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Biomedical subjects

E Carvalho

Publications and source records attributed to E Carvalho.

At least 37 records · Page 2Linked to original sources
PubMed1995

Characterization of human and rat immortalized clones parotid acinar cells with respect to specific proteins and their mRNAs, and receptor-linked adenylate cyclase.

This study reports the isolation and characterization of a rat nontumorigenic parotid acinar cell clone (2RSG), a human nontumorigenic parotid acinar cell clone (2HPC8), and a human tumorigenic acinar clone (2HP1G). The levels of alpha-amylase mRNAs detected when using alpha-amylase cDNA of 1176 and 702 bp for hybridization were higher in 2RSG and 2HPC8 cells than their respective whole parotid glands. The level of these mRNAs decreased in 2HP1G cells. In contrast to alpha-amylase mRNAs levels, the alpha-amylase activity in cultured acinar cells was extremely low in comparison to whole glands, irrespective of species or cell status. The levels of proline-rich protein (PRP) mRNA and parotid secretory protein (PSP) mRNA detected when using PRP cDNA of 600 bp and PSP cDNA of 805 bp for hybridization were higher in 2RSG cells than those in rat parotid glands; the reverse was observed in 2HPC8 cells and human parotid glands. The levels of PRP mRNA and PSP mRNA in 2HPC8 and 2PH1G acinar cells were similar. The level of mRNA was not detectable in murine neuroblastoma cells (NBP2) using the same alpha-amylase cDNA, PRP cDNA and PSP cDNA for hybridization. The PSP level in rat parotid gland was lower than that found in 2RSG cells; the reverse was observed in 2HPC8 cells and human parotid glands. The level of PSP in 2HP1G cells was higher than that found in 2HPC8 cells. Isoproterenol increased the cAMP level in 2RSG, 2HPC8, and 2HP1G clones, being most effective in 2RSG cells, and least effective in 2HPG cells. Prostaglandin E1 (PGE1) also increased cAMP level, being most effective in 2HPC8 cells and ineffective in 2HP1G cells, suggesting that the PGE1 receptor-linked adenylate cyclase becomes inactive upon transformation. These results suggest that the three clonal acinar cells from rat and human parotid glands reported here can be useful in comparative studies on regulation of growth, differentiation, and transformation.

Adenylyl Cyclases↗
PubMed1994

Differential regulation of phenotypic expression in a pluripotential neuroblastoma cell line.

Our laboratory has recently been involved in investigating factors which influence plasticity of neurotransmitter phenotypic expression both in vivo and in culture. Our previous studies have shown that precursor neuroblasts are pluripotential with respect to neurotransmitter phenotype and respond differentially to microenvironmental signals. In the present study, we examined phenotypic expression in neuroblastoma cells, P2 clone, using the activities of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH) as neuronal markers for the cholinergic and catecholaminergic phenotypes, respectively. Cells were plated and grown for 4 days in culture, harvested and frozen for assay of ChAT and TH. Basal activity of ChAT was 2.47 +/- 0.22 nmoles Ach formed /h/mg protein and that of TH was 5.23 +/- 0.41 pmoles CO2 formed /h/mg protein in control cultures. When intracellular cAMP levels were increased by addition to the growth medium of 10 micrograms/ml prostaglandin E1 (PGE1; a receptor-mediated enhancer of adenylate cyclase activity) or 200 micrograms/ml RO20-1724 (an inhibitor of cyclic nucleotide phosphodiesterase) the activity of TH was increased 340- and 423-fold, respectively. In marked contrast, the activity of ChAT was not affected by either agent. Double staining immunocytochemical examination demonstrated that both ChAT and TH were colocalized in the same cell. The molecular mechanism whereby catecholaminergic expression exclusively is affected in this cell model is currently under investigation. We conclude that (1) P2 neuroblastoma is a pluripotential cell line, (2) phenotypic expression in a homogenous cell population, such as P2 neuroblastoma, is differentially regulated. Moreover, this cell line is a unique model for studying the molecular mechanisms of phenotypic expression and neuronal plasticity.

4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone↗
PubMed1994

Establishment and characterization of immortalized clonal cell lines from fetal rat mesencephalic tissue.

