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Biomedical subjects

N Maggiano

Publications and source records attributed to N Maggiano.

At least 37 records · Page 2Linked to original sources
PubMed2000

Laser scanning cytometry can complement the flow cytometric DNA analysis in paraffin-embedded cancer samples: a paradigmatic case.

Archival studies on paraffin-embedded tumor samples are often complicated by difficulty obtaining a reliable diploid DNA standard. Nontumor cells, e.g., inflammatory and stromal cells, most often found interspersed among tumor cells, would represent a solution to this problem. Unfortunately, there is an inherent difficulty to positively identifying tumor cells in paraffin-embedded specimens. Using an aneuploid paraffin-embedded breast cancer sample, we show here that laser scanning cytometer (LSC) in conjunction with flow cytometry can help to address this issue. Following standard protocols, the tissue was deparaffinized and rehydrated, and the nuclei mechanically isolated before being exposed to propidium iodide. An aliquot served for single-parameter flow cytometric analysis, and the remaining cells were cytocentrifuged onto a microscope slide and LSC analysis was performed. The DNA histogram profiles generated by the two approaches were comparable and both showed the presence of cell populations with different DNA content. To assess the nature of these subsets, we performed a correlated measurement of DNA content and chromatin organization at the single-cell level by LSC. This allowed the identification of several subsets of nuclei. Slides were then stained with Giemsa and the nature of these subsets was assessed morphologically by exploiting the relocating capability of LSC. Inflammatory and stromal cells, residual diploid epithelial cells, and hyperdiploid tumor cells-each characterized by a peculiar coordinate pattern of DNA content and chromatin organization-could be positively identified. Diploid, nontumor cells can then be used as an internal standard for DNA ploidy.

Breast Neoplasms↗
PubMed2000

Quercetin inhibits p21-RAS expression in human colon cancer cell lines and in primary colorectal tumors.

Immunocytochemical studies have revealed that 10 microM quercetin reduced the steady state levels of p21-ras proteins in both colon cancer cell lines and primary colorectal tumors. These findings were confirmed by Western blot and flow cytometric analysis showing that the inhibition of p21-ras expression by quercetin was time- and concentration-dependent. Twenty-four-hour treatment with 10 microM quercetin reduced p21-ras levels to about 50% of control values. Quercetin was similarly effective in inhibiting the expression of K-, H-, and N-ras proteins. Moreover, the effect of quercetin on ras oncogene expression was not dependent on the cell cycle position of colon cancer cells and appeared to be specific and not merely a consequence of overall inhibition of protein synthesis. Northern blot analysis revealed that quercetin produced in colon cancer cells an early (30 min) reduction of the steady state levels of K-, H-, and N-ras mRNAs. This reduction was also present after 6 hr of flavonoid treatment. These effects of quercetin suggest a possible chemopreventive role for this compound in colorectal carcinogenesis.

Anticarcinogenic Agents↗
PubMed2000

Beta-carotene antagonizes the effects of eicosapentaenoic acid on cell growth and lipid peroxidation in WiDr adenocarcinoma cells.

The effects of combinations between eicosapentaenoic acid (EPA) and beta-carotene on cell growth and lipid peroxidation were investigated in human WiDr colon adenocarcinoma cells. EPA alone was able to inhibit the growth of WiDr cells in a dose- and time-dependent manner. Such an inhibition involved fatty acid peroxidation, as shown by the remarkable increase in the levels of Malondialdehyde (MDA) in EPA-treated cells. Beta-carotene was capable of reducing the growth inhibitory effects of EPA and the levels of MDA in a dose- and a time-dependent manner. In addition, EPA increased beta-carotene consumption in WiDr cells. This study provides evidence that beta-carotene can antagonize the effects of EPA on colon cancer cell growth and lipid peroxidation.

Adenocarcinoma↗
PubMed2000

Proliferating cell nuclear antigen labelling index in localised pigmented villo-nodular synovitis and its relationship to the size of nodules.

