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M L Mancianti

Publications and source records attributed to M L Mancianti.

At least 19 recordsLinked to original sources
PubMed2003

Measuring stem cell frequency in epidermis: a quantitative in vivo functional assay for long-term repopulating cells.

Epidermal stem cells play a central role in tissue homeostasis, wound repair, tumor initiation, and gene therapy. A major impediment to the purification and molecular characterization of epidermal stem cells is the lack of a quantitative assay for cells capable of long-term repopulation in vivo, such as exists for hematopoietic cells. The tremendous strides made in the characterization and purification of hematopoietic stem cells have been critically dependent on the availability of competitive transplantation assays, because these assays permit the accurate quantitation of long-term repopulating cells in vivo. We have developed an analogous functional assay for epidermal stem cells, and have measured the frequency of functional epidermal stem cells in interfollicular epidermis. These studies indicate that cells capable of long-term reconstitution of a squamous epithelium reside in the interfollicular epidermis. We find that the frequency of these long-term repopulating cells is 1 in 35,000 total epidermal cells, or in the order of 1 in 104 basal epidermal cells, similar to that of hematopoietic stem cells in the bone marrow, and much lower than previously estimated in epidermis. Furthermore, these studies establish a novel functional assay that can be used to validate immunophenotypic markers and enrichment strategies for epidermal stem cells, and to quantify epidermal stem cells in various keratinocyte populations. Thus further studies using this type of assay for epidermis should aid in the progress of cutaneous stem cell-targeted gene therapy, and in more basic studies of epidermal stem cell regulation and differentiation.

Animals↗
PubMed1996

Dosimetry studies utilizing the urolase right-angle firing neodymium:YAG laser fiber in the human prostate.

BACKGROUND AND OBJECTIVE: Until recently, little or no objective data have been available to support either the choice of power setting or the timing of laser applications to achieve optimal tissue ablation in the human prostate. The objective of this study was to define quantitative dosimetry curves for the Urolase right angle laser fiber in human prostates. STUDY DESIGN MATERIALS AND METHODS: Transurethral Neodymium:YAG laser application was performed with the Urolase right-angle laser fiber in adult human prostates prior to planned radical surgery. Depth and volume of prostatic tissue coagulation for single, continuous laser applications were measured at variable power settings from 20 to 60 watts while holding total energy delivery constant. Then, holding the power setting constant at 40 watts, the extent of tissue coagulation was measured for variable treatment times from 60 to 120 seconds. RESULTS: Peak tissue coagulation was observed at 40 watts up to a maximum of 14 mm tissue penetration and 4.23 cc volume coagulated following a single spot laser application. The mean depth of tissue coagulation at 40 watts power setting was 13.5 mm, with a mean volume of tissue coagulation of 3.68 cc. The mean depth of tissue penetration at 40 watts was more than 25% greater than that observed at 60 watts, and the mean volume of tissue coagulation was 190% greater than that observed at 60 watts. As treatment time was increased from 60 to 90 seconds, extent of tissue coagulation increased significantly. However, beyond 90 seconds continuous laser application at 40 watts, a plateau in tissue effects was observed, with minimal increase in tissue coagulation between 90 and 120 seconds. Histologic examination of prostates removed acutely showed heat-induced damage to both stromal and glandular epithelial elements in laser-treated areas. At 1 year, the prostatic urethra was lined with a normal transitional epithelium, and mild periurethral fibrosis with focal squamous metaplasia was seen. CONCLUSION: Using the Urolase right-angle laser fiber, this study suggests that 40 watts power setting and 90 seconds continuous application time with a Neodymium:YAG laser source represent optimal treatment parameters to maximize prostatic tissue coagulation.

Adult↗
PubMed1994

1,25-Dihydroxyvitamin D3 potentiates the keratinocyte response to calcium.

