Biomedical subjects

G Ghanem

Publications and source records attributed to G Ghanem.

At least 37 records · Page 2Linked to original sources

PubMed1999

Expression of the MC1 receptor gene in normal and malignant human melanocytes. A semiquantitative RT-PCR study.

Alpha melanocyte-stimulating hormone (MSH) and related proopiomelanocortin-derived (POMC) peptides bind to the melanocortin 1 receptor (MC1-R) of mammalian melanocytes and stimulate proliferation and melanogenesis. POMC transcripts and alpha-MSH-like immunoreactivity have been found in melanoma cells and a possible autocrine loop involving MC1-R and POMC-derived products has been proposed. Therefore, the alpha-MSH/MC1-R system plays a major role in the biology of melanocytes, and provides targets for melanoma diagnosis and therapy. However, the relative levels of MC1-R expression in normal melanocytes (NM) and melanoma cells are unknown, and it is still debated whether or not all human melanomas express the MC1-R. We describe a semiquantitative RT-PCR assay for MC1-R expression, using a competition vector generated by deleting 164 bp of the native gene. The competitor was employed to analyse a panel of human melanoma cells, tumour samples, giant congenital nevus cells (CNM) and normal melanocytes (NM). All samples were positive for MC1-R expression, but expression of the receptor gene did not correlate with that of tyrosinase. Expression levels were about 10 and 20 times higher for surgical specimens and cultured melanoma cells, respectively, than for NM, but comparable for CNM and NM. Thus, high MC1-R expression is a frequent event in malignant melanocytes, and might lead to a higher activity of the alpha-MSH/MC1-R system in melanoma cells as compared to normal melanocytes, for equal local concentrations of the hormone or related melanocortins.

Binding, Competitive↗

PubMed1998

Modulation of ICAM-1 expression by alpha-MSH in human melanoma cells and melanocytes.

Alpha-MSH, a proopiomelanocortin (POMC)-derived peptide, is known to be produced in the pituitary, the skin, and melanoma tumors and to possess many biological effects, mainly on melanocyte pigmentation and growth. Moreover, the melanocyte expresses adhesion molecules, including ICAM-1. The latter has been reported to play a role in melanoma spread and associated metastatic process. We conducted a study in order to evaluate the possible effect of MSH on ICAM-1 expression in human cultured malignant and normal melanocytes. Our data show that alpha-MSH inhibits ICAM-1 expression stimulated by TNF in a concentration-dependent manner, both at the protein and gene expression level. Ninety percent inhibition was obtained with 10 nM MSH, while 50% inhibition was achieved with 1 nM. Endogenous cAMP elevation with forskolin as well as an exogenous cAMP stable analogue (Sp-cAMPS) produced the same inhibitory effect. A screening of malignant melanocytes showed that inhibition of ICAM-1 expression could be achieved only in those cells expressing detectable MSH receptors and seemed to correlate with the number of binding sites. In conclusion, our data strongly suggest alpha-MSH as a potent inhibitor of ICAM-1 expression in malignant melanocytes acting through MSH receptor stimulation and subsequent cAMP increase.

Adult↗

PubMed1998

alpha-Melanotropin immunoreactivity in human melanoma exudate is related to necrosis.

We have previously reported high immunoreactive alpha-MSH (IR-alpha-MSH) concentrations in melanoma patients' plasma, as well as significant amounts in melanoma metastases and cells grown in culture. Necrosis within the melanoma tumour leads to a massive proteolysis of intracellular proteins and release of cell content: this might significantly contribute to the elevated IR-alpha-MSH plasma levels measured in melanoma patients. To test this hypothesis, we studied the necrosis-related release of MSH from human melanoma cells, using a specific radioimmunoassay. The studies of fine-needle biopsies indicated that most of the human melanoma tumour exudates tested contained very high MSH concentrations (> 500 pg/ml; 14/15), while plasma levels were generally normal (< or = 25 pg/ml; 10/15). The level in an exudate from a non-melanoma tumour type was < 40 pg/ml. In vitro studies showed that release of the IR-alpha-MSH was time- and temperature-dependent, and related to cell death.