Proliferating cell nuclear antigen (PCNA) is one of the cell cycle-related proteins directly involved in DNA synthesis. It is a marker of cellular proliferation and has been shown to correlate with ploidy and proliferative activity of cells. Its expression has been used to estimate the growth fraction of human cancer and its prognostic value. Pigmented villo-nodular synovitis (PVNS) is characterised by a nodular lesion in the paratendinous synovial tissue or, less frequently, in a joint. Whether PVNS is a neoplastic or inflammatory lesion remains controversial. We have studied immunohistochemical PCNA expression with pc10 monoclonal antibody in 16 paraffin sections, in 16 cases of localised PVNS, or giant cell tumour of tendon sheath. We have found significant correlation between the size of the lesions and PCNA-LI (labelling index).

Adolescent↗
PubMed2000

Phenotypic change of human cultured meningioma cells.

One objection to using cell cultures for studying the proliferation of tumors is the potential for phenotypic changes that may occur in vitro. Here, we compared the antigen pattern expression of cultured meningioma cells with that of the primary tumor. Cell cultures established from 9 intracranial meningiomas and deparaffinized sections of the resected tumors were analyzed for immunophenotyping with the following antibodies: vimentin, cytokeratin, epithelial membrane antigen, S-100, neuron-specific enolase, synaptophisin, factor VIII-related antigen, CD4, CD31, CD34, CD45RB, CD68-PGM1, CD68-KP, and myeloid/histiocyte antigen (MAC387). Overall, the cultured meningioma cells retained the main feature of the primary tumor, being positive both for mesenchymal antigens and for epithelial antigens. Interestingly, the cultured meningioma cells abundantly expressed the CD68 antigens at early passage. The CD68 antigens, which are normally found on hematopoietic cells like macrophages and monocytes, were not detectable on meningioma cells in situ. Our results show that phenotypic changes on human meningioma cells may occur in vitro. This phenomenon suggests caution when transposing the in vitro results to the in vivo condition.

Aged↗
PubMed2000

Evidence that hydrogen sulphide can modulate hypothalamo-pituitary-adrenal axis function: in vitro and in vivo studies in the rat.

The gas hydrogen sulphide (H2S) is normally produced in large amounts in the central nervous system during the metabolism of sulphur-containing aminoacids. H2S was recently shown to influence long-term potentiation in the rat hippocampus; this finding suggested that the gas may act as a neuromodulator in the brain. We therefore tested the effect of the gas on the release of corticotropin-releasing hormone (CRH) from rat hypothalamic explants. CRH immunoreactivity in the incubation media was taken as a marker of peptide release. We found that the addition of NaHS to incubation media was consistently associated with a concentration-dependent decrease in KCl-stimulated CRH release, whereas basal secretion was unaffected. Increased endogenous H2S production may be also obtained using an indirect precursor of H2S formation, S-adenosyl-L-methionine (SAMe). The latter mimicked the effects of NaHS, since it reduced potassium-stimulated CRH release. In vivo, SAMe showed no effect on hypothalamo-pituitary-adrenal (HPA) function under resting conditions, but inhibited stress-related glucocorticoid increase.

Adrenal Glands↗
PubMed2000

Oxygen-induced retinopathy in the newborn rat: morphological and immunohistological findings in animals treated with topical timolol maleate.

Oxygen-induced retinopathy (OIR) similar to that seen in premature infants can be produced in newborn rats by exposure to 80% oxygen for the first 5 days of life, with subsequent recovery for 7 days in a room air environment. Previous studies have shown that the latter phase is associated with degeneration of retinal astrocytes. In the absence of astrocytes, the vascular endothelial growth factor (VEGF) is produced by the neurons of the inner retina. In this study, immunohistochemical methods were used to assess retinal expression of VEGF protein in ratlings exposed to the above protocol, with and without simultaneous treatment with topical timolol maleate. Intraocular pressure (IOP) and arterial pressure were measured. The severity of OIR was also evaluated in retinal flat mounts after India ink perfusion. The mean IOP was 8.25 +/- 0.32 mm Hg in untreated ratlings and 5.15 +/- 0.24 mm Hg in timolol-treated animals. OIR in retinas from the timolol group was less severe than that seen in untreated ratlings. VEGF protein expression was almost completely absent in the latter group. Retinas from timolol-treated animals expressed VEGF protein, though the level was inferior to that found in controls raised under room air conditions. In conclusion, these data seem to indicate that experimental OIR can be attenuated by a reduction of the IOP during and after oxygen supplementation. It is possible that these effects are due to improved ocular perfusion pressure, which would mitigate the hypoxic insult to the retina during the room air recovery period.

