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Biomedical subjects

P Vago

Publications and source records attributed to P Vago.

At least 19 recordsLinked to original sources
PubMed1998

Attempt at hair cell neodifferentiation in developing and adult amikacin intoxicated rat cochleae.

Recent studies have shown that an attempt at auditory hair cell neodifferentiation occurs in vivo in the rat organ of Corti after amikacin intoxication during the last stages of cochlear maturation. Atypical cells, with morphological characteristics reminiscent of very immature sensory hair cells, were transiently observed after outer hair cell losses. The aim of the present study was to assess (i) if this attempt at hair cell neodifferentiation was related to the degree of maturity of the organ of Corti and (ii) to characterise morphological and molecular changes in the scarring epithelium. We therefore investigated, using electron and confocal microscopy, morphological and molecular changes in cochleae from rats treated with amikacin at two different periods: from post natal day (PND) 1 to PND 8, when the organ of Corti is very immature; and from PND 30 to 37, when the organ of Corti is morphologically and functionally mature. In both groups, transient atypical cells were observed, attesting that the attempt at hair cell neodifferentiation is not strictly related to the immaturity of the cochlea. The results also suggest that Deiters cells are involved in the appearance of atypical cells, possibly through a transdifferentiation process. Finally, it appears that non-sensory epithelial cells from the outer spiral sulcus progressively colonize the region of pre-existing outer hair cells.

Amikacin↗
PubMed1998

Characterization of atypical cells in the juvenile rat organ of corti after aminoglycoside ototoxicity.

Hair cell regeneration is well documented in the inner ear sensory epithelia of lower vertebrates and birds and may occur in the vestibular organs of mammals. By contrast, hair cell loss in the mature mammalian cochlea is considered irreversible. However, recent reports have suggested that an attempt at hair cell regeneration could occur in vivo in aminoglycoside-lesioned cochleas from neonatal rats. After amikacin treatment, atypical cells with apical specialization reminiscent of early differentiating stereocilia are transiently present at the apex of the intoxicated cochleas but fail to differentiate as hair cells in later stages. In the present study, we used electronic microscopy, histochemistry, and confocal microscopy to investigate the cellular rearrangements in the amikacin-lesioned organ of Corti of rat pups. In addition, we used 5-bromo-2'-deoxyuridine immunocytochemistry to determine whether mitotic processes are involved in the formation of the atypical cells. The morphologic and molecular data suggest that atypical cells are not recovering hair cells, but share characteristics of immature hair cells and supporting cells. Proliferative cells were absent from the region occupied by atypical cells, suggesting that the latter did not arise through mitotic processes. Altogether, the present results support the hypothesis that atypical cells arise through direct transformation of some of the supporting cells that reorganize during hair cell degeneration.

Amikacin↗
PubMed1998

Amikacin intoxication induces apoptosis and cell proliferation in rat organ of Corti.

Recently, an attempt at cochlear hair cell neodifferentiation has been reported in amikacin-treated rats. In the present study, we aimed to ascertain whether hair cell losses are mediated by apoptosis and whether cell proliferation occurs in damaged intoxicated cochleas. The results show that apoptosis is responsible for hair cell losses and that cell proliferation occurs in the region of the outer spiral sulcus but not in the region of Deiters cells and pre-existing hair cells. We suggest that cell proliferation maintains a certain homeostasis in the number of non-sensory cells and participates in epithelial scar formation. Neodifferentiated cells therefore probably arise from direct transdifferentiation, which could be triggered by phagocytosis of apoptotic bodies.

Amikacin↗
PubMed1997

Cloning of the genes encoding two murine and human cochlear unconventional type I myosins.