Humans↗

PubMed1998

alpha-Melanocyte stimulating hormone inhibits tumour necrosis factor-alpha stimulated intercellular adhesion molecule-1 expression in normal cutaneous human melanocytes and in melanoma cell lines.

alpha-Melanocyte stimulating hormone (alpha-MSH) was found significantly to reduce tumour necrosis factor-alpha (TNF-alpha) stimulated upregulation of intercellular adhesion molecule-1 (ICAM-1) in normal adult cutaneous melanocytes. The maximum inhibitory response to alpha-MSH was obtained at around 10(-10) mol/L alpha-MSH when cells were coincubated with alpha-MSH and TNF-alpha for 24 h. alpha-MSH had little or no effect on basal ICAM-1 expression in melanocytes and the effects of alpha-MSH could be mimicked with 3-isobutyl-1-methylxanthine (IBMX). Preliminary data in three human melanoma cell lines also showed alpha-MSH and forskolin to be effective in significantly reducing TNF-alpha stimulated ICAM-1 expression over 24 h. The extent of the inhibition varied from cell line to cell line and was greatest in those cells with the highest number of alpha-MSH receptors. These data suggest that alpha-MSH has the ability to oppose the action of the pro-inflammatory cytokine TNF-alpha on melanocytes and melanoma cells.

1-Methyl-3-isobutylxanthine↗

PubMed1998

Comparison of HPLC and stereologic image analysis for the quantitation of eu- and pheomelanins in nevus cells and stimulated melanoma cells.

The aim of the study was to compare two methods of quantitating eumelanins and pheomelanins, pigments synthesized by melanocytes. One is based on the high performance liquid chromatography quantitation of specific degradation products of each melanin type. The other requires image analysis, transmission electron microscopy, and stereology. In a previous study, we showed good correlations between both methods for total melanin but not for eumelanins or pheomelanins. We describe here the same comparison in more pigmented cells (nevus cells and stimulated HBL melanoma cells). Transmission electron microscopy micrographs were image analyzed to generate several primary parameters. Stereology was used for estimating melanosomal maturation, intracellular melanin content, and the number of melanized melanosomes per cell, for total melanin, eumelanins, or pheomelanins. Our results showed a good correlation between both methods for total melanin, eumelanins, and pheomelanins with an r equal to 0.99, 0.91, and 0.93, respectively, when all the points were used in the linear regression analyses. In the melanoma cell group (HBL cells cultured in media of different compositions), the chemical and morphometric estimations were not parallel in the case of eumelanins and pheomelanins. In addition, the stereologic and high performance liquid chromatography pheomelanins to eumelanins ratios were still not correlated. These results demonstrate the relevancy of the stereologic method, but the low level of melanization, the possible lack of specificity of melanogenesis in melanoma cells, and a problem of sensitivity of the stereologic method in this context seem to be obstacles in obtaining better results. The utilization of normal human melanocytes could give some answers to our hypotheses.

Chromatography, High Pressure Liquid↗

PubMed1998

Comparison of high performance liquid chromatography and stereological image analysis for the quantitation of eumelanins and pheomelanins in melanoma cells.

The aim of the study was to compare two methods quantifying eumelanins and pheomelanins, pigments synthesized by melanocytes. One is based on the high performance liquid chromatography (HPLC) quantitation of specific degradation products of each melanin type. The other requires image analysis, transmission electron microscopy (TEM), and stereology. This study was carried out in cultured human melanoma cells and for each line, melanins were measured by HPLC and cells were fixed and embedded as pellets for TEM. Ultrathin sections were treated or not by the alkali elution method allowing the elimination of pheomelanins. The obtained micrographs were analyzed with our image analysis program permitting the estimation of several primary parameters. Stereology was used for estimating melanosomal maturation, intracellular melanins content, and number of melanized melanosomes per cell, for total melanin, eumelanins, or pheomelanins. Our results show a good correlation between both methods for total melanin, particularly when using the cytoplasmic volume density of melanin (r=0.93). Moreover, we report that the number of melanized melanosomes per cell and not the melanosomal maturation is responsible for the differences in total melanin content observed between the different cell lines. However, none of the stereological melanization parameters was correlated in the case of eumelanins or pheomelanins. In order to demonstrate the utter relevancy of this stereological approach, utilization of more pigmented melanoma cells, comparative study of HPLC and stereology, in normal epidermal melanocytes and a new evaluation of the alkali elution method in appropriate animal models would help us to explain the present results.

