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Biomedical subjects

D Navarro

Publications and source records attributed to D Navarro.

At least 37 records · Page 2Linked to original sources
PubMed1998

Antibody response to human cytomegalovirus (HCMV) glycoprotein B (gB) in AIDS patients with HCMV end-organ disease.

Human cytomegalovirus (HCMV)-specific antibody responses in HIV-1 infected individuals either with or without HCMV end-organ disease were examined to determine the whether development of HCMV disease was associated with a particular deficit in the antibody response. Antiwhole HCMV, anti-glycoprotein B (gB), and neutralizing antibody levels were higher in HIV-1 infected individuals than in healthy immunocompetent subjects, particularly in patients with AIDS either with or without HCMV-associated disease. Irrespective of location and spread of HCMV disease, patients who had received anti-HCMV therapy prior to sampling exhibited significantly higher anti-gB and neutralizing antibody titers than those who remained untreated. Likewise, patients with HCMV disease who were antigenemic or viremic had significantly lower anti-gB and neutralizing antibody titers than those who tested negative in either assay. Patients with untreated HCMV disease had significantly lower antibody titers than AIDS patients without disease. Analysis of the IgG subclass antibody responses to gB revealed no significant differences among HIV-1 infected individuals. These results suggest that levels of detectable anti-gB and HCMV neutralizing antibodies are inversely related to systemic viral load. Thus, antibodies with such specificities may be relevant in preventing the establishment of HCMV-associated disease or in modulating its progression.

AIDS-Related Opportunistic Infections↗
PubMed1998

A comparative study on the efficacy of the new quinolone alatrofloxacin in the treatment of experimental legionellosis in guinea pigs.

The in vivo efficacy of trovafloxacin, intraperitoneally administered as alatrofloxacin (CP-116,517), was assessed and compared with that of erythromycin, alone or in combination with rifampicin, in a model of Legionella pneumophila pneumonia in guinea pigs. Trovafloxacin (5 mg/kg administered as alatrofloxacin once daily for 7 days) gave a survival rate of 100% in infected animals. Clearance of bacteria and of bacteria-induced lesions from lungs was achieved by day 6 post-inoculation. The lungs of trovafloxacin-treated animals remained free of bacteria at day 28 post-challenge. Trovafloxacin proved as effective as erythromycin administered intraperitoneally, but was superior to erythromycin alone. or in combination with rifampicin, when given orally.

Administration, Oral↗
PubMed1998

The two native estrogen receptor forms of 8S and 4S present in cytosol from human uterine tissues display opposite reactivities with the antiestrogen tamoxifen aziridine and the estrogen responsive element.

We have investigated the capability of the different native ER forms, present in cytosols from human uterine tissues, of reacting with the antiestrogen [3H]Tamoxifen aziridine ([3H]TA) and with the Estrogen Responsive Element (ERE). Cytosols from uterine leiomyoma (myoma) prepared in buffer containing 40 mM molybdate and protease inhibitors, labelled with [3H]estradiol and analyzed in low-salt sucrose gradient showed 8S and 4S ER forms. The same cytosols labelled with [3H]TA only showed a 4S ER form, whereas the ERE only reacted with fractions from the 8S peak. The band of ERE reaction in the EMSA assay showed a lower relative mobility than the band labelled with [3H]TA, but both bands contained immunoreactive ER of 65 kDa. Electrophoretic mobility of the [3H]TA-labelled band in that system was not affected by cytosol treatment with cross-linkers or SDS, which suggests that it is a monomeric protein. The [3H]TA-binding 4S ER form was found in all studied myoma samples, as well as in human endometrium or myometrium, but not in rat tissues. These results suggest that the 8S and 4S ER form were already present before cytosol from human uterine tissues comes into contact with the molybdate buffer. They both contain the same ER molecule of 65 kDa, either in the free form or as an oligomer. Only the ER dimers, which have been described both in the cytosolic 8S form and in the nuclear 4-5S form, react with the ERE. [3H]TA only binds to the 4S ER monomer probably because its binding site is concealed in the 8S form under these experimental conditions. The opposite reactivity of the 8S and 4S ER forms with [3H]TA and the ERE support the hypothesis that they may constitute separate entities with a different physiological role.