Several lines of evidence indicate a crucial role for unconventional myosins in the function of the sensory hair cells of the inner ear. We report here the characterization of the cDNAs encoding two unconventional type I myosins from a mouse cochlear cDNA library. The first cDNA encodes a putative protein named Myo1c, which is likely to be the murine orthologue of the bullfrog myosin I beta and which may be involved in the gating of the mechanotransduction channel of the sensory hair cells. This myosin belongs to the group of short-tailed myosins I, with its tail ending shortly after a polybasic, TH-1-like domain. The second cDNA encodes a novel type I myosin Myo1f which displays three regions: a head domain with the conserved ATP- and actin-binding sites, a neck domain with a single IQ motif, and a tail domain with the tripartite structure initially described in protozoan myosins I. The tail of Myo1f includes (1) a TH-1 region rich in basic residues, which may interact with anionic membrane phospholipids; (2) a TH-2 proline-rich region, expected to contain an ATP-insensitive actin-binding site; and (3) a SH-3 domain found in a variety of cytoskeletal and signaling proteins. Northern blot analysis indicated that the genes encoding Myo1c and Myo1f display a widespread tissue expression in the adult mouse. Myo1c and Myo1f were mapped by in situ hybridization to the chromosomal regions 11D-11E and 17B-17C, respectively. The human orthologuous genes MYO1C and MYO1F were also characterized, and mapped to the human chromosomal regions 17p18 and 19p13.2-19p13.3, respectively.

Amino Acid Sequence↗
PubMed1997

Identification of preferentially expressed cochlear genes by systematic sequencing of a rat cochlea cDNA library.

107 expressed sequence tags (ESTs) from a rat cochlea cDNA library were identified by systematic sequencing coupled to database selection and RT-PCR analysis of novel sequences. This approach led us to select a clone, pCO8, showing no significant homology with any database sequence, that corresponds to a mRNA whose expression is restricted to the cochlea, except for traces detected in brain. Additional clones with novel sequences enriched in the cochlea were also found. ESTs bearing significant homologies with database sequences (63 out of 107) were classified according to the putatively encoded protein. They include tissue-specific genes not previously described in the cochlea as well as known genes from other species. We performed in situ hybridization in cochlear tissues to localize the pCO8 mRNA and that of clone pCO6 which is 100% homologous to the delayed rectifier potassium channel drk1. We found that both mRNAs were exclusively expressed in the cellular body of the primary auditory neurons from the spiral ganglion of the cochlea. These results indicate that this approach is an efficient way to identify novel genes that could be of importance in cochlear function.

Animals↗
PubMed1997

Does the organ of Corti attempt to differentiate new hair cells after antibiotic intoxication in rat pups?

In the adult mammalian cochlea, post-injury hair cell losses are considered to be irreversible. Recent studies in cochlear explants of embryonic rodents show that the organ of Corti can replace lost hair cells after injury. We have investigated this topic in vivo during the period of cochlear development. Rat pups were treated with a daily subcutaneous injection of 500 mg/kg amikacin for eight consecutive days between postnatal day 9 (PND 9) and PND 16. During this period the organ of Corti is not fully mature, but hair cells are hyper-sensitive to aminoglycoside antibiotics. Scanning and transmission electron microscopy was used to evaluate morphological changes in the organs of Corti during the treatment and at different post-treatment periods, up until PND 90. A massive loss in outer and inner hair cells was observed at least as early as PND 14. A prominent feature in the apical part of cochleas at PND 21 and 35 was the transient presence of small atypical cells in the region of pre-existing outer hair cells. These atypical cells had tufts of microvilli reminiscent of nascent stereociliary bundles. A second striking observation was the replacement of degenerating inner hair cells by pear-shaped supporting cells throughout the cochlea. These cells were covered with long microvilli, and their basal pole was contacted by both afferent and efferent fibers, as in the early stages of inner hair cell maturation. At PND 55 and 90, these features were not clearly observed due to further cytological changes in the organ of Corti. It is possible that an attempt at hair cell neodifferentiation could occur in vivo after an amikacin treatment in the rat during the period of cochlear hyper-sensitivity to antibiotic.

Amikacin↗
PubMed1996

Distribution of actin and tubulin in outer hair cells isolated from developing rat cochlea: a quantitative study.

