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Biomedical subjects

A Silva

Publications and source records attributed to A Silva.

At least 145 records · Page 8Linked to original sources
PubMed1991

Endoglucanase A gene fusion vectors for monitoring protein secretion and glycosylation in yeast.

We have constructed a set of replicating and integrating vectors that allow expression and secretion of Clostridium thermocellum endoglucanase A in Saccharomyces cerevisiae using the alpha-factor or the invertase promoters and secretion signals. The enzyme expressed in yeast was enzymatically active regardless of its degree of glycosylation and was released into the culture medium. One advantage of using this experimental system is that secretion of the reporter enzyme can be detected in individual colonies facilitating the isolation of mutants. A second advantage is that transit through the secretory pathway, as judged from the extent of glycosylation, can be easily monitored by staining for enzyme activity in agar replicas of polyacrylamide gels.

Cellulase↗
PubMed1991

Phase II trial of carboplatin or iproplatin in cervical cancer.

From July 1984 to November 1987, 89 patients with recurrent measurable squamous-cell cancer of the uterine cervix were randomized in a single institution to receive treatment with either carboplatin (CBDCA) or iproplatin (CHIP). Objective response rates were similar: 2 complete regressions (CRs) and 10 partial regressions (PRs) were recorded both in the 46 evaluable patients treated with CBDCA (response rate, 26.1%; 95% confidence interval, 15-41%) and in the 40 evaluable patients treated with CHIP (response rate, 30%; 95% confidence interval, 17-47%). The median duration of response was 5.5 months for CBDCA and 6 months for CHIP; the median survival was 7.5 and 7.6 months, respectively. Both drugs were given in an outpatient setting and myelosuppression (thrombocytopenia) was the predominant toxicity. Analysis of all toxic events yielded additional interesting observations: the occurrence of moderate to severe platelet nadirs beyond cycle 1 was confined to CHIP, a higher incidence of gastrointestinal toxicity during treatment with CHIP, and five moderate to severe complaints of asthenia (recorded as neurologic events) during CHIP therapy versus only one during treatment with CBDCA. Because of its antitumor activity and its toxicologic advantage, a future role for CBDCA in the treatment of cervical cancer appears likely.

Antineoplastic Agents↗
PubMed1991

[Treatment of uncomplicated urinary tract infections: cefuroxime vs cotrimoxazole].

Forty females (ages 15-74) with community-acquired, uncomplicated urinary tract infections were studied. Clinical and microbiological efficacy of cefuroxime (250 mg td) and trimethoprim sulfamethoxazole (160/800 mg td) used for 7 days were evaluated. The microorganisms found in the pre-treatment urinary cultures were: Escherichia Coli (85%), Klebsiella Pneumoniae (12.5%), and E. Agglomerans (2.5%). They were all susceptible to cefuroxime, and 42.5% were resistant to trimethoprim sulfamethoxazole (Kirby-Bauer). These findings show that trimethoprim sulfamethoxazole should not be used empirically while waiting for the results of urinary cultures, and that cefuroxime is a good alternative in these cases. Clinical cure was observed in all, and bacteriological cure in 75% of the pts treated with both antimicrobial agents. Relapses and reinfections were detected during follow-up emphasizing the importance of intra and post-treatment urinary cultures.

Adolescent↗
PubMed1991

[Echo-Doppler in patients undergoing mitral commissurotomy. Comparison of open and closed commissurotomy].