Animals↗
PubMed1998

Tamoxifen aziridine binding to cytosolic proteins from human breast specimens is negatively associated with estrogen receptors, progesterone receptors, pS2, and cathepsin-D.

[3H]Tamoxifen Aziridine ([3H]TAZ) is a derivative of the antiestrogen tamoxifen that covalently labels the Estrogen Receptor (ER), and perhaps other uncharacterized proteins. In a previous article we described that [3H]TAZ binds to a cytosolic protein from human uterine tissues that shares some, but not all, the ER properties. Here we have extended these studies to [3H]TAZ binding to cytosol proteins from human breast cancer specimens, and studied its quantitative association with other molecular markers and clinico-pathological variables. Cytosols were obtained in hypotonic buffer containing 20 mM molybdate and protease inhibitors, incubated with [3H]TAZ, and subjected to Sucrose Gradient Analysis (SGA). A [3H]TAZ labeled peak that consistently migrated with the 4S fractions was found in most of the assayed cytosols (range of 0 to 1278 fmol/ mg p.). The 4S peak of [3H]TAZ was partially inhibited by both estrogens and antiestrogens. When [3H]E2 was used instead of [3H]TAZ, only an 8S peak was detected. [3H]TAZ was covalently bound to a protein with an apparent MW of 65 kDa, as determined by SDS-PAGE and fluorography. The mean of [3H]TAZ binding was significantly higher in the subgroups of samples classified as ER-, PR-, pS2- or cathepsin D-, than in the respective positive subgroups (P < 0.01 in all the cases). [3H]TAZ binding was not associated with clinico-pathological variables, except that its mean was significantly larger in tumors larger than 5 cm than in smaller tumors. These results, and those previously reported, suggest that: 1) [3H]TAZ labels a cytosolic protein present in human breast cancers and uterine tissues that does not share all the ER properties, and 2) the [3H]TAZ binding by breast cancer cytosols is negatively associated with markers of estrogenic dependency, and its quantification may provide valuable information on antiestrogen responsiveness of a given tumor.

Biomarkers, Tumor↗
PubMed1998

Candida albicans fibrinogen binding mannoprotein: expression in clinical strains and immunogenicity in patients with candidiasis.

A 58 kDa cell wall-associated fibrinogen binding mannoprotein (mp58), previously characterized by our group in a Candida albicans laboratory strain (ATCC 26555), was found to be also present in the cell wall of clinical isolates of this fungus. Most strains examined appear to have functional mp58 species, as detected by their ability to bind fibrinogen. Western immunoblot analysis, with a monovalent polyclonal antibody generated against the mp58 species from strain ATCC 26555, revealed differences in recognition patterns depending on the strain tested and the culture conditions used. Serum samples from normal and Candida infected individuals were examined for the presence of antibodies against mp58 by Western immunoblotting. None of the sera from control individuals and patients suffering from superficial candidiasis contained antibodies against mp58. However, positive reactivity with this antigen and other cell wall constituents was detected for all sera from patients with confirmed systemic candidiasis. Together, these results suggest that mp58 could play an active role during infection and may be useful as a specific antigenic marker for candidiasis.

Antigens, Fungal↗
PubMed1997

Antimicrobial susceptibility of Streptococcus pneumoniae isolated from pediatric carriers in Spain.

The in vitro activity of several beta-lactam agents, macrolides, and cotrimoxazole was investigated against 53 Streptococcus pneumoniae isolates recovered from healthy children. The rates of resistance to penicillin or amoxicillin, cefaclor, and cefuroxime were 30%, 51%, and 37%, respectively. No cefotaxime-resistant isolates were found. Rates of resistance to erythromycin, clarithromycin, and cotrimoxazole were 22.6%, 13.2%, and 83%, respectively. Pneumococci with divergent antimicrobial susceptibility profiles (susceptible or moderately resistant vs. resistant isolates) coexisted in 32% samples, with divergencies more often involving beta-lactam agents and/or macrolides. In five of these samples, isolates belonged to different serotypes.