In the organ of Corti, outer hair cells (OHCs) are sensory effectors responsible for the high sensitivity and sharp tuning of the cochlea. Whilst the distribution and organization of actin and tubulin in adult OHCs have been extensively studied, less is known about developing OHCs. In this study we use a quantitative cytometric approach on rat isolated OHCs to measure the distribution of these cytoskeletal proteins from the first stages of development (postnatal day 5) to the adult stage. We report a general decrease in both actin and tubulin concentrations during OHC maturation. Actin first decreases in the apical domain, and then in the medio-basal domain. In the apical domain, this could be related to the physiological reduction in the number of stereocilia occurring during ciliogenesis. In the medio-basal domain, the decrease, accompanied by a redistribution of actin toward the lateral wall, is possibly related to the general reorganization of cytoplasmic organelles, to the maturation of the cortical lattice, and to cell growth. Tubulin concentration decreases regularly in both the apical and the medio-basal domains. This developmental change in tubulin concentration could be due to the regression of the kinocilium that occurs by the end of ciliogenesis, and, as argued for actin, to the general reorganization of cytoplasmic organelles, and to cell growth.

Actins↗
PubMed1996

Morphological indications of hair cell neodifferentiation in the organ of Corti of amikacin treated rat pups.

Postnatal rats were treated with amikacin during the period of cochlear supra-normal sensitivity to ototoxic antibiotics, i.e. from day 9 to day 16 when the organ of Corti is achieving its maturation. Scanning and transmission electron microscopies were used to evaluate morphological changes in the organs of Corti at different post-treatment periods, up until postnatal day 90. A massive loss in outer (OHCs) and inner (IHCs) hair cells was observed from as early as day 17. A prominent feature in the apical part of cochleas at days 21 and 35 was the transient presence of small atypical cells in the region of pre-existing OHCs. These atypical cells did not have the appearance of degenerating OHCs, but had tuffs of microvilli reminiscent of nascent stereociliary bundles. A second striking observation was the presence of pear-shaped cells in the place normally occupied by IHCs. These cells had neither cuticular plate nor stereocilia, but their basal pole was contacted by both afferent and efferent fibers, as in the early stages of IHC maturation. Such efferent contacts are very rare in the adult cochlea. These findings, not previously reported in antibiotic poisoned cochleas of adult animals, suggest that an attempt at sensory cell neodifferentiation occurs in vivo in the developing organ of Corti intoxicated with aminoglycoside antibiotics.

Amikacin↗
PubMed1995

Structural and ultrastructural aspects of isolated immature cochlear outer hair cells maintained in short-term culture.

Immature outer hair cells (OHCs), isolated from developing rat cochlea without using proteolytic enzymes, were maintained in short-term culture in a clot of coagulated plasma. Cell viability was assessed by a laser scanning image cytometer, using double-fluorescent labeling. Light and transmission electron microscopy was used to study the morphology of isolated cells. Ten to 60 healthy OHCs were obtained from one cochlea, either as single isolated cells or clusters containing 2-10 cells from the same row. Although dead cells were observed only 1 h after dissociation, there were still viable cells after 6 h. Isolated OHCs were not perfectly cylindrical, due to the immaturity of their cortical structures. One hour after dissociation the ultrastructural organization of the isolated cells was generally well preserved, but this was followed by dilatation of the Golgi apparatus and endoplasmic reticulum. Specific changes in isolated OHCs were also observed at the subsurface cisternae and cuticular plate. Although degenerating OHCs generally showed a classic pattern of necrosis, certain morphological features reminiscent of apoptosis were also observed. This study emphasises the difficulty involved in investigating isolated immature OHCs in vitro and provides a basis for future research into the physiological requirements of isolated immature OHCs.

Animals↗
PubMed1993

Airway macrophages from patients with asthma do not proliferate.

BACKGROUND: Macrophages are involved in asthma, but their pulmonary turnover is unknown. We compared the ability of bronchoalveolar lavage (BAL) and bronchial macrophages to proliferate in normal subjects and patients with asthma. METHODS: BAL cells from eight patients with asthma and eight normal volunteers were separated with a discontinuous Percoll gradient (Pharmacia Fine Chemicals, Uppsala, Sweden). In a first experiment, nuclei of each alveolar macrophage (AM) fraction, stained with propidium iodide, were analyzed for DNA content with a flow cytometer, and the proportions of cells in the G0/G1, S, and G2 + M phases were determined. In a second experiment, expression of Ki-67-related antigen was sought on AMs by immunocytochemistry. Macrophages from 10 patients with asthma and 10 normal volunteers were studied in biopsy specimens by means of immunohistochemistry with a panmacrophage monoclonal antibody (HAM-56) and a monoclonal antibody against proliferating cell nuclear antigen. RESULTS: The proportions of BAL AMs in the different phases of the cell cycle were similar in normal subjects and patients with asthma for all fractions, and the percentage of cells in S and G2 +/- M phases ranged from 7.3% to 11.3%. Under 1% of BAL AMs expressed Ki-67-related antigen. None of the macrophages present in the biopsy specimens expressed proliferating cell nuclear antigen. CONCLUSIONS: This study does not indicate that an important source of airway macrophages is local proliferation.