OBJECTIVE: 1. To evaluate with Echo-Doppler the medium/long term results of mitral commissurotomy. 2. To compare the results of open mitral commissurotomy to those of closed commissurotomy. PATIENTS AND METHODS: The Echo 2D-Doppler study has been performed in 117 patients (104 females and 13 males), randomly selected among patients previously submitted to open (62 d.) or closed (55 d.) mitral commissurotomy, with greater than or equal to 12 months follow-up. Pulsed and continuous Doppler recordings were obtained in all 117 patients, and the following parameters were evaluated: Maximum Mitral Gradient (MG); mitral valvular area calculated by half-pressure time (MVA); presence of mitral regurgitation greater than or equal to grade 2 (MR); evaluation of systolic Pulmonary Artery pressure (PAP) by the gradient RV/RA + 14mmHg, in the patients with tricuspid insufficiency; acceleration time (ACT), ejection time (EJT) and the relation ACT/EJT in pulmonary artery. To compare the results of open commissurotomy, to those of closed commissurotomy, and in order to minimize the pre-operative differences between the patients submitted to each of these interventions, only the patients in the same NYHA functional class before surgery (class III), and in sinus rhythm have been selected. In this way two groups were compared: group A-24 patients with open commissurotomy and group F-37 patients with closed commissurotomy. RESULTS: in the total of patients the results were: MG-9.8 +/- 4.2 mmHg; MVA-2.3 +/- 0.95 cm2; MR 16 pts. (13.7%); PAP obtained in 30 pts. (25.6%) -27.47 +/- 1.18 mmHg; the ACT, the EJT and the relation ACT/EJT in PA were respectively 115.63 +/- 34 ms, 309.9 +/- 38.5 ms, 0.37 +/- 0.1. The Doppler parameters obtained respectively in group A and in group F were: MG 8.96 +/- 4.5 mmHg and 10.38 +/- 3.82 mmHg (ns); MVA 2.62 +/- 1.01 cm2 and 2.08 +/- 0.84 cm2 (ns); MR 8.3% and 15.6% (ns) (Fig.3); the PAP have been calculated on 5(20.8%) patients in group A and on 10(27%) patients in group F and their respective values were 30.78 +/- 0.48 mmHg and 28.26 +/- 1.11 mmHg (ns); ACT 109.09 +/- 41.55 ms and 116.67 +/- 33.22 ms (ns); EJT 297.27 +/- 72.94 ms and 308.7 +/- 41.58 ms; ACT/EJT 0.35 +/- 0.12 and 0.38 +/- 0.10 (ns). CONCLUSION: Both open and closed commissurotomy revealed to be good therapeutic alternatives to mitral stenosis with identical long term results as evaluated by Echo-Doppler. The results of percutaneous mitral valvuloplasty should be compared with these in the future.

Adult↗
PubMed1990

A novel functional cell surface dimer (Kp43) expressed by natural killer cells and T cell receptor-gamma/delta+ T lymphocytes. I. Inhibition of the IL-2-dependent proliferation by anti-Kp43 monoclonal antibody.

In the present study we describe a novel functional cell surface molecule, designated as Kp43, which is expressed among leukocytes by NK cells, TCR-gamma/delta + T lymphocytes, and some CD8+ CD56+TCR-alpha/beta + T cell clones. The Kp43 Ag is a 70-kDa disulfide-linked dimer, which migrates in SDS-PAGE under reducing conditions as a single 43-kDa band. Two-color immunofluorescence staining of fresh PBL revealed that only a fraction of CD16+, and of TCR-gamma/delta + T lymphocytes expressed the Ag. The analysis of TCR-alpha/beta + T cell clones showed that a small proportion (2 out of 20) weakly expressed Kp43 together with the CD8 and CD56 molecules. By immunoperoxidase staining of different tissues the anti-Kp43, reactivity was detected exclusively in lymphoid organs, where a minority of scattered cells was stained, and in some liver sinusoidal cells. Essentially all NK cells acquired Kp43 when stimulated with a B lymphoblastoid cell line. By contrast, the pattern of distribution of Kp43 remained stable upon in vitro culture of T-gamma/delta lymphocytes, thus delineating two subsets according to its expression. In lymphokine-activated killer populations, obtained by culturing either PBL or NK cells with high concentration of IL-2, most CD16+ and CD56+ cells became Kp43+. The Kp43-specific mAb inhibited the IL-2-dependent proliferative response of cultured NK and TCR-gamma/delta + T cells without affecting their non-MHC-restricted cytotoxicity. The partial inhibitory effect, which was mediated as well by pepsin digested F(ab')2 fragments, was lost upon reduction to Fab. The anti-Kp43 mAb did not interfere with the specific binding of IL-2 to its surface receptors. Altogether the data point out that the Kp43 dimer is involved in the regulation of the IL-2-dependent proliferative response of NK cells and a subset of TCR-gamma/delta + T lymphocytes.