Chromatography, High Pressure Liquid↗

PubMed1998

Detection and serotyping of Mediterranean plum pox virus isolates by means of strain-specific monoclonal antibodies.

Plum pox virus (PPV) is a major threat to the expanding Mediterranean stone fruit industry. In order to control the plum pox disease it is of utmost importance to detect early PPV foci and to identify the PPV isolates involved. A survey was therefore carried out in Albania, Cyprus, Egypt, Greece, Italy and Turkey by a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with the following monoclonal antibodies (MAbs): 5B (universal), 4DG5 (PPV-D-specific), AL (PPV-M-specific), TUV and AC (PPV-C-specific), and EA24 (PPV-El Amar-specific). A hundred and seventy Mediterranean PPV isolates were tested for strain type. PPV-M was detected in Albania, Cyprus, Greece, Italy, and Turkey; PPV-D was detected in Albania and Italy, whereas samples with natural mixtures of both strains were found in a couple of orchards in Albania. Seven PPV isolates from apricots in two Egyptian localities were recognized only by MAb EA24. In conclusion, DAS-ELISA with a combination of the universal MAb5B and the MAbs specific to the four PPV serotypes currently known (M, D, C and El Amar) is an efficient tool for a simple, sensitive and routine detection of PPV and discrimination of its serotypes.

Antibodies, Monoclonal↗

PubMed1998

Quantification of eumelanin and pheomelanin: stereologic image analysis method.

OBJECTIVE: To develop an ultrastructural stereologic image analysis method allowing quantification of intracellular melanization. STUDY DESIGN: First, in the field of image analysis, a newly elaborated segmentation method, SEM2, was compared with a previously described method based on gray level histograms. Only SEM2 allows the segmentation of melanin in micrographs of poorly melanized melanocytes. Second, in the field of stereology, estimation of cell volume remains problematic in the case of mixed cell populations. This problem is approached by the comparison of stereologic alternatives and cytochemistry (L-3, 4-dihydroxyphenylalanine reaction) in epidermal melanocytes and melanoma cells from several in vitro experiments. The cytochemical approach was found to be the best choice. RESULTS: Concerning the quantification of eumelanin and pheomelanin, the alkali elution method, permitting the specific dissolution of pheomelanin on ultrathin sections, was validated in normal human follicular melanocytes. CONCLUSION: These results allow us to envisage the stereologic quantification of eumelanin and pheomelanin at the ultrastructural level. At present our method is undergoing evaluation by comparison with the present method of reference based on high-performance liquid chromatography.

Adolescent↗

PubMed1997

Tumor type and vascularity: important variables in infusional brachytherapy with colloidal 32P.