Extracellular calcium (Cao) stimulates the differentiation of keratinocytes; 1,25 dihydroxyvitamin D3 1,25(OH)2D3) does likewise. Since 1,25(OH)2D3 regulates calcium flux in other cells, we hypothesized that 1,25(OH)2D)3 acted through and promoted the effects of calcium on keratinocyte differentiation. To test this hypothesis, we evaluated the effects of calcium and 1,25(OH)2D3 alone and in combination on the mRNA and protein levels of involucrin and transglutaminase in neonatal human keratinocytes. Cao alone increased these mRNA levels in a dose-dependent fashion (0.03 to 1.2 mM) over a 24-h period. This increase in mRNA levels was associated with a stimulation by calcium of involucrin and transglutaminase gene transcription. However, by 72 h, the mRNA levels of involucrin and transglutaminase decreased. At 0.03 mM Cao, 1,25(OH)2D3 showed a dose-dependent stimulation of involucrin and transglutaminase mRNA for up to 48 h and potentiated the initial (4-h) stimulation by Cao of involucrin and transglutaminase mRNA. As for calcium alone, this increase in mRNA was associated with an increase in transcription of the involucrin and transglutaminase genes. However, by 24 h of exposure to both calcium and 1,25(OH)2D3, a dose-dependent fall in mRNA levels was seen. The mRNA levels of involucrin and transglutaminase were stable for 24 h when neonatal human keratinocytes were grown in serum-free keratinocyte growth medium containing 0.03 or 1.2 mM Cao alone. However, the mRNAs of both genes underwent rapid degradation when neonatal human keratinocytes were treated with 1,25(OH)2D3, especially in high Cao. 1,25(OH)2D3 and Cao increased the protein levels of involucrin and transglutaminase activity in a synergistic fashion throughout the 48-h time course. These data support the hypothesis that 1,25(OH)2D3 promotes calcium-induced differentiation at the level of both gene expression and mRNA stability.

Calcitriol↗
PubMed1993

Growth regulation of cultured human nevus cells.

Cells isolated from congenital melanocytic nevi and cultured in vitro have growth characteristics that resemble their premalignant stage in situ. A serum-free, chemically defined medium has been developed that allows continuous growth of established nevus cultures for up to several months. Like primary melanoma cells, nevus cells in high-calcium-containing W489 medium require insulin for growth. In contrast to melanoma cells, nevus cells in serum-free medium require the presence of alpha-melanocyte-stimulating hormone, which enhanced intracellular levels of cyclic adenosine monophosphate. In contrast to the requirements of normal human melanocytes from newborn foreskin, congenital nevus cells grow with less dependency on basic fibroblast growth factor (bFGF). Nevus cultures contain bFGF-like activity, and they express bFGF mRNA. Nevic cells of compound nevi also express bFGF mRNA in situ but only in the junctional areas. These results indicate that bFGF plays an important growth regulatory role for nevus cells in vitro and in vivo.

Base Sequence↗
PubMed1991

Uncoupling of the calcium-sensing mechanism and differentiation in squamous carcinoma cell lines.

Increases in the intracellular calcium (Cai) levels, induced either by extracellular calcium or by calcium ionophores, stimulate the terminal differentiation of normal human keratinocytes in culture (NHK). Despite extensive differences in phenotypic expression, squamous carcinoma cell lines (SCC lines) display only partial terminal differentiation even in the presence of normal extracellular calcium. Therefore, in this study, we evaluated whether the inability of SCC lines to differentiate normally is due to a defect in achieving adequate levels of Cai. Membrane-bound transglutaminase activity and involucrin levels of the various SCC lines were lower than those of NHK and correlated with their low extent of cornified envelope formation. Ionomycin, a calcium ionophore, acutely increased cornified envelope formation of NHK 60- to 70-fold, but only initiated a 1- to 5-fold increase in SCC lines. Yet resting Cai levels in and the Cai response to various agents of SCC lines were similar or higher than those of NHK. Extracellular calcium evoked a rapid, transient and a slower, sustained increase of Cai. Extracellular ATP increased Cai by a rapid release from intracellular sources. Ionomycin, on the other hand, increased Cai from both intracellular compartments and extracellular sources. Thus, these studies indicate that the abnormalities in differentiation among SCC lines do not appear to involve their calcium-sensing mechanism. An uncoupling of the Cai changes to the synthesis of the precursor molecules required for differentiation may be responsible for the defect in differentiation displayed by these SCC lines.