Antibodies, Monoclonal↗
PubMed1990

Synergy of tumor necrosis factor with protein kinase C activators on T cell activation.

The ability of tumor necrosis factor (TNF)-alpha to activate T lymphocytes in combination with other stimuli has been studied. TNF was strongly co-mitogenic with low doses of anti-CD3 antibodies or phorbol esters (those which are strong activators of protein kinase C, PKC) but poorly with phytohemagglutinin or concanavalin A. No synergism was seen with the calcium ionophore A23187. TNF was co-mitogenic with several phorbol esters known to activate PKC but was uneffective with inactive phorbol esters such as methyl-phorbol 12-myristate 13-acetate. Furthermore, H-7 a known inhibitor of PKC, inhibited the proliferative response of T cells induced by esters plus TNF. This effect took place at low doses of TNF and was also observed with purified T lymphocytes indicating that the effect of TNF was not dependent on accessory cells. This proliferative effect of TNF was inhibited by an anti-interleukin 2 receptor (IL2R) antibody, MAR 108, which blocks IL2 binding to its receptor. Although PKC activation induced CD25 (IL2R) expression but very little IL2 synthesis, TNF did not synergize by augmenting the synthesis of this lymphokine in peripheral blood lymphocytes stimulated with phorbol esters. By contrast, TNF strongly increased the membrane level of CD25 and to a lesser extent that of the activation antigen, 4F2, over the levels already induced by phorbol esters on T cells. More interestingly, TNF significantly increased the number of high-affinity IL2R on purified T cells in the presence of phorbol 12,13-dibutyrate. Our results indicate that TNF is co-mitogenic with those stimuli which strongly activate PKC and suggest that TNF may play a role on T cell activation increasing the number of effective IL2/IL2R interactions when these are limiting.

Antigens, CD↗
PubMed1990

Further characterization of monoclonal antibodies to lipopolysaccharide of Salmonella Minnesota strain R595.

We have shown here that despite the use of monoclonal antibodies with well-defined epitope-specificities, and despite testing them in the most simple animal model available (i.e., mixing of homologous LPS with Mab prior to injection), we are not yet able to explain why some of the antibodies were effective and others not. For some of the clones (e.g., clone 20), an even better definition of binding sites is currently taking place in an attempt to obtain this understanding. We also do not yet understand why clone 20 was not effective in the mucin model, while using much lower amounts of injected antibody, and much higher challenge doses, this Mab was effective against E. coli in the gentamicin-treated mouse model. Very clear is, however, that in order to be protective in the latter model, Mabs are not required to be specific for lipid A. In the future it will be essential to develop procedures which measure specific interaction between smooth LPS/bacteria and antibodies to the LPS core region. In addition, it will be of great help when the chemical structure of non-substituted, rough-form LPS, as occurring in smooth LPS preparations, would be defined. This applies also to O-substituted core molecules.

Animals↗
PubMed1990

Production and characterization of human gamma interferon from Escherichia coli.

The production of human gamma interferon as intracellular inclusion bodies in Escherichia coli, which simplified the purification process, is described. An expression plasmid carrying lipoprotein and the tryptophane promoters in tandem was used. Preparation of highly pure interferon was achieved using high resolution chromatography after denaturation and renaturation steps. Structural characteristics of this protein were verified by mass spectrometric analysis. Additional control tests have shown the suitability of the final product for clinical purposes.

Amino Acid Sequence↗
PubMed1990

Plasmodium falciparum: two antigens of similar size are located in different compartments of the rhoptry.