PURPOSE: This study investigated the role of histologic tumor characteristics, in comparison with a normal tissue, and of tumor vascularization on the uptake and retention of colloidal 32P used in infusional brachytherapy of solid cancers. The cytotoxicity of colloidal 32P was also evaluated for two tumors of different radiosensitivity, a melanoma, and a squamous cell carcinoma. METHODS AND MATERIALS: An in vitro analysis of colloidal 32P uptake was carried out on a human melanoma cell line, HBL, a human squamous cell carcinoma, SCC1, and normal fibroblasts, F-NBB. Tumor retention of colloidal 32P was studied in vivo for the HBL and the SCC1 tumors implanted subcutaneously in nude mice. Tumor vascular density was determined by microscopic study of Masson's trichrome slides of HBL and SCC1 tumors of about 1 cm diameter. RESULTS: In vitro studies showed that the time required for maximal cell uptake of colloidal 32P was only 10-20 min for the SCC1 and HBL tumors, while it took at least 60 min for the fibroblasts. After intratumoral injection of macroaggregated albumin (MAA), followed by 50 microCi of colloidal 32P, Bremsstrahlung imaging performed at 6 and 24 h showed that the activity remained in the HBL tumor while some of the radiocolloids leaked from the SCC1 tumor and was trapped in the reticuloendothelial system of the liver. Organ activity counting confirmed this finding: 32P activity was three to four times higher in the HBL than in the SCC1 tumor, whereas the activity in the liver, insignificant in the HBL mice (less than 0.1 microCi/g), was as high as 24 microCi/g in the SCC1 mice. This phenomenon may be explained by the difference in tumor vascular density, estimated for the HBL to be about four times less than that of the SCC1 tumor (5.7 vs. 21.4 blood vessels per mm2 for the HBL and the SCC1 tumors, respectively). CONCLUSION: Intratumoral infusion of colloidal 32P may be a useful complement of radiation therapy in the treatment of nonresectable but accessible solid tumors. Tumor vascularization must be taken into account for a successful vascular blockade by MAA prior to the infusion of colloidal 32P.

Animals↗

PubMed1997

Immunoreactive alpha-melanotropin as an autocrine effector in human melanoma cells.

Melanotropin is a peptide having several functions, including the stimulation of melanogenesis and the modulation of proliferation of melanocytes and melanoma cells. It acts through binding to high-affinity receptors of the melanocortin-1 subtype, exclusively expressed in cells of the melanocytic lineage. Elevated levels of immunoreactive alpha-melanotropin were previously reported in melanoma cell lines, tumours and plasma from patients with melanoma. Here, we show that this high ectopic production of melanotropin is restricted to melanoma and non-pituitary tumours with the same neuroectodermic origin. The occurrence of a melanotropin-specific autocrine loop was further investigated in human melanoma cells. Immunoreactive alpha-melanotropin was spontaneously released from a melanoma cell line (HBL) expressing melanotropin receptors on the cell surface. This release was significantly increased in the presence of melanotropin-related peptides such as corticotropin-(4-10)-peptide and beta-melanotropin, competing for binding to the melanotropin receptor and was directly correlated to the displacement potential of these peptides. Both spontaneous and induced releases of immunoreactive alpha-melanotropin could be blocked at low temperatures, suggesting the involvement of intracellular protein movement in the release mechanism. The release of immunoreactive alpha-melanotropin was not significant in melanoma cells expressing very low levels of melanotropin receptors (IGR3) or in non-melanoma cells (SCC1). However, upon expression of the melanocortin-1 receptor cDNA into IGR3 cells, spontaneous and competition-induced releases of immunoreactive alpha-melanotropin were both increased and also blocked at low temperatures. This observation further underlines a role for the melanotropin receptor in the release of immunoreactive alpha-melanotropin. These experiments indicate that an autocrine loop between the melanocortin-1 receptor and immunoreactive alpha-melanotropin may be functional in human melanoma cells.

Adult↗

PubMed1997

Pilomatrix carcinoma.

A 43-year-old man presented with a tumour of the medial part of the right eyebrow. A biopsy was performed and histological examination revealed a malignant transformation of a 'calcifying epithelioma of Malherbe'. A secondary wide excision was performed. Clinical and pathological findings of this extremely rare tumour are reviewed. Factors influencing the prognosis are proposed, based on a compilation of the literature.

Adult↗

PubMed1997

In vitro cytotoxic effect of difluoromethylomithine increased nonspecifically by peptide coupling.

Difluoromethylomithine (DFMO)-peptide conjugates were synthesized as prodrugs to improve the cytotoxic efficacy of DFMO. All conjugates inhibited cell growth in different cell lines more effectively than DFMO itself. The best cytotoxic effect was achieved in all cell lines by DFMO-Glu-His-Phe-Arg-Trp-Gly-OMe, where the carrier peptide is a melanotropin hormone fragment. Although this conjugate is capable of displacing labeled melanotropin from its receptor, its cytotoxic effect on the receptor-positive human melanoma cell line has not been proven to be receptor-mediated. The differences in the cytotoxicities of the congeners seem to be influenced, at least in part, by the nature of the carrier molecule.