Adolescent↗
PubMed1997

New directions in diagnostics.

BACKGROUND: Infectious diseases are still a significant clinical problem in children, and accurate identification of the causal pathogen plays an important role in clinical management. The availability of an etiologic diagnosis enables the clinician to make appropriate therapeutic decisions and to avoid the indiscriminate use of antibiotics. The availability of a microbiologic diagnosis and the susceptibility profile of the pathogen allows the prompt initiation of suitable antibiotic treatment. However, the usefulness of current culture and identification methods is limited by the time needed and by their sensitivity and specificity. Also some microorganisms are difficult or impossible to grow in the laboratory. OBJECTIVES: To review the newer and more rapid diagnostic techniques that are becoming available and consider their application in the diagnosis of specific infections. DISCUSSION: Immunoassays have many advantages and it is hoped that new optical immunoassays will overcome the problems of poor sensitivity. Nucleic acid amplification techniques have enormous potential in the diagnosis of infectious diseases because of their high specificity and sensitivity and the speed with which the results can be obtained. However, there are still a number of difficulties that must be overcome before these methods can be widely adopted for routine testing. These techniques may be particularly relevant for the rapid diagnosis of streptococcal pharyngitis, where throat culture is slow and beset by a number of factors which reduce its accuracy. Polymerase chain reaction methods have been developed for many respiratory pathogens, including Mycoplasma pneumoniae, Chlamydia pneumoniae and Mycobacterium tuberculosis, and are likely to play an increasingly important part in diagnosis. In bacterial meningitis culture is still the gold standard and molecular techniques have not yet been developed to the point where they can be used in routine diagnosis. Nucleic acid techniques are likely to be very valuable in the diagnosis of streptococcal pharyngitis and viral central nervous system infections in the near future.

Bacterial Infections↗
PubMed1997

Humoral immune response to functional regions of human cytomegalovirus glycoprotein B.

Sera from patients with primary human cytomegalovirus (HCMV) infections, both acute and convalescent phase, and from HCMV-seropositive healthy subjects were analyzed to determine whether the sera would recognize antigenic domains on HCMV glycoprotein B (gB) that function in virion infectivity and spread of virus from cell to cell. The intact gB molecule, amino-terminal derivatives of different lengths, and internal deletion derivatives were expressed in eukaryotic cells and reacted by immunofluorescence with the sera. All convalescent-phase sera and most sera from healthy seropositive individuals reacted with full-length gB and with an amino-terminal derivative containing 687 amino acids (aa), gB-(1-687); approximately half of the sera recognized an amino-terminal derivative of 447 aa, gB-(1-447), and one-third reacted with the shortest deletion derivative of 258 aa, gB-(1-258). Of the acute-phase sera, 77% recognized intact gB and gB-(1-687), 32% recognized gB-(1-447), and 14% recognized gB-(1-258). Deletion of aa 548 to 618 dramatically reduced the percentage of reactive sera, whereas deletion of aa 411 to 447 had a minor impact on reactivity of sera. To investigate the epitope specificity of human antibodies to gB, we carried out competition experiments between human sera with neutralizing activity and selected monoclonal antibodies (mAbs) to conformational epitopes on gB. We found that antibodies in human sera preclude syncytium formation in UB15-11 glioblastoma cells constitutively expressing gB and compete with certain murine mAbs that block virus entry into cells and transmission of infection from cell to cell. Our results show that HCMV-immune human sera contain antibodies to functional regions on gB, and the abundance of these antibodies in convalescent-phase sera suggests that they may play a central role in limiting dissemination of virus in the host.

Acute Disease↗
PubMed1997

[Penetration of meropenem in gram-negative bacilli. Differences in activity with imipenem].