This investigation reports for the first time the establishment of immortalized clones of dopamine-producing nerve cells in culture. Freshly prepared single-cell suspensions from fetal (12-day-old) rat mesencephalic tissue were transfected with plasmid vectors, pSV3neo and pSV5neo, using an electroporation technique. Cells were plated in tissue culture dishes which were precoated with a special substrate and contained modified MCDB-153 growth medium with 10% heat inactivated fetal bovine serum. The immortalized cells were selected by placing the transfected cells in a selection medium (modified MCDB-153 containing 400 micrograms/ml geneticin). The survivors showed the presence of T-antigens and were non-tumorigenic. Two cell lines, 1RB3 derived from cells transfected with pSV3neo, and 2RB5 derived from cells transfected with pSV5neo revealed only 1 to 2% tyrosine hydroxylase (TH)-positive cells. Repeated single-cell cloning of these cell lines by a standard technique failed to increase the number of TH-positive cells in any clones. Using three cycles of growth, alternating between hormone-supplemented, serum-free medium and serum-containing medium produced a cell line (1RB3A) that was very rich in TH-positive cells. The recloning of 1RB3A yielded clones some of which contained over 95% TH-positive cells. These cells produced homovanillic acid, a metabolite of dopamine, and may be useful not only for neural transplant but also for basic neurobiological studies.

Animals↗
PubMed1994

Establishment of human parotid pleomorphic adenoma cells in culture: morphological and biochemical characterization.

This study reports the establishment of alpha-amylase-producing human parotid pleomorphic adenoma cell lines (2HP and 2HP1) which have been maintained in culture for over 1 yr. The procedures required preparation of cellular clumps from tumor tissue and plating them on plasma clot or precoated dishes. During the initial phase of growth they required modified MCDB-153 medium without serum. When cells showed signs of degeneration they were changed to MCDB-153 medium containing first 2% and then 10% heat inactivated fetal bovine serum. Although cells grew well in MCDB-153 containing 10% serum, the epithelial cell morphology was not distinct. Therefore, the growth and morphology of cells grown in MCDB-10% serum were compared with those in RPMI growth medium containing 10% fetal bovine serum and F12 containing 10% agammaglobulin newborn bovine serum. Although the growth of cells was a little slower in F12 medium than those in MCDB and RPMI, the epithelial cell morphology was maintained better than in other growth media. The cells of 2HP and 2HP1 produce low levels of alpha-amylase and relatively high levels of alpha-amylase mRNAs of 1176 and 702 bp and contain neurofilament-160, a neuronal-specific marker. The cells of 2HP1 are tumorigenic when tested in athymic mice, but the cells of 2HP are not. The establishment of amylase-producing human parotid adenoma cell lines of different characteristics in culture provides a new opportunity to study the mechanisms of differentiation and transformation, and regulation of alpha-amylase in these cells.

Adenoma, Pleomorphic↗
PubMed1994

Establishment and characterization of immortalized cell lines from rat parotid glands.

This study reports for the first time the establishment of immortalized cell lines from normal adult rat parotid glands. The freshly prepared cellular clumps obtained from parotid glands of isoproterenol-treated rats were incubated in 0.2% trypsin solution without EDTA. These clumps were transfected with plasmid vectors pSV3neo and pSV5neo by electroporation and calcium phosphate-Co-DNA-precipitation techniques. The untransfected and transfected cellular clumps were plated in precoated dishes containing modified MCDB-153 medium. Epithelial cells grew from the clumps that were attached. All epithelial cells from untransfected culture died within 6 to 8 wk. Two cell lines which were isolated from transfected cultures subsequently grew on regular tissue culture dishes. One of them, which was isolated from pSV5neo transfected cultures, exhibited non-epithelial cell morphology, but at confluency, many cells mature to acinar-like cells containing numerous granules. The other cell line (2RS), which was isolated from pSV3neo transfected culture, contained cells of non-epithelial and epithelial morphology. During the initial phase of the growth, MCDB-153 medium was essential; however, at a later time, RPMI medium was better than MCDB-153 or F12 medium for maintaining morphology and growth of these cells. The immortalized cells grew in RPMI with a doubling time of about 25 h, synthesize T-antigen, alpha-amylase mRNAs of 1176 and 702 bp, and alpha-amylase and were non-tumorigenic. These amylase-producing cells can be a useful model to study the mechanisms of regulation of growth and differentiation in these cells.

Animals↗
PubMed1994

Modification of the expression of adenosine 3',5'-cyclic monophosphate-induced differentiated functions in neuroblastoma cells by beta-carotene and D-alpha-tocopheryl succinate.