Antineoplastic Agents↗

PubMed1997

Comparison of TRPs from murine and human malignant melanocytes.

Most of our knowledge of the mammalian tyrosinase related protein (TRP) activities is derived from studies using murine melanoma models, such as B16 or Cloudman S-91 melanocytes. Owing to the high degree of homology between the murine and human enzymes, it has been assumed that their kinetic behaviour could be similar. However, the protein sequences at the metal binding sites of the murine and human enzymes show some differences of possible functional relevance. These differences are more significant in the metal-A site than in the metal-B site. By using three human melanoma cell lines (HBL, SCL, and BEU), we have studied the catalytic abilities of the human melanogenic enzymes in comparison to those obtained for the counterpart murine enzymes isolated from B16 melanoma. We have found that TRP2 extracted from all cell lines show dopachrome tautomerase activity, although the activity levels in human malignant melanocytes are much lower than in mouse cells. Reconstitution experiments of the human enzyme indicate that TRP2 has Zn at its metal binding-sites. Although mouse tyrosinase does not show DHICA oxidase activity, and this step of the melanogenesis pathway is specifically catalyzed by mouse TRP1, the human enzyme seems to recognize carboxylated indoles. Thus, human tyrosinase could display some residual DHICA oxidase activity, and the function of human TRP1 could differ from that of the murine protein. Attempts to clarify the nature of the metal cofactor in TRP1 were unsuccessful. The enzyme contains mostly Fe and Cu, but the reconstitution of the enzymatic activity from the apoprotein with these ions was not possible.

Animals↗

PubMed1997

Characterization of the nuclear deoxyribonucleic acid content and nuclear morphometry in 71 primary cutaneous melanomas.

BACKGROUND: While the determination of nuclear deoxyribonucleic acid (DNA) content (DNA ploidy level) and nuclear morphometry characterization has proved to be of prognostic value in melanocytic lesions, there are several ways of performing these determinations. OBJECTIVE: To identify which of 9 DNA ploidy- and 2 nuclear morphometry-related variables are of prognostic and/or diagnostic value in 71 primary melanomas. METHODS: Histological typing, Breslow depth determination, the evaluation of Clark's level of invasion and the 11 quantitative variables (calculated in Feulgen-stained nuclei using computer-assisted microscope analysis) determined for each melanoma were submitted to discriminant analysis. RESULTS: The discriminant analysis of image cytometric variables enabled specific cell subpopulations to be identified in histological and the Breslow-related groups, but not in the Clark-related ones. CONCLUSION: The characterization of melanoma heterogeneity by means of the identification of specific DNA ploidy level-related cell subpopulations in specific Breslow-related groups enables the problem of intra- and interobserver variability in Breslow depth determination to be reduced and therefore can help dermatologists in their daily routine.

Adult↗

PubMed1996

Cysteine deprivation promotes eumelanogenesis in human melanoma cells.

Melanocytic cells can produce two types of pigment, pheomelanin or eumelanin. We used two types of human melanoma cell lines to explore the regulation of pigmentation by biochemical and enzymatic studies. These two cell lines were previously designated as either pheomelanotic or of mixed type when cultured in a medium rich in cysteine. We analyzed the effects of L-cysteine depletion on melanin synthesis and the involvement of the tyrosinase-related proteins in the production of both eumelanin and pheomelanin. Cultures were exposed to L-cysteine concentrations ranging from 206 to 2.06 microM, and the following parameters were measured: tyrosine hydroxylase activity, intracellular L-cysteine and glutathione concentrations, eumelanin and pheomelanin formation, and tyrosinase-related protein-1 and -2 mRNA levels. Extracellular L-cysteine depletion significantly increased tyrosine hydroxylase activity and promoted both eumelanogenesis and visible pigmentation in both lines. In contrast, pheomelanogenesis was increased only in the pheomelanotic cell line. Whereas eumelanogenesis was apparent upon L-cysteine depletion, tyrosinase-related protein-1 expression was not induced in the pheomelanotic cells, and tyrosinase-related protein-2 expression remained unchanged. Thus, tyrosinase-related protein-1 mRNA expression seems to be concomitant with eumelanogenesis when the L-cysteine concentration is high, but does not appear essential for eumelanogenesis at low L-cysteine concentrations. The mechanisms governing pheomelanin to eumelanin balance are dependent on L-cysteine, glutathione, and tyrosinase-related protein-1 expression, but none of these factors alone appears to be dominant in directing the synthesis of a particular type of melanin.