The outer membrane of gramnegative bacteria cell-wall contain channels formed by proteins known as porins, which facilitate the penetration of molecules into the cell. Imipenem and meropenem possess an important intrinsic activity against most gramnegative bacteria due to their high affinity for the penicillin-binding proteins PBP-2 and/or PBP-3. Meropenem is slightly more active against certain species of Enterobacteriaceae, Pseudomonas aeruginosa and nonfermenters bacilli. In this sense the differences in the activity between the two carbapenems may be attributable to differences in their affinity for PBPs, differences in their resistance to beta-lactamases hydrolysis, or to the differences in the capacity to employ certain porin channels. OprF is the main porin channel involved in the beta-lactam penetration of bacteria, though OprC and OprD2 may also contribute to the penetration of carbapenems into P. aeruginosa. However, evidences suggest that although impenem requires the presence of OprD2, meropenem may use other pathways for penetration. In Escherichia coli both carbapenems use ompF and ompC, and no specific porin channels have been detected. Only in the case of Enterobacter cloacae may exceptions exists among Enterobacteriaceae.

Bacterial Outer Membrane Proteins↗
PubMed1996

Validation of a differential PCR and an ELISA procedure in studying HER-2/neu status in breast cancer.

HER-2/neu oncogene status and total cellular p185HER-2 content were simultaneously analyzed in 415 invasive breast-cancer specimens by differential PCR and ELISA respectively. Mathematical analysis of the data led us to establish a cut-off value of 1.7 for the ratio between the intensity of the HER-2/neu gene band and the reference gene band, to consider the HER-2/neu gene amplified, and of 260 fmol/mg protein, to consider p185HER-2 over-expressed. Of the 415 tumors studied, 15% showed a diverse degree of HER-2/neu gene amplification. Of these tumors, 87% showed over-expression of the p185HER-2. Of the remaining 352 specimens that did not display HER-2/neu gene amplification, 97% showed no p185HER-2 over-expression (p < 0.0001). In 40 selected samples with a p185HER-2 level lower than 260 fmol/mg protein, the degree of p185HER-2 phosphorylation was very low or undetectable. Conversely, 38 of 46 selected tumors with a p185HER-2 level higher than 260 fmol/mg protein exhibited a considerable degree of p185HER-2 phosphorylation (p < 0.0001). Our data suggest that: (i) differential PCR and ELISA, which are relatively simple procedures, give similar information on HER-2/neu status in breast cancer; and (ii) given the large series analyzed, the cutoff values established can be considered as safe values for determining whether, in a given tumor, the HER-2/neu oncogene is amplified or p185HER-2 is over-expressed.

Base Sequence↗
PubMed1996

Relationship between outer membrane protein profiles and resistance to ceftazidime, imipenem, and ciprofloxacin in Pseudomonas aeruginosa isolates from bacteremic patients.

Outer membrane protein (OMP) profiles of 122 Pseudomonas aeruginosa isolates recovered from the blood of bacteremic patients were analyzed to relate alterations in the expression of OMPs with porin activity to resistance to imipenem, ceftazidime, and ciprofloxacin. Imipenem-resistant isolates lacked or expressed reduced amounts of porin OprD. In contrast, alterations of OMP profiles were absent in most ceftazidime-resistant isolates. Six of 12 ciprofloxacin-resistant isolates had normal OMP profiles. The remaining isolates showed alterations in the expression of either OprC, OprF, or OprD. In addition, imipenem- and ceftazidime-resistant isolates displayed a beta-lactamase activity compatible with that of a group 1 chromosomal cephalosporinase.

Anti-Infective Agents↗
PubMed1995

Mutated forms of human cytomegalovirus glycoprotein B are impaired in inducing syncytium formation.

Human cytomegalovirus (HCMV) glycoprotein B (gB) promotes virion entry into cells by fusing the virion envelope with the cellular membrane. We recently reported that UB cells (U373 glioblastoma cells constructed to produce HCMV gB constitutively) form multinucleate syncytia that are dependent on the density of gB in the plasma membrane. In this report, we describe the properties of a clonal cell line, UB31-B3, that expressed a spontaneously mutated form of gB which lacked the fusion-inducing function of the wild-type molecule, and three UB cell lines that were constructed to investigate the effect of specific mutations in gB on syncytium formation. Flow cytometry analysis with a pool of monoclonal antibodies (mAbs) showed that the UB cells contained a high density of gB, which was associated with the cell surface. Immune precipitation experiments with UB31-B3 cells showed that the mutant gB reacted with all of the mAbs to the ectodomain of gB but with none of those to the cytoplasmic carboxy terminus, and that it was 35 kDa smaller than wild-type gB. Nucleotide sequence analysis showed that a termination codon had been introduced after amino acid lysine at position 669 in the ectodomain of UB31-B3 gB, generating a truncated glycoprotein. UB31-B3 gB was not secreted into the medium and was stably anchored in the plasma membrane, which suggested that a hydrophobic stretch of amino acids from 629 to 652 in the ectodomain may serve as a membrane anchor for this truncated form. Analysis of the UB cell lines expressing deleted forms of gB showed that deletion of all or part of the cytoplasmic and transmembrane domains reduced or abolished syncytium formation. In contrast, deletion of a major neutralizing region in the ectodomain of gB did not alter syncytium formation. Results of these studies indicate that different regions of the gB molecule participate in syncytium formation.