OBJECTIVE: The role of beta-carotene and vitamin E in modifying the effect of cell differentiating agent has not been studied. This study has investigated the effects of beta-carotene and d-alpha-tocopheryl succinate (alpha-TS) on adenosine 3',5'-cyclic monophosphate (cAMP) induced differentiated functions in murine neuroblastoma cells (NBP2) in culture. METHODS: Prostaglandin E1 (PGE1), a stimulator of adenylate cyclase, and 4-(3-butoxy-4-methoxy-benzyl)-2-imidazolidinone (R020-1724), an inhibitor of cyclic nucleotide phosphodiesterase, were used to induce differentiation in NB cells. RESULTS: Both beta-carotene and alpha-TS markedly enhanced the level of morphologic differentiation (neurite formation) induced by both PGE1 and R020-1724. However, beta-carotene and alpha-TS by themselves were ineffective. These vitamins increased tyrosine hydroxylase (TH) activity. However, beta-carotene did not significantly affect PGE1- and R020-1724-stimulated rise in TH activity. alpha-TS at a higher concentration inhibited PGE1- and R020-1724-stimulated increase in TH activity. None of the above treatments affected basal choline acetyltransferase (ChAT) activity. beta-carotene and alpha-TS caused a transient increase in cAMP level, and they also enhanced the effect of PGE1 and R020-1724 on cAMP level in a transient manner. CONCLUSION: These results suggest that beta-carotene and alpha-TS modify the effects of cAMP stimulating agents on differentiation of NB cells in culture.

4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone↗
PubMed1993

[The efficacy of isosorbide dinitrate administered in an intravenous bolus in acute cardiogenic pulmonary edema].

STUDY OBJECTIVE: To evaluate the efficacy, safety and tolerability of intravenous (i.v.) isosorbide dinitrate (ISDN) administered as a bolus in the treatment of cardiogenic acute pulmonary edema (CAPE). DESIGN: Clinical, prospective, open, noncontrolled trial. SETTING: Emergency room. PATIENTS AND INTERVENTIONS: Twenty two patients (15 male and 7 female), aged 54 to 80 years (68.4 +/- 6.4) with severe respiratory distress consistent with CAPE were included. The cause of CAPE was chronic ischemic cardiopathy in 13 patients, acute myocardial infarction in four, hypertensive cardiopathy in three and mitral valve disease in two. Patients were excluded from the study because of shock or systolic blood pressure equal or lower than 100 mmHg, severe aortic stenosis, hypertrophic cardiomyopathy and non-cardiogenic pulmonary edema. All patients were placed in the sitting position and received oxygen. Initial therapy consisted of an i.v. bolus of 5 to 10 mg of ISDN. Clinical data were recorded at admission and after 5, 10, 15 and 30 minutes. A new i.v. bolus of ISDN and/or another drug was administered at 5 minutes, when necessary. RESULTS: Fifteen patients treated exclusively with ISDN (in three a second i.v. bolus was necessary) improved markedly. In the remaining seven patients that needed other drugs, the improvement was not so impressive. The mean total dose of i.v. ISDN was 10.34 +/- 3.48 mg. Although all data showed a trend towards improvement, just the following were statistically significant (p < 0.05): pH increased from 7.26 +/- 0.13 to 7.32 +/- 0.9, systolic blood pressure decreased from 192.7 +/- 34.8 mmHg to 155.0 +/- 24.4 mmHg (-19%) and diastolic blood pressure decreased 110.5 +/- 12.7 mmHg to 93.2 +/- 9.1 mmHg (-16%). CONCLUSIONS: In this trial, iv ISDN administered as a bolus in doses ranging from 5 to 20 mg, was effective and safe as a first line agent in the treatment of CAPE. No serious adverse reaction were reported.

Acute Disease↗
PubMed1993

[Primary hyperaldosteronism--4 clinical cases].

We describe four cases of primary hyperaldosteronism whose initial presentation was a moderate hypertension. Serum potassium and plasmatic aldosterone values were high although plasmatic renin levels were normal. The captopril test (Lyons version), abdominal CT and iodocholesterol (NP-59) scan proved useful to exclude essential hypertension. A good therapeutical results was achieved in all cases by unilateral adrenalectomy. After surgery, diagnosis was confirmed in all cases by histological studies. At one year follow-up, all patients were asymptomatic, with no hypertension without therapeutic and the serum potassium and plasmatic aldosterone and renin values were normal.

Adenoma↗
PubMed1993

[Fever in the first 48 hours of an acute myocardial infarct treated with fibrinolytics--an indicator of nonreperfusion of the coronary vessels?].