Two previously described antigens, AMA-1 and QF3, which are located in the rhoptries of Plasmodium falciparum merozoites have polypeptides of similar relative molecular masses. On immunoblots, antibodies to both antigens recognized polypeptides of relative molecular mass 80,000 and 62,000 in all isolates tested. Two-dimensional electrophoresis showed that the isoelectric points of the two antigens were different. QF3 being more basic than AMA-1. AMA-1 was soluble in Triton X-114 whereas QF3 partitioned into the aqueous phase after temperature-dependent phase separation. In immunoelectron microscopic studies. QF3 was found in the body of the rhoptry whereas AMA-1 was consistently found in the neck of the rhoptry. Both antigens gave a punctate double-dot pattern in mature schizonts and merozoites when visualized by fluorescence microscopy, but AMA-1 antibodies also appeared to label the merozoite surface. QF3 was also detected in ring-infected erythrocytes whereas AMA-1 was not. Synthesis of both antigens was first observed in mature trophozoites and immature schizonts. Pulse-chase experiments showed that the Mr 80,000 polypeptide of the AMA-1 gene was subject to immediate processing to the Mr 62,000 product. This cleavage pattern was not stage specific. The Mr 80,000 polypeptide of QF3 was derived from a short-lived Mr 84,000 precursor polypeptide. Processing of the Mr 80,000 polypeptide to an Mr 62,000 polypeptide was restricted to the period of merozoite maturation and reinvasion. Hence AMA-1 and QF3 are different antigens with polypeptides of similar size but located in different compartments of the merozoite rhoptries.

Animals↗
PubMed1990

Endothelin immunoreactivity in mice with gram-negative bacteraemia: relationship to tumour necrosis factor-alpha.

1. To investigate the role of endothelin in Gram-negative bacteraemia and the possible involvement of tumour necrosis factor-alpha in its pathophysiology, we measured plasma and tissue (lung, kidney and spleen) immunoreactive endothelin levels in Gram-negative bacteraemic mice, with and without passive immunization by anti-(tumour necrosis factor-alpha) antibody. 2. Plasma immunoreactive endothelin levels were greatly increased after the Escherichia coli injection. Pretreatment with anti-(tumour necrosis factor-alpha) antibody did not suppress elevated plasma immunoreactive endothelin levels (P greater than 0.1). 3. Lung tissue immunoreactive endothelin levels in mice were increased 16 h after the E. coli injection and were not affected by prior passive immunization with anti-(tumour necrosis factor-alpha) antibody. Immunoreactive endothelin in spleen and kidney was undetectable (less than 34 fmol/g wet weight). 4. Injection of rMu tumour necrosis factor-alpha into mice did not increase plasma immunoreactive endothelin levels. 5. Antibody to endothelin given 30 min after a 90% lethal dose challenge with E. coli did not affect mortality. 6. We conclude that the rise in plasma and tissue endothelin that occurs in Gram-negative bacteraemia is independent of tumour necrosis factor-alpha.

Animals↗
PubMed1990

[Hemorrhagic cardiac tamponade without heart rupture during a myocardial infarct. A clinical case].

Cardiac tamponade during acute myocardial infarction is a life-threatening complication that can be confounded with right ventricular infarction. The most frequent cause of this complication is cardiac rupture. We report here a patient with acute myocardial infarction that developed cardiac tamponade on day 7, after receiving late systemic thrombolysis. The diagnosis was suspected with echocardiography and confirmed with hemodynamic measurements. The tamponade was partially relieved with pericardiocentesis but afterwards required emergency surgery. No cardiac rupture was found but an hemorrhagic infarction. We conclude that in this case the hemorrhagic tamponade was probably related both to late thrombolysis and to post infarction pericarditis.

Adult↗
PubMed1990

OX48, a monoclonal antibody against a 70,000 MW rat activation antigen expressed by T cells bearing the high-affinity interleukin-2 receptor.