Adolescent↗
PubMed1991

Effects of castration, Depo-testosterone and cyproterone acetate on lymphocyte T subsets in mouse thymus and spleen.

The effects of testosterone on the relative proportion of Thy 1.2, CD4 (L3T4) and CD8 (Lyt-2) cells in thymus and spleen were studied after castration and administration of Depo-testosterone (DT) separately or together with cyproterone acetate (CA) (an antiandrogen) in BDF1 mice. Injection of 0.5 mg/100 g body weight of DT during 2 weeks decreased significantly the number and proportion of double positive (DP) (CD4+ CD8+) and increased the percentage of single positive (SP) CD4+ (CD4+ CD8-), whereas there was a slight decrease in the Thy 1.2+ cells in the thymus. In parallel, we observed an increase in CD8+ (CD4- CD8+) cells in the spleen. The androgen deprivation after 3 weeks of castration induced a decrease in the percentage of CD4+ cells in thymus and both CD4+ and CD8+ cells in spleen. Injection of CA (0.5 mg/100 g body weight) had the same qualitative effects as DT on the proportion of lymphocyte T subsets in castrated mice. However, the combined activities of DT and CA were greater than either alone. These data indicate the main role of testosterone in the distribution of CD4+ and CD8+ cells in male mice. The similar effects of CA and DT in the lymphoid organs may suggest a difference between androgen receptors of sexual and lymphoid organs.

Androgen Antagonists↗
PubMed1991

[Cytometric profile of the DNA content in pathologies of the uterine cervix].

Through flow cytometry, we have analysed DNA content of cervical cells recovered by scrapping the uterine cervix in 1) 103 women without human papillomavirus (HPV) infection and cervical intraepithelial neoplasia (CIN), 2) 42 patients with HPV infection without CIN, and 45 patients with CIN. We have observed four different DNA patterns: 1) normal DNA pattern, 2) increased and heterogeneous DNA pattern (IH), 3) increased S G2 + M phases, and 4) DNA aneuploid pattern. Statistical calculation has emphasized the significant correlation linking flow cytometric DNA pattern with cytologic diagnosis. HPV infection without CIN was associated with IH DNA pattern and CIN with increased S G2 + M phases or DNA aneuploid pattern. These results point out the interest of flow cytometric analysis of DNA content in uterine cervix pathology and in more extend in infectious or preneoplastic pathology.

Carcinoma in Situ↗
PubMed1991

Nycthemeral variations on LPS- and BCG-induced PMN migration in normal mice.

This study was performed to determine whether temporal variations could exist in polymorphonuclear neutrophil (PMN) migration induced by LPS or BCG in mice. LPS- and BCG-impregnated cell traps were implanted at 6 different times of the day and removed after 480 min. The PMN number per square unit (mm2) was counted and the results were expressed as means +/- S.E. in 8-10 mice. The results showed that nycthemeral variations occurred in both LPS- and BCG-induced PMN migration. Between 5.00 and 9.00 h a maximum value of 4210 +/- 270 and 1920 +/- 486 PMN/mm2 were obtained with BCG and LPS respectively; at 17.00 h a minimum value of 1300 +/- 270 with BCG and 396 +/- 127 PMN/mm2 with LPS were observed. There was no significant nycthemeral variation in saline-induced PMN migration.

Animals↗
PubMed1990

[Cytometric profiles of DNA content in cervical intraepithelial neoplasia (CIN)].