Adenosine Triphosphate↗
PubMed1991

Limited tissue distribution of the intestinal brush border myosin I protein.

A myosinlike 105-110-kilodalton calmodulin-binding protein, brush border myosin I, found in the intestinal brush border has been linked to two seemingly disparate but possibly interacting functions of the brush border, namely, microvillar motility and vitamin D regulated calcium transport. If brush border myosin I were to function primarily as a myosinlike molecule powering cellular or microvillar motility, one might expect it to be found in a variety of tissues with microvilli such as the renal brush border and bile canaliculus. On the other hand, a more specialized function such as participation in vitamin D regulated calcium transport might dictate a more restricted tissue distribution for brush border myosin I. To determine the tissue distribution of brush border myosin I, we purified this protein to apparent homogeneity, generated antisera to it, and used the antisera to localize the protein within the intestinal epithelial cell by immunocytochemistry. We then screened a variety of other tissues (brain, lung, heart, liver, spleen, pancreas, kidney, and skeletal muscle) both for calmodulin-binding proteins as well as for brush border myosin I using Western blots and immunofluorescence. Our results indicate that the intestinal brush border myosin I is limited in its distribution to the intestinal brush border.

Animals↗
PubMed1990

Calcium regulation of growth and differentiation of normal human keratinocytes: modulation of differentiation competence by stages of growth and extracellular calcium.

In this study we examined the different aspects of the pathway leading to the differentiation of keratinocytes as a function of time in culture and calcium concentration of the culture medium. Human neonatal foreskin keratinocytes were grown in a serum-free, defined medium containing 0.07, 1.2, or 2.4 mM calcium and assayed for the rate of growth and protein synthesis, involucrin content, transglutaminase activity, and cornified envelope formation at preconfluent, confluent, and postconfluent stages of growth. We observed that keratinocytes grown to postconfluence in all calcium concentrations showed an increased protein/DNA ratio and an increased rate of membrane-associated protein synthesis. Extracellular calcium concentrations did not have a clear influence on these parameters. However, preconfluent and confluent keratinocytes grown in 0.07 mM calcium showed markedly retarded differentiation at all steps, i.e., involucrin synthesis, transglutaminase activity, and cornified envelope formation. Within 1 week after achieving confluence, these keratinocytes began synthesizing involucrin and transglutaminase and developed the ability to form cornified envelopes. Cells grown in 1.2 and 2.4 mM calcium synthesized involucrin and transglutaminase prior to confluence and were fully competent to form cornified envelopes by confluence. Thus external calcium-regulated keratinocyte differentiation is not an all or none phenomenon, but rather it is the rate at which keratinocytes differentiate that is controlled by calcium. We conclude that either or both higher extracellular calcium concentration and the achievement of cell-cell contacts lead to a coordinate increase of at least two precursors--involucrin content and transglutaminase activity--required for cornified envelope formation. We speculate that a critical level of cytosolic calcium, achieved by increased extracellular calcium or by achievement of intercellular communication established by cell-cell contact, may trigger mechanisms required for initiation of keratinocyte differentiation.

Calcium↗
PubMed1990

Expression and structure of human SPARC transcripts: SPARC mRNA is expressed by human cells involved in extracellular matrix production and some of these cells show an unusual expression pattern.