Administration, Topical↗
PubMed2000

Growth inhibitory effects and radiosensitization induced by fatty aromatic acids on human cervical cancer cells.

Evidences have been reported that phenylacetic (PA) and phenylbutyric (PB) fatty aromatic acids can exert tumor growth inhibition in vitro and in vivo. Moreover, clinical trials also showed some activity for these drugs to modulate the expression of genes implicated in tumor growth, metastasis, immunogenicity, and to potentiate the efficacy of cytotoxic agents. The aim of the study was to examine the effects of PA and PB on the growth as well as sensitization to cisplatin and radiation in human cervical cancer cells. The effects of PA and PB on the proliferative activity and apoptosis induction in cervical tumor tissue was investigated. Both PA and PB exhibited a time- and dose-dependent antiproliferative activity in SW756 and ME180 cell lines: after 72-h treatment, the IC50 (concentration able to inhibit 50% of cell growth) of PB was 1.9 +/- 0.2 mM and 1.5 +/- 0.2 mM in SW756 and ME180 cells, respectively, while the IC50 of PA was 13.0 +/- 1.7 mM and 10.0 +/- 1.2 mM in SW756 and ME180 cells, respectively. In tumor tissue biopsies obtained from patients affected by squamous cervical cancer, both drugs resulted in a marked reduction of the percentage of bromodeoxyuridine-labeled cells compared with untreated samples [19.0 +/- 1.63% in untreated tissues with respect to 1.30 +/- 0.54% and 4.20 +/- 2.50% of stained cells after treatment with PA (30 mM) (P < 0.0001) and PB (5 mM) (P < 0.0001), respectively]. Moreover, analysis of the staining with M30 monoclonal antibody revealed that PA (30 mM) and PB (5 mM) were able to produce a marked increase in the number of stained apoptotic nuclei with respect to untreated samples. Finally, PB and PA were shown to enhance the sensitivity of SW756 to radiation and to exert an additive effect when combined with cisplatin. A significant reduction of the processed form of p21ras and rhoB proteins in the membrane fraction of cells exposed to PA and PB was observed. When farnesol, which is able to circumvent the enzymatic step inhibited by PA and PB, was added to the medium only a partial reversal of the growth inhibition and potentiation of sensitivity to radiation induced by PA and PB were found. In conclusion, the growth inhibitory properties of fatty aromatic acids suggest that these molecules could represent the prototype of a new class of compounds with some therapeutic potential in cervical cancer.

Antimetabolites, Antineoplastic↗
PubMed2000

Inherited macrothrombocytopenia with distinctive platelet ultrastructural and functional features.

We report a family with inherited macrothrombocytopenia and characteristic large membrane complexes in the platelets. Two affected subjects had platelet counts of 40 and 65 x 10(9)/L respectively as assessed by contrast phase microscopy. Ultrastructural studies revealed giant spheroid platelets with characteristic large membrane complexes and/or giant vacuoles containing platelet organelles. Immunohistochemical studies of actin and tubulin showed a disorganization of the microtubule and actin systems. These abnormalities were absent in leukocytes, indicating a platelet-specific cytoskeleton disorder. Platelet autoantibodies were repeatedly absent. Nevertheless, in the peripheral blood we observed several figures of platelet phagocytosis by macrophages and neutrophils. The in vitro aggregometric response of platelets to ADP, collagen, thrombin, ristocetin was present, but shape change was absent. The urinary excretion of thromboxane A2 metabolites of the affected subjects were approximately 2 standard deviations above control values, in spite of a reduced maximal biosynthetic capacity of thromboxane from giant platelets assessed in vitro during whole blood clotting. This inherited platelet disorder shows structural and functional features which allow to distinguish it from other syndromes associated with giant platelets. We also propose to include ultrastructural and cytoskeletal studies in the diagnosis as well as in the classification of inherited giant platelet disorders.

