 Graves' patients after 6-12 months of methimazole treatment. ABRs remained abnormal in all the hypothyroid patients despite their having been on L-thyroxine treatment for 6-12 months and were euthyroid for at least 5 months before the study was repeated. These findings suggest that ABR abnormalities are indicative only of a nonspecific injury in the bulbo-ponto-mesencephalic centers. Alterations of ABRs in thyroid diseases are not specific in relation to hyper- or hypothyroidism. Lastly, there is a relationship between ABR abnormalities and the degree of hypothyroidism, even if ABR alterations are not always reversible after long-term therapy.
Characterization and cellular localization by monoclonal antibodies of the 60 kDa mannose specific lectin of human promyelocytic cells, HL60.
Myelomonocytic lineage cells express an M(r) 60,000 mannose specific lectin, MR60 (Pimpaneau et al. (1991), Carbohydr Res 213: 95-108). Under non-reducing conditions, this protein migrates as a 120,000 protein. MR60 does not contain any N-glycan moiety cleavable by the action of N-glycanase. MR60 induces a sugar selective aggregation of beads coated with glycosylated albumin: beads bearing alpha-D-mannosyl residues are aggregated while beads bearing alpha-D-glucosyl residues are not. A monoclonal antibody Lec101B, specific for MR60, recognizes a single M(r) 60,000 protein by Western blotting. This monoclonal antibody does not label the cell surface of cells expressing MR60, but decorates intracellular vesicles upon permeabilization of these cells.
[Recent advancements in the clinical and surgical therapy of allergic rhinitis].
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Immunoactivity of pidotimod against episodes of recurrent tonsillitis in childhood.
The therapeutic efficacy of the synthetic immunostimulant pidotimod ((R)-3-[(S)-(5-oxo-2-pyrrolidinyl) carbonyl]-thiazolidine-4-carboxylic acid, PGT/1A, CAS 121808-62-6) was evaluated in a double-blind placebo-controlled study in parallel groups in the management of recurrences in 235 children with recurrent tonsillitis. The ambulant study provided for 15 days of treatment with two oral vials of pidotimod 400 mg or placebo daily, in accordance with a randomisation list, 60 days of treatment with one oral vial of pidotimod 400 mg or placebo daily, and a 90-day follow-up period. The total trial period was 165 days. In addition to evaluating the number of tonsillitis recurrences which occurred during the 75 days of treatment and the 90-day follow-up period, the number of days on which the principal symptoms of the illness were present and on which drugs such as antibiotics or anti-inflammatory agents were used concomitantly, as well as the number of days' absence from school, were analyzed. The findings showed that, taking the treatment phase and the three-month follow-up period together, pidotimod significantly reduces the incidence of inflammatory upper airways episodes. The very low incidence of adverse effects, which was the same as that in the placebo group, confirmed the excellent safety of the product.
Rapallo Conference. New frontiers in lung cancer.
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Human plasma kallikrein processing: proteolysis as an alternative control.
Human plasma kallikrein (HuPK) was detected in normal non-activated and dilution-activated plasma by immunoblotting, using polyclonal antibodies. In non-activated plasma, the predominatly detected protein corresponds to prokallikrein (Mr 80,000-90,000). Activated plasma, besides kallikrein, contains larger proteins (Mr > 130,000) that possibly represent complexes between kallikrein and proteinase inhibitors. Plasma also contains species (Mr 43,000) which corresponds to kallikrein heavy chain. In activated plasma, monoclonal antibodies against kallikrein heavy chain detected, besides these same bands described above, two additional bands (Mr 30,000 and 20,000) possibly correspondent to fragments of kallikrein heavy chain.
Allergic rhinoconjunctivitis: diagnostic and clinical assessment.