A mouse SPARC cDNA clone was used to elucidate the expression of SPARC mRNA in normal diploid human cells as well as in tumor cells. Among 40 cell lines examined, 19 showed expression. The mRNA transcribed by the majority of the expressors are 2.1 kb with a trace amount of 3 kb. However, three cell types, undifferentiated basal keratinocytes, their differentiated derivatives, and breast adenocarcinoma cells, showed an expression pattern distinct from the typical one, having abundant 3-kb mRNA but no detectable 2.1-kb mRNA. The mRNA was translated and the product secreted. This expression pattern was not observed before in human cells and was not found in tumor cells of keratinocytes, squamous carcinoma cells, or many other adenocarcinoma cells. We showed by Northern hybridization that the SPARC-expressing melanocytic melanoma cell lines produced laminin, a component of extracellular matrix. Other cell types expressing the SPARC mRNA were also reported to synthesize extracellular matrix components. Thus, our results indicate an association between SPARC gene expression and production of extracellular matrix. However, the opposite is not true since non-SPARC-producers may or may not produce extracellular matrix. For example, A431 cell line, which does not express SPARC mRNA, is known to produce extracellular matrix components while the normal diploid melanocytes and undifferentiated embryonal carcinoma cells, which do not express SPARC mRNA, do not produce extracellular matrix component.

Adenocarcinoma↗
PubMed1990

Phenotypes and interactions of human melanocytes and keratinocytes in an epidermal reconstruction model.

The morphologic and antigenic phenotype of normal human melanocytes and keratinocytes was investigated in monolayer and 3-dimensional cultures in an effort to develop an epidermal model that resembles the normal human epidermis. When cultured for several passages in optimal growth medium, pure cultures of either cell type could be established as demonstrated by light and electron microscopy and with monoclonal antibodies defining melanocyte- and keratinocyte-associated antigens. Three-dimensional growth of keratinocytes on polycarbonate filters was induced by increasing calcium concentrations in the culture medium and exposing cultures to air. After 30 to 35 days incubation, the 3-dimensional keratinocyte cultures reached a total of 12 to 25 layers and keratinocytes of various stages of differentiation formed three morphologically and antigenically different strata. The basal layer of these constructs consisted of ovoid cells with desmosomes and hemidesmosome-like structures. These cells expressed low molecular weight cytokeratins similar to basal cells in situ. The intermediate layer, representing the stratum spinosum in situ, contained flat cells with keratohyaline granules and many desmosomes. These cells expressed gp 80 kilodaltons, gp 40 to 50 kilodaltons, involucrin, and filaggrin. The upper layer, the stratum corneum equivalent, contained large, flattened cells with keratohyaline granules. The majority of these cells were anucleate. When melanocytes were cocultured with keratinocytes in monolayer or in epidermal reconstructs, they assumed a multidendritic morphology and donated pigment to surrounding keratinocytes. The majority of pigmented cells localized singly within the basal layer of the reconstructs and their dendrites were intimately associated with keratinocyte plasma membranes. Pigment donation to keratinocytes appeared to occur through the uptake of melanosome-containing dendrite fragments and phagocytosis of individual melanosomes by keratinocytes. It is hypothesized that keratinocytes produce unique microenvironmental factors that regulate the melanocytic phenotype.

Cell Communication↗
PubMed1990

Malignant melanoma simulants arising in congenital melanocytic nevi do not show experimental evidence for a malignant phenotype.

Proliferative neoplasms that resemble malignant melanoma may develop in large congenital melanocytic nevi, prenatally or in the neonatal period, although these lesions rarely show the progressive growth or behavioral characteristics of melanoma. This report describes the genetic, biologic, and immunologic characteristics of six tissue culture cell lines derived from two neoplasms present in congenital melanocytic nevi in two newborn infants. Both neoplasms had clinical and histologic features of malignant melanoma. Despite these features, cells from all lines were phenotypically benign, as evidenced by a normal karyotype, their expression pattern of pigment cell-associated antigens, absence of melanoma-associated ganglioside GD2, their mitogenic response to the tumor-promoting phorbol ester 12-0-tetradecanoyl phorbol-13-myristate, their inability to grow anchorage independently in soft agar, and prolonged but finite life span. The cells did not produce tumors in nude mice, but they remained viable at the injection site for over 7 months.

Antibodies, Monoclonal↗
PubMed1989

Transformation of normal human melanocytes and non-malignant nevus cells by adenovirus 12-SV40 hybrid virus.