Antibodies, Monoclonal↗
PubMed1994

Function of human cytomegalovirus glycoprotein B: syncytium formation in cells constitutively expressing gB is blocked by virus-neutralizing antibodies.

We report that U373 glioblastoma cells constitutively producing human cytomegalovirus (HCMV) glycoprotein B (gB), the product of open reading frame UL55 of the HCMV genome, formed syncytia that contained 5 to 25 nuclei. Flow cytometry with a panel of monoclonal antibodies (mAbs) to the extracellular domain of HCMV gB showed that these cells expressed high densities of gB in the plasma membrane. We studied the properties of five clonal UB cell lines and the results are as follows. Reactivity of a panel of mAbs to HCMV gB showed that UB cells forming syncytia expressed gB with all of the conformational and sequential epitopes contained in the viral gB made in HCMV-infected cells. Syncytium formation in UB cells was independent of low pH and proteolytic cleavage of gB and was blocked by drugs that inhibit glycosylation and translocation of gB to the cell surface. Infected UB cells formed more syncytia than infected U373 cells, virions entered UB cells more rapidly, and higher virus titers were produced. Incubation of UB cells with complement-independent neutralizing antibodies to gB, which prevent virion entry into cells, cell-to-cell transmission of infection, and fusion of HCMV-infected glioblastoma cells, significantly reduced syncytium formation in UB cells. These results show that overlapping functional domains on HCMV gB promote fusion of the virion envelope with the cell surface, fusion of infected U373 cells, and syncytium formation in UB cell lines expressing high densities of gB in the plasma membrane. Our findings indicate that the functional regions of gB can be subjected to detailed analysis without constructing viral mutants by expressing mutated forms of gB with site-directed changes that preclude syncytium formation of U373 cells expressing these gene products.

Antibodies, Viral↗
PubMed1994

A comparative study on cell wall antigens and cell surface hydrophobicity in clinical isolates of Candida albicans.

Characterization of common cell surface-bound antigens in Candida albicans strains, particularly those expressed in the walls of mycelial cells might be useful in the diagnosis of systemic candidiasis. Hence, antigenic similarities among wall proteins and mannoproteins from C. albicans clinical serotype A and B isolates, were studied using polyclonal (mPAbs) and monoclonal (MAb 4C12) antibodies raised against wall antigens from the mycelial form of a common C. albicans serotype A laboratory strain (ATCC 26555). Zymolyase digestion of walls isolated from cells of the different strains studied grown at 37 degrees C (germination conditions), released, in all cases, numerous protein and mannoprotein components larger than 100 kDa, along with a 33-34 kDa species. The pattern of major antigens exhibiting reactivity towards the mPAbs preparation was basically similar for all the serotype A and B isolates, though minor strain-specific bands were also observed. The immunodeterminant recognized by MAb 4C12 was found to be absent or present in very low amounts in C. albicans isolates other than the ATCC 26555 strain, yet high molecular weight species similar in size (e.g., 260 kDa) to the wall antigen against which MAb 4C12 was raised, were observed, particularly in wall digests from serotype A strains. Cell surface hydrophobicity, an apparently important virulence factor in C. albicans, of the cell population of each serotype B strain was lower than that of the corresponding serotype A counterparts, which is possibly due to the fact that the former strains exhibited a reduced ability to form mycelial filaments under the experimental conditions used.