In allergic rhinoconjunctivitis not only the nasal mucosa but also the conjunctiva and, in severe cases, the cornea are affected by reactive phenomena. This pathological process is often encountered in clinical practice, its incidence ranging from 10% to 30% of the whole population, as reported by many authors. Up to now, the pathogenetic mechanisms of allergic reactivity are not completely understood. In our opinion this is due to insufficient standardization of the diagnostic procedures; even the clinical picture of such a pathological process seems to be insufficient, especially with regard to the analogies between the nasal and conjunctival pathologies. To a great extent this depends on the different clinical pictures both in otorhinolaryngology and ophthalmology. Our study has been carried out on 98 patients suffering from rhinoconjunctivitis, and presents an accurate documentation with regard to the existence of analogies in reactivity at the immunoallergic level, in both the nose and conjunctiva. These are evident when we refer not only to specific routine allergological tests but, mainly, to local examinations. The evaluation of the selected data enables us to formulate a single classification of allergic rhinoconjunctivitis, taking into account the common reactive phenomena.
Authors respond to questions regarding pressure measurements in recent article.
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Action of plant proteinase inhibitors on enzymes of the kallikrein kinin system.
Serine proteinase inhibitors, in the seeds of several Leguminosae from the Pantanal region (West Brazil), were studied using bovine trypsin, Factor XIIa and human plasma kallikrein. The inhibitors were purified from Enterolobium contortisiliquum (Mr = 23,000), Torresea cearensis (Mr = 13,000), Bauhinia bauhinioides (Mr = 20,000), Bauhinia mollis (Mr = 20,000) and Bauhinia pentandra (Mr = 20,000). E. contortisiliquum inhibitor inactivates all three enzymes, whereas the T. cearensis inhibitor inactivates trypsin and Factor XIIa, but does not affect plasma kallikrein. B. bauhinioides and B. pentrandra inhibitors, on the other hand, inactivate trypsin and plasma kallikrein but only the B. pentandra inhibitor affects Factor XIIa, and B. mollis inhibitor causes trypsin inactivation only. Calculated Ki values were between 10(-7) and 10(-9) M. Chymotrypsin, like trypsin, is also inhibited, but with lower affinity. The trypsin inhibitors, isolated from E. contortisiliquum, B. pentandra, B. bauhinioides and B. mollis seem to be of the Kunitz type; the inhibitor purified from T. cearensis is of the Bowman-Birk type.
Human plasma kallikrein. Immunoreactivity and activity on natural and synthetic substrates.
Human plasma kallikrein (HuPK) is a serine protease found in mammalian plasma. Following limited proteolysis, the enzyme is activated and forms two chains. The light chain occurs with molecular weight 36,000 or 33,000 and contains the active site. The heavy chain occurs with molecular weight of 45,000 and contains the binding site for high-molecular-weight kininogen. Both chains were prepared from active HuPK, following mild reduction with dithiothreitol and carboxymethylation with iodoacetamide. The light chain was isolated in SBTI-Sepharose and its kinetic properties were determined with synthetic derivatives of arginine-p-nitroanilides, to investigate any possible alteration of the active site. These studies showed that substrate modifications affected the hydrolytic activity more than the binding capacity. The ability to cleave high-molecular-weight kininogen, as observed for intact kallikrein, was reduced in beta-kallikrein and absent in the light chain, even when equimolar amounts of both light and heavy chains were tested together. Anti-kallikrein antiserum formed immunoprecipitates not only with kallikrein itself, but also with the separated chains. The immunoreactivity of the light-chain was not identical with that of kallikrein. Immunoselected specific antibodies for both chains, depending on the selectivity, reacted with the heavy chain and kallikrein, or with the light chain and kallikrein. These antibodies were shown to be effective in binding radio-iodinated HuPK in the radioimmunoassay developed for intact kallikrein.
[Indications and advantages of vocal rehabilitation of laryngectomized patients using tracheoesophageal prosthesis].
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Topological mapping of 13 epitopes on a subunit of Androctonus australis hemocyanin.