UNLABELLED: Fever in the first days of acute myocardial infarction (AMI) is a very common clinical feature, being its prognostic value unquestionable. As infarction area reduction implies a less important fever reaction in the first days of AMI, we believe that thrombolytic therapy would result in a decline of body temperature of patients so treated. That is why we tried to identify such a correlation, and demonstrate the value of normal body temperature as indicative of reperfusion. We studied retrospectively 68 patients (10 F and 58 M, 57.1 +/- 9.6 years) survivors of AMI (I-II KK classes), with (TT) or without (NT) thrombolytic therapy. In NT group, there was an axillary temperature (AX T) higher than 37 degrees C at the first 24 hours in 21 patients (62%); TT group only had 10 patients (30%) with AX T over 37 degrees C (p < 0.01). NT group CK mean peak was 856 +/- 610 U.I./l in patients having AX T > 37 degrees C, and 436 +/- 233 U.I./l when AX T was < or = 37 degrees C (p < 0.05); in TT group there was no difference between CK peak means when AX T was > or < or = 37 degrees C (1508 +/- 1210 U.I./l vs 1406 +/- 1149 U.I./l, respectively) (NA). We established statistic difference between AX T of 15 patients which CK peak was reached after 10 hours over onset of AMI (37.59 +/- 0.36 degrees C) and those (19 p) with CK peak before 10 hours (37.17 +/- 0.60 degrees C) (p < 0.05). NT group presented then more febrile patients than did TT group. CONCLUSIONS: in NT group there was a positive relation between AX T and CK peak level; AX T > 37 degrees C was less frequent in TT group and was as much light when CK peak was more precocious. These results suggest that in thrombolytic treated patients the absence of fever in the first 48 hours may constitute one more coronary reperfusion criterion.

Adult↗
PubMed1992

Spontaneous development of protective anti-T cell receptor autoimmunity targeted against a natural EAE-regulatory idiotope located within the 39-59 region of the TCR-V beta 8.2 chain.

The development of experimental autoimmune encephalomyelitis (EAE) in Lewis rats is mediated by V beta 8.2+ T cells specific for myelin basic protein. One consequence of this biased expression of V beta 8.2 is the spontaneous development of regulatory T cells and antibodies against residues 39-59 of the V beta 8.2 sequence. Moreover, a synthetic V beta 8.2-39-59 peptide could induce protection against and speed recovery from EAE. T cells and antibodies specific for V beta 8.2-39-59 could transfer protection from EAE. Recently, we reported that the protective T cell epitope is subsumed within the V beta 8-44-54 sequence. We now report that protection induced by V beta 8-44-54 lasted at least 102 days and produced "split tolerance," enhancing anti-myelin basic protein antibody titers but reducing anti-myelin basic protein T cell frequency. The shorter V beta 8-44-54 peptide induced a distinct set of antibodies that did not cross-react with the longer V beta 8.2-39-59 peptide, although both specificities could stain V beta 8.2+ T cells and were equally protective against EAE. However, the V beta 8.2-39-59 peptide, but not the V beta 8-44-54 peptide, would appear to represent the natural idiotope: antibodies to V beta 8.2-39-59 that develop spontaneously during EAE could be boosted to higher titers only by the V beta 8.2-39-59, but not by other TCR peptides from the V beta 8.2 sequence, including V beta 8-44-54 that contains the functional T cell epitope. These results suggest that natural processing of the TCR V beta-chain favors the formation of a peptide that resembles the V beta 8.2-39-59 sequence. The B cell epitope present on the V beta 8-44-54 sequence was evident only in the absence of residues 39-43 and 55-59, suggesting that the two peptides possess distinct conformations. However, the V beta 8-44-54 B cell epitope is most likely expressed on the V beta 8.2+ T cells, either as a low affinity determinant on the intact TCR alpha/beta heterodimer or as a cryptic epitope bound in the cleft of surface MHC molecules.

Amino Acid Sequence↗
PubMed1992

Evidence of drainage of tungsten particles introduced in the pleural space through the visceral pleura into the lung parenchyma.

Studies of pleural clearance of calcium tungstate particles were made in the dog. By using scanning electron microscopy and elemental microanalysis, we show that mesothelial cells of the visceral leaflet of the pleura are also involved in the clearance of particles present in the pleural space. The histological study of lung parenchyma shows many macrophages loaded with tungsten particles, and we conclude that this way may be an important pathway for the transmission of pathologic processes from the pleural space to the lung.

Absorption↗
PubMed1991

Inflammatory macrophages in the dog contain high amounts of intravesicular ferritin and are associated with pouches of connective tissue fibers.