Blood Platelets↗
PubMed2000

n-3 PUFA dietary supplementation inhibits proliferation and store-operated calcium influx in thymoma cells growing in Balb/c mice.

The antitumor effect of daily individual administration of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (2 g/kg body weight) in Balb/c mice bearing a transplantable thymoma was investigated. Mice received oleic acid (control group), EPA and DHA ethyl esters starting 10 days before tumor inoculation. Analysis of phospholipid composition of neoplastic cell revealed that EPA and DHA levels were significantly increased (63 and 22% increase) after EPA and DHA treatments, respectively. Conversely, decreased levels of arachidonic acid were found in both cases (19 and 24% decrease in EPA and DHA groups, respectively). EPA and DHA delayed the appearance of macroscopic ascites (100% of animal, from 7 to 28 days), prolonged animal survival (100% of animal, from 22 to 32 and 33 days, respectively) and reduced the percentage of proliferating tumor cells detected by immunostaining of proliferation cell nuclear antigen (PCNA) (80 and 85% decrease, respectively). Moreover, the regulatory effects of these dietary n;-3 fatty acids on the influx of Ca(2+), activated by depletion of intracellular stores with thapsigargin (Tg), were investigated. By using a Ca(2+)-free/Ca(2+)-reintroduction protocol and Fura-2 as fluorescent indicator of intracellular free Ca(2+)([Ca(2+)](i)), we observed that EPA and DHA treatments markedly decreased Tg-induced rise in [Ca(2+)](i) (49 and 37% decrease, respectively). This effect was related to the inhibition of the store-operated Ca(2+) influx, as confirmed also by Mn(2+) influx experiments. The inhibitory action of EPA and DHA on the store-operated Ca(2+) influx could explain, at least in part, their antitumoral activity, as this Ca(2+) mobilization pathway appears to be involved in the cell signaling occurring in non-excitable cells to evoke many cellular processes, including cell proliferation.

Animals↗
PubMed1999

Cell proliferation, differentiation, and apoptosis are modified by n-3 polyunsaturated fatty acids in normal colonic mucosa.

Supplementation with low doses of eicosapentaenoic (EPA) or docosahexaenoic (DHA) acid was used here to investigate changes in epithelial proliferation, differentiation, and apoptosis in normal rat colonic mucosa. ACI/T rats received by oral administration low doses of purified EPA or DHA ethyl esters (1 g/kg body weight) and colonic mucosa was analyzed for cell proliferation, differentiation, and apoptosis. n-3 Polyunsaturated fatty acid incorporation into membrane phospholipids was investigated as reflections of fatty acid metabolism. Both EPA and DHA suppressed colonocyte proliferation and increased the numbers of differentiating and apoptotic cells without modification of the crypt morphology and the number of cells per crypt columns. A significant incorporation of the supplemented fatty acids into total phospholipids was observed. This enrichment was accompanied by a decreased content in arachidonic acid. The observation that EPA and DHA do not alter crypt morphology although they modify cell turnover in normal colonic mucosa suggests a possible use of these fatty acids as dietary chemopreventive agents.

Animals↗
PubMed1999

hMSH2 and GTBP expression in advanced stage epithelial ovarian cancer.