A topological localization of epitopes on the surface of the Aa6 subunit of Androctonus australis hemocyanin has been carried out. First, immunocomplex strings composed of native hemocyanin and monoclonal antibodies were examined in the electron microscope and submitted to an image processing by correspondence analysis. The average images were then compared to a three-dimensional model of the 24-mer suggesting that 11 of the 13 epitopes are located in three zones of the subunit surface. Second, the overlaps between the epitopes were then studied by polyacrylamide gel electrophoresis, competitive binding inhibition, and immunoelectron microscopy. Four groups of epitopes were identified. One group was capable of binding exclusively to the free subunit. The other three groups were identical to those found in immunoelectron microscopy. The data are consistent with the existence of a small number of immunodominant regions on the surface of the Aa6 subunit.
Prognostic value of the Jass histopathologic classification in left colon and rectal cancer: a multivariate analysis.
Variables correlated to survival were studied in 121 patients who had undergone potentially curative surgery for left colon and rectal cancer. To investigate the prognostic value of the parameters both univariate and multivariate analysis were carried out. Minimum follow-up was 5 years. Multivariate analysis showed that while disease stage (p less than 0.0001) and site of primary tumor (p less than 0.0006) independently influenced survival, type of surgical procedure and histopathologic grade had no impact on survival. Jass histopathologic classification predicted survival for patient group I and IV whereas no significant relationship was observed for group II and III, which, in our series, were the most frequently encountered groups. Of the three parameters considered for Jass classification, tubule configuration, pattern of tumor growth and lymphocytic infiltration, only the latter was significantly correlated to survival (p less than 0.005). Different results were obtained when the prognostic values of Jass group was investigated separately for the 49 patients with adenocarcinoma of the left colon and the 72 patients with rectal cancer. Further investigation is required before routine clinical application of the Jass classification can be recommended.
Clinical application of electroglottography.
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Improving clinical efficacy of computed tomographic scan in the preoperative assessment of patients with non-small cell lung cancer.
The criterion of choice for computed tomographic scan identification of metastatic mediastinal nodes is not clearly fixed. This prospective study was designed to define the most suitable computed tomographic criterion for detection of nodal metastasis, enabling improvement of the test's clinical efficacy. One hundred twenty-three patients with potentially operable non-small cell lung cancer underwent mediastinal evaluation by computed tomographic scan and cervical mediastinoscopy followed by thoracotomy with mediastinal node dissection. There were 116 men and seven women; the mean age was 59.3 +/- 9.1 years. Forty-six tumors were classified after operation as stage I, 20 as stage II, 27 as stage IIIa, and 30 as stage IIIb. Mediastinal nodes were classified as metastatic according to the following computed tomographic scan criteria: (1) shorter axis 1 cm or larger; (2) shorter axis 1.5 cm or larger (nodes less than 1 cm were classified as negative and those 1 to 1.5 cm as indeterminate); and (3a) shorter axis 1 cm or larger, plus evidence of central necrosis or discontinued capsule, or (3b) shorter axis 2 cm or more, regardless of the nodal morphologic condition. The highest sensitivity rate was achieved by using criterion 1 (90%) and the poorest by criterion 3 (75%). The greatest specificity rate was obtained by applying criterion 3 (90%) and the lowest by criterion 1 (54%). The prediction by using computed tomographic criterion 3 correlated better with pathologic findings than that derived by adopting the criterion 1 or 2. When mediastinal nodes were identified as negative according to criterion 1, 2, or 3, the complete resection rate was 92%, 92%, or 95%, respectively, rendering cervical mediastinoscopy unnecessary. When mediastinal nodes were classified as positive, the resectability rate was 55%, 27%, or 13%, respectively. In these instances cervical mediastinoscopy allowed identification of different degrees of mediastinal involvement; it proved to be the most useful procedure for preoperative selection of those patients with N2 tumors who are amenable to a complete resection. In conclusion, the use of computed tomographic criterion 3 does improve the clinical efficacy of the test, by sparing a large number of unnecessary mediastinal explorations, without increasing the rate of useless thoracotomies.