The cervical cytologic screening is responsible for the overall decline in the incidence of cervical cancer. Despite this apparent success, the cytologic assessment by light microscopy of cervical smear has recently been shown to have deficiencies particularly in cervical intraepithelial neoplasia (CIN). Now, this pathology of the uterine cervix is increasingly becoming recognized as a major health problem in young women because of its high frequency and its association with the development of cervical carcinoma. Flow cytometry (FCM) is widely used as a rapid method to analyse DNA content of a great number of cells on a cell by cell basis. The authors have applied FCM to CIN in order to reinforce the cytologic screening of cervical smears. Three abnormal DNA patterns have been observed: increased and heterogeneous DNA pattern; increased S and G2+M phases; abnormal DNA content. The statistical calculation has emphasized the significant relation linking these DNA patterns with the cytologic diagnosis and the severity of CIN. Therefore, FCM is a powerful complementary tool in cervical pathology.

Carcinoma in Situ↗
PubMed1990

[Qualitative approach of DNA content analysis by flow cytometry].

One step DNA staining procedures are very easy to use. Reduced staining time and small amount of needed components explain the world wide use of these protocols. The heterogeneity of the results obtained with these technics during a study of different mice organs has let the authors to think that there was a close relationship between this heterogeneity and DNA staining technics. The authors have observed the same discordances with some human tissues (parathyroid, prostatic adenoma...). They disappear when the same cells are stained with a procedure that associates to propidium iodide and RNAse a pretreatment of nuclei by trypsin and spermin. These molecules are DNA stabilizing components. So one can develop the hypothesis that the abnormalities observed with the one step procedures are due to a decondensated state of DNA that allows the fixation of a higher amount of stain. The main point will be to find a direct relationship with different physiological or pathological states. Now, it is necessary to sensibilise users against the risk of false abnormal DNA content detection through the one step procedures.

Animals↗
PubMed1989

[Flow cytometry. Principles and methods for cell studies].

Through flow cytometry cells are investigated by optical, electrical and electronic components. On a rate of several hundred cells per second, quantitative multiparametric cells studies are realized on a cell by cell basis. Cells in monodisperse suspension are drawn by a stream of fluid which aligns them before their interception by a beam of light, generally produced by Laser. The generated optical signals (diffused lights and emitted fluorescences), related to biological properties of the cells, are conveyed to photomultipliers which transform light energy into electric currents. The signals are quantified by electronic and processing components which can drive cell sorting. Flow cytometry is playing an increasingly significant role in the practice of the clinical laboratory in several areas i.e. oncology (DNA content measurement) or immunology (lymphocytes phenotyping).

Electronic Data Processing↗
PubMed1988

[Inter-organ differences of the cytometric DNA content in mice: relation of the staining method].

With one step DNA staining methods including cell membrane lysis and RNase treatment, we regularly observed a higher fluorescence emission in liver nuclei compared to bone marrow nuclei in C57BL/6 mice. Therefore this study was conducted in order to emphasize such a phenomenon in other organs and to assess if higher fluorescence emission was related to higher DNA content or staining procedure failure. Liver, bone marrow and testis were removed from Swiss, BDF and C57BL/6 mice. The following samples were prepared: 1) liver cells with TRBC (TRBC = Trout Red Blood Cells = internal standards), 2) bone marrow cells with TRBC, 3) testis cells with TRBC and 4) mixtures of liver, bone marrow and testis cells. The staining procedures were: A) one step pH 10 procedure described by Vindelov (Virchows Arch. B. Cell Path., 1977, 24, 227-242), B) same procedure with twice RNase concentration, C) first method with twice NP 40 concentration and D) three steps procedure including Trypsin and Spermine treatment (Vindelov et al., Cytometry, 1983, 3, 323-327). In protocols A, B and C, "Diploid cells/TRBC" ratio differed significantly between liver, bone marrow and testis nuclei. Moreover, 3 distinct populations of diploid cells were present in samples 4. In protocol D, "Diploid cells/TRBC" ratio were identical between liver, bone marrow and testis nuclei. In samples 4, only 1 population of diploid cells has been observed. This study shows that DNA stabilization by polyamine and protein degradation by protease could act on Propidium Iodide fixation and/or fluorescence emission, with significant differences according to the origin of the cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