We have studied the subcellular distribution of ferritin in inflammatory macrophages present in regional lymph nodes from dogs subjected to a pulmonary inflammatory reaction. The inflammatory reaction was induced by intrabronchial instillation of calcium tungstate (CaWO4), a water-insoluble powder. Ferritin was identified by electron microscopy, and its electron density was enhanced by the use of a modified Perls method. From day 14 on after the CaWO4 deposition, tungsten-positive lymph node macrophages showed a massive accumulation of ferritin. Most of the ferritin was stored in membrane-bounded vesicles that showed heterogeneous concentrations of the protein. A significant complement of ferritin was also detected in the cytoplasmic ground substance of phagocytes. The cell surface of the ferritin-rich, tungsten-positive macrophages showed deep infoldings that encompassed small pockets of connective tissue fibers. These features were not observed in control samples or in lymph nodes from dogs subjected to CaWO4-induced inflammation for periods shorter than 1 week. Our data indicate that inflammatory macrophages greatly increase their content of ferritin macrophages greatly increased their content of ferritin and that ferritin is stored predominantly by a membrane-bounded vesicular compartment. This is in contrast with suggestions that the inflammation-induced increase in macrophage iron is restricted to the labile pool of iron and it does not involve the iron bound to ferritin molecules. Our observation of nodules of connective-tissue fibers in intimate topographical association with ferritin-rich macrophages may indicate that the increase in intracellular ferritin in the macrophage is in some way related to the secretion of factors by the phagocyte that will stimulate fibrillogenesis by neighboring fibroblats.

Animals↗
PubMed1990

Antibodies specific for VB8 receptor peptide suppress experimental autoimmune encephalomyelitis.

Recent studies from our laboratory have shown, for the first time, that a synthetic peptide from that TCR VB chain used preferentially by encephalitogenic T cells induced the formation of protective, MHC class I-restricted T cells and prevented the development of EAE in Lewis rats. In this report we 1) demonstrate that immunization with the TCR-VB8-39-59 peptide generated peptide-specific antibodies that protect against experimental autoimmune encephalomyelitis induced by either of the two distinct encephalitogenic epitopes of basic protein, and 2) characterize the production and biologic functions of rat and rabbit antibody responses to the TCR peptide. The antibodies in both species increased in titer over time, were highly specific for the immunogen by direct reaction and inhibition assays, stained only VB8+ T cells, and suppressed clinical signs and to lesser extent the number of histologic lesions of experimental autoimmune encephalomyelitis mediated by VB8+ T cells. Coupled with our previous work, these results indicate that both humoral and cellular responses to the TCR-VB8-39-59 peptide can contribute independent immunoregulatory effects on encephalitogenic T lymphocytes that use common V region genes in response to epitopes of myelin basic protein.

Amino Acid Sequence↗
PubMed1990

Time course and distribution of tungsten-laden macrophages in the hilar lymph nodes of the dog lung after experimental instillation of calcium tungstate into the left apical bronchus.

We sprayed a tungsten powder (CaWO4) into the airway of a single lobe (left apical) of the dog lung in order to study: (a) the kinetics of particle translocation from the bronchoalveolar lining to hilar lymph nodes, and (b) the sorting in lung lymph nodes of inhaled microcrystals. We found that the transport of the tungsten particles to the regional lymph node takes at least 24 hours and reaches its peak at day 7. In situ detection of tungsten by elemental particle analysis of lymph node sections by scanning electron microscopy allowed precise mapping of the marker in the node; the method was complemented by light microscopy and thin-section electron microscopy of the same nodes. Virtually all of the lymph node tungsten was located inside macrophages. The first tungsten-positive macrophages seen in the regional lymph nodes (day 1 to day 3) were restricted to the subcapsular space. This was followed by massive filling of the same sinus and of the narrow interfollicular areas by the particle-laden macrophages (day 3 to day 7). The even distribution of the tungsten-bearing phagocytes found in these anatomical regions of the node indicated that the subcapsular area in the dog was a continuous domain rather than the segmented region observed in nodes of common laboratory animals such as the rat. By day 7 after tungsten instillation, a moderate number of tungsten-positive macrophages was also detected in the paracortical region of the node. Finally, the presence of tungsten-bearing macrophages was extended to the outer lymph node medulla (day 7 to day 14); here, the macrophages were located in association with cords of plasmacytes and showed interdigitations with these lymphocytes. Only minimal amounts of tungsten were detected inside lymphoid follicles in association with dendritic cells. Some of the tungsten initially deposited in the airway of the apical left lung lobe was detected in contralateral hilar lymph nodes. We conclude that: (i) particle translocation from the alveolus to regional lymph nodes is a slow process that is mediated by pulmonary macrophages, in agreement with the findings of Harmsen et al Science 230:1277, 1985); (ii) in the lymph node, particle-bearing macrophages are sorted through narrow interfollicular sinuses into the outer medulla where they interact extensively with plasma cells; (iii) the migrating macrophages cannot penetrate the follicular domains of the node; minute quantities of exogenous particles may, nevertheless, be transferred from macrophages to follicular dendritic cells; and (iv) contralateral drainage may be a feature of the lymphatic system in the lung.

Animals↗