Animals↗
PubMed1994

Age-related changes in the induction of tyrosine aminotransferase by dexamethasone: correlation with the low-affinity glucocorticoid binding sites.

Rat liver membranes contain Low-affinity glucocorticoid binding sites (LAGS), capable of binding with low affinity (Kd approximately 100 nM) endogenous glucocorticoids. Unlike the glucocorticoid receptor (GR), the LAGS level undergoes abrupt changes throughout life. The investigation of these changes may be useful in determining whether the LAGS are involved in the cellular response to glucocorticoids. For this purpose, we have studied glucocorticoid induction of tyrosine aminotransferase (TAT), and its relationship with the LAGS level in adrenalectomized and fasted rats of different ages. No significant differences in the GR level, or in its Kd and activation, were observed among rats of 1, 3, and 12 months of age. On the other hand, the LAGS level showed an important variation with age, from almost undetectable in 1-month-old rats, to a maximum value in 3-month-old rats. With respect to TAT activity, an increase with age in the threshold of response to dexamethasone (DEX) administration was observed. The smallest dose of DEX capable of provoking a significant TAT induction rose from 0.1 microgram/kg body wt. in 1-month-old rats to 10 micrograms/kg body wt. in 12-month-old rats. However, the smallest dose of DEX able to elicit the maximal response was 10 micrograms/kg body wt. in all the assayed ages. This dose provoked a 40% decrease in the GR level, but did not significantly modify the LAGS content. From these results, we conclude that there is an age-related change in the threshold of response to DEX that cannot be explained by the GR-glucocorticoid interaction. The possibility that the LAGS modulate the cell response to glucocorticoids arises from the coincidence of this change with that observed in the LAGS concentration throughout life.

Adrenal Glands↗
PubMed1994

Stanozolol and danazol, unlike natural androgens, interact with the low affinity glucocorticoid-binding sites from male rat liver microsomes.

Some 17 alpha-alkylated androgens used as anabolic agents, such as stanozolol (ST) and danazol (DA), have specific effects on the liver that are not exerted by testosterone. This gives rise to the possibility that a steroid-binding protein, other than the androgen receptor, could modulate the intracellular actions of these agents. Male rat liver microsomes contain a homogeneous population of [3H]dexamethasone ([3H]DEX)-binding sites which we have denominated low affinity glucocorticoid-binding sites (LAGS). Because glucocorticoids, progestagens, and the synthetic estrogen ethynyl estradiol compete with [3H]DEX for binding to the LAGS, we aimed to study the possible interactions between androgens and the LAGS. To investigate whether several androgens had the capability of interacting with the LAGS, we performed competition experiments. The LAGS had no affinity for testosterone or methyltrienolone (R1881). However, some 17 alpha-alkylated androgens (DA (IC50, 116 nM) > ST >> fluoxymesterone > mestaline > methandriol >> methandrostenolone > methyltestosterone) were able to compete with [3H]DEX binding to liver microsomes. ST and DA were potent inhibitors of [3H]DEX binding to liver microsomes. They decreased both the affinity and the number of [3H]DEX-binding sites, increased the dissociation rate of [3H]DEX from the LAGS, and provoked a time- and dose-dependent inactivation of the [3H]DEX-binding site. These results strongly suggest that ST and DA exert a negative allosteric modulation on [3H]DEX binding to the LAGS. The in vivo administration of ST (but not other androgens) to male rats provoked a time- and dose-dependent decrease in the LAGS level. Full recovery of the LAGS concentration required at least 8 h and was blocked by protein synthesis inhibitors. Such results suggest that ST irreversibly inactivates the [3H]DEX-binding site in vivo as it does in vitro. Taken together, these observations are indicative of an irreversible interaction between some 17 alpha-alkylated androgens and the LAGS both in vitro and in vivo and suggest that ST may be an important pharmacological tool that can be used in the elucidation of the molecular structure of the LAGS. These results also mean that the LAGS are a steroid-binding entity able to distinguish between natural androgens and 17 alpha-alkylated testosterone derivatives used as anabolic agents.

Animals↗