The monoclonal antibody (mAb) OX48 recognizes a 70,000 MW cell-surface protein present in a small percentage of activated rat T cells and in CD8+ rat x BW5147 interleukin-2 (IL-2)-dependent T-cell hybridomas, but not in resting spleen cells or in IL-2-independent T-cell hybrids. OX48 antibody added simultaneously with concanavalin A (Con A) to resting spleen cells inhibits the cell proliferation and reduces the IL-2 production. However, addition of IL-2 does not restore the mitogenic response. Growth of rat blast T cells or IL-2-dependent hybrids is not affected by the OX48 antibody. There is a close correlation between the expression of high-affinity IL-2 receptors (IL-2R) and the OX48 antigen in T-cell hybridomas. In spite of this striking correlation, OX48 mAb does not inhibit the binding of 125I-IL-2 to the IL-2-dependent hybrids, and is unable to immunoprecipitate any of the proteins chemically cross-linked to 125I-IL-2. Therefore, the OX48 molecule represents a new rat activation antigen, undefined in other species, and probably involved in the early steps of T-cell activation.

Animals↗
PubMed1989

IL-2 protects T cell hybrids from the cytolytic effect of glucocorticoids. Synergistic effect of IL-2 and dexamethasone in the induction of high-affinity IL-2 receptors.

IL-2-independent CD8+ rat x BW5147 T cell hybridomas are highly sensitive to treatment with 10(-6) M dexamethasone. This glucocorticoid analog induces a rapid DNA fragmentation with a pattern similar to that observed during glucocorticoid-induced killing of mouse thymocytes, which suggests the activation of a similar specific endonuclease. Among these hybrids, we select variants expressing low affinity IL-2R, as measured by IL-2 binding assay and by the cell surface expression of the IL-2Rp55 Ag (rat CD25 recognized by OX-39 mAb). These OX-39+ IL-2 independent hybrids (named V type) are protected from the toxic action of dexamethasone by IL-2. The addition of IL-2 to V type cells induces the expression of a low number of high affinity IL-2R, which is strongly potentiated by the simultaneous addition of dexamethasone. Furthermore, dexamethasone is strongly synergistic with IL-2 in the induction of mRNA p55/IL-2R, which could be observed 6 h after the treatment. These data suggest that the utilization of the IL-2-R signaling pathway may induce an effective protection against glucocorticoid toxicity in mature T cells. Finally, we proved that the upregulation of IL-2R by IL-2 is strongly potentiated by glucocorticoids, which implies a new role for these agents in the immune system.

Animals↗
PubMed1989

Loss of interleukin 2 dependence in cloned interleukin 2-dependent rat T lymphocyte x BW5147 hybridomas is not associated with segregation of a specific pair of rat chromosomes.

A fusion between the mouse AKR thymoma BW5147 and a culture of homogeneously OX8+ (CD8) rat T lymphoblasts yield interleukin (IL) 2-dependent T cell hybridomas when selected in HAT medium supplemented with IL 2-containing supernatants of concanvalin A-activated cells and dexamethasone. IL 2-independent variants can be selected from cloned IL2-dependent hybrids in the absence of conditioned medium. Karyotype analysis was used to test a previously proposed hypothesis according to which IL2-independent variants arise through loss of a specific cytotoxic T lymphocyte (rat) chromosome carrying a gene responsible for IL2 dependence. Comparison of karyotypes of several independently derived hybrids with those of their IL 2-independent variants showed that the hybrids contain at least one homologue of all rat chromosomes, and that no pair of rat chromosomes is consistently absent in the IL 2-independent variants.

Animals↗
PubMed1989

Growth requirements of T cell hybridomas obtained by the fusion between a mouse cytolytic T cell line and the rat tumor C58NTD.

T23 are hybrids derived from the fusion between an IL-2-dependent mouse cell line, C10 and the rat lymphoma C58NTD. Supernatants from exponentially growing T23 cells induce the growth of CTLL2, and IL-2-dependent cell line, suggesting that these hybrids secrete interleukin 2. Addition of recombinant IL-2 to slowly growing T23 cells increases the rate of growth. Using an 125I IL-2 binding assay, a low number of cell surface IL-2 receptors were detected. T23 hybrids contain mouse but not rat IL-2 receptor genes as revealed by Southern blot analysis. These receptors are functional because the growth of exponentially growing hybrids is inhibited by an anti-mouse IL-2 receptor antibody. These data suggest an autocrine-like mechanism as responsible for the growth of these T cell hybridomas.

Animals↗