Cysteine↗

PubMed1996

Action of cAMP on expression and release of adhesion molecules in human endothelial cells.

The expression of E-selectin induced by tumor necrosis factor (TNF) on the surface of human umbilical vein endothelial cells (HUVEC) was partially inhibited by an increase in the level of adenosine 3',5'-cyclic monophosphate (cAMP), produced by forskolin or cholera toxin combined with the type IV phosphodiesterase inhibitor rolipram and the protein kinase A agonist phosphorothioate analogue of cAMP SpcAMPS. The same agents had no significant effect on the constitutive and TNF-stimulated expression of intercellular adhesion molecule 1 (ICAM-1), whereas the effect on vascular cell adhesion molecule 1 (VCAM-1) expression was variable depending on cell culture conditions. The stimulatory effects of phorbol 12-myristate 13-acetate and bacterial lipopolysaccharide (LPS) on E-selectin expression were also downregulated by the forskolin-rolipram combination and by SpcAMPS. Inhibition of the surface expression of E-selectin was associated with a decrease of the total amount of the protein in the cell lysate and a reduced mRNA level, with no significant effect on mRNA stability. In anesthetized rats, the terbutaline-rolipram combination reduced the rolling of leukocytes induced by LPS in the mesenteric microcirculation. In addition to their partial inhibitory effect on the TNF-induced surface expression of E-selectin on HUVEC, the forskolin-rolipram combination and SpcAMPS strongly inhibited the release of soluble E-selectin from these cells; the release of soluble ICAM-1 and VCAM-1 was unaffected by these agents. Isoproterenol reduced the release of soluble E-selectin, whereas it had no significant effect on the cell surface expression of the protein. This study underscores the potential anti-inflammatory effect of a rise in the endothelial cAMP level.

Animals↗

PubMed1995

[First Lebanese series of percutaneous mitral commissurotomies].

From January 1993 to January 1994, we realized at Risk Hospital 11 percutaneous mitral commissurotomies (PMC). This first Lebanese series comprised 9 women and 2 men. The mean age was 36 y (18-73 y). Ten patients were en class III of the NYHA and one in class IV (pregnant woman on the end of the 7th month). The predilatation evaluation was done by transthoracic echocardiography for the just 2 patients and by transthoracic with transesophageal multiplane echocardiography for the 9 others. The mean gradient was at 20 mmHg (10-24 mmHg) and the mean mitral area at 1 cm2 (0.65-1.5 cm2). We used the Inoue balloon for all these procedures with a stepwise technique and a color echo-doppler control between inflations. We obtain bicommissural opening in 8 patients and unicommissural opening in 3 patients. The mean gradient post dilatation was at 4 mmHg (3-8 mmHg) and the mean mitral area at 2.3 cm2 (1.5-2.8 cm2). No mitral regurgitation > 2/4 was noted. After a general review, we concluded the PMC is at present the treatment of choice of non or discrete calcified mitral stenosis and this procedure has to be taken on charge by the Ministry of Health in Lebanon.

Adolescent↗

PubMed1995

[Coronary artery-cardiac fistulas. Review of the literature. Two accidentally discovered cases].

We report two cases of coronary artery fistula discovered accidentally during coronary-angiography. One case between the left anterior descending and the pulmonary artery and another case between the circumflex and the left ventricule. Both cases were associated with hypertrophic cardiomyopathy. There was a spontaneous closure of the fistula in the first case. A brief review of the literature is presented because this anomaly is being discovered frequently because of the possibility of diagnosis with transesophageal echocardiography.

Adult↗