Defects in DNA mismatch repair have been associated with both hereditary and sporadic forms of human cancer. Most of the attention has been focused on the incidence and genetics of mismatch repair defects, while little is known about the expression levels of the mismatch repair proteins and their significance in cancer cell biology. In this study, both the expression levels of hMSH2 and GTBP proteins were investigated by Western blotting in 20 untreated epithelial ovarian cancers. For these analyses, a commercial anti-hMSH2 monoclonal antibody and a newly generated mouse monoclonal anti-GTBP antibody were used. hMSH2 and GTBP proteins were detected by Western blotting in 19 out of 20 (95%) samples analysed and were found to be directly correlated (r= +0.51, P = 0.025). hMSH2 expression was significantly higher in ovarian cancer cells originating from solid tumours than from ascites (H = 4.5, P = 0.033), whereas GTBP content did not significantly differ according to the origin of cancer cells. No statistically significant differences were found in the distribution of hMSH2 and GTBP levels according to the age of the patients, grade of differentiation, histotype and extent of surgical debulking. The amount of hMSH2 protein was demonstrated to be significantly lower in stage IV than in stage III patients (H = 7.35, P = 0.007). Moreover, significantly lower hMSH2 levels were observed in non-responding patients compared to patients who achieved complete or partial response to cisplatin-based chemotherapy (H = 4.88, P = 0.027). Conversely, GTBP levels were not distributed differently according to stage of disease and chemotherapy response. Our study suggests a possible involvement of hMSH2 in ovarian cancer cell biology and susceptibility to chemotherapy. The possible biological and/or clinical role of GTBP expression in ovarian cancer patients remains to be elucidated.

Adult↗
PubMed1999

Detection of synaptophysin-producing cells in human thymus by immunohistochemistry and nonradioactive in situ hybridization.

We investigated human thymic tissue by immunohistochemistry and in situ hybridization for the presence of synaptophysin-producing cells. Our results indicate that anti-synaptophysin antibody detected immunoreactive material in nerve fibers around vessels located in major thymic septa, in a relevant number of cortical epithelial cells, and in scattered epithelial cells in the medulla. The epithelial nature of synaptophysin-positive cells was documented by the co-expression of cytokeratins as revealed by double immunofluorescence. In situ hybridization studies revealed the presence of synaptophysin mRNA in cells mainly located in the cortex, the specific fluorescent signals being localized in the cell cytoplasm. Western blot analysis using an affinity-purified polyclonal antibody revealed an immunoreactive band of about 38 kD in the extracts from unfractionated thymic tissue and from epithelial cell-enriched fractions. No staining was observed in isolated thymocytes. The expression of synaptophysin in epithelial cells of the thymic cortex suggests that this protein may be involved in secretory activities related to T-cell maturation.

Blotting, Western↗
PubMed1998

Growth-inhibitory effect of tamoxifen and quercetin and presence of type II estrogen binding sites in human laryngeal cancer cell lines and primary laryngeal tumors.

Quercetin and tamoxifen, in a range of concentrations between 0.01 and 5 microM, exert a dose-dependent inhibition on the anchorage-dependent and anchorage-independent cell growth of Hep2 and CO-K3 laryngeal cancer cell lines. Cell cycle analysis revealed that the growth-inhibitory effect was associated with a block of the cells at the G2/M checkpoint of the cell cycle followed by DNA fragmentation. This suggests that the failure of cells to proceed through the G2/M checkpoint can be a trigger for apoptosis. The induction of apoptosis by quercetin and tamoxifen was confirmed immunocytochemically by the in situ nick end labeling (TUNEL) reaction. These compounds also exerted a dose-dependent growth-inhibitory effect on primary tumor cells, as assessed by colony-forming assay and bromodeoxyuridine labeling. Laryngeal cancer cell lines and primary tumor cells expressed Type II estrogen binding sites (Type II EBS) with binding characteristics similar to those of Type II EBS in other tumor cells. Since the affinities of quercetin and tamoxifen for Type II EBS were correlated with their growth-inhibitory potential while ipriflavone neither interacted with these sites nor inhibited cell growth, the possibility exists that the action of these compounds is mediated, at least in part, by the interaction with Type II EBS. In conclusion, our data indicate that quercetin and tamoxifen could be potentially useful in laryngeal cancer treatment.

Apoptosis↗