Infection of normal human melanocyte and nevus cultures with an adenovirus 12-Simian Virus 40 hybrid virus (Ad12-SV40) produced transformed cells that expressed SV40-T antigen. The Ad12-SV40 cells exhibited rapid cell proliferation to high cell densities and efficient growth in soft agar, but none of 15 transformed melanocyte and nevus cultures formed tumors when injected s.c. or under the renal capsule into athymic nude mice. While the Ad12-SV40-transformed cells lost certain properties associated with the melanocytic phenotype, i.e., pigmentation, tyrosinase activity and melanosome content, the expression of melanoma-associated antigens, including nerve growth factor receptor, p97 melano-transferrin, and chondroitin sulfate proteoglycan, remained stable. The transformed melanocytes acquired the ability to express HLA-DR antigen, which is found on nevus and melanoma cells. Total ganglioside patterns in Ad12-SV40-transformed cells changed to reflect more advanced stages of tumor progression. Transformed melanocytes, like nevus and melanoma cells, showed increased GD3 content and transformed nevus cells increased GD2 which is a feature of malignant melanoma cells. Ad12-SV40-transformed human melanocytes and nevus cells are useful tools for studying tumor progression under experimental conditions.

Adenoviruses, Human↗
PubMed1989

Tumor progression in melanoma: the biology of epidermal melanocytes in vitro.

The propagation of pigmented cells derived from normal skin, common and precursor nevi, and primary and metastatic melanoma in tissue culture has allowed the study of tumor progression under experimental conditions. In accordance with Clark's hypothesis, which is based on histopathological observations, cells isolated from different stages of tumor progression show a stepwise development of a malignant phenotype as defined by different biological parameters: life span in culture, anchorage-independent growth, increased growth autonomy from exogenous growth factors, expression of melanoma-associated antigens, progressively severe chromosomal abnormalities, and tumorigenicity in nude mice. Qualitative and quantitative differences exist between normal melanocytes and nevus cells on the one hand, and between VGP primary and metastatic melanoma cells on the other. Little information, however, is available on cells from dysplastic nevi and RGP primary melanoma cells. Preliminary results suggest that the biologic, immunologic and genetic characterization of these cells from the intermediate stages of tumor development will significantly increase our understanding of the pathogenesis of melanoma.

Antibodies, Monoclonal↗
PubMed1988

Regulatory factors that determine growth and phenotype of normal human melanocytes.

Normal human melanocytes, unlike malignant melanoma cells, required at least three growth-promoting agents, i.e., phorbol ester for protein kinase C activation and the growth factors basic fibroblast growth factor (bFGF) and insulin, for growth in chemically defined W489 medium. Cell growth was further stimulated by addition of agents that increase intracellular levels of cyclic adenosine 3',5'-monophosphate (cAMP) to the medium. Among these agents, the pituitary hormones alpha-melanocyte-stimulating hormone (alpha-MSH) and follicle-stimulating hormone were the most potent, whereas bacterial toxins, including cholera, tetanus, and pertussis toxin and their subunits either were less mitogenic or gave variable results depending on the culture tested. Medium containing phorbol ester PMA, growth factors bFGF and insulin (or insulin-like growth factor-I), and synthetic alpha-MSH supported melanocyte growth for more than 5 months with doubling times between 5 and 8 days. Two copper-binding proteins, ceruloplasmin and tyrosinase, were mitogenic when added to medium and ceruloplasmic induced a long bi- to tripolar-shape of cells. Addition of 1 mM dibutyryl cAMP to the medium led to the formation of dendrites in all cells, with an average of 28 extensions per cell. Although cell growth was inhibited by dibutyryl cAMP, cells were not terminally differentiated and continued to proliferate. Dendritic melanocytes showed a 2.2-fold increase in activity of the tyrosine kinase pp60c-src. The induction of dendritic processes in melanocytes by dibutyryl cAMP or sodium butyrate was reversible and appears to reflect the expression of the mature melanocytic phenotype in situ.

Bucladesine↗
PubMed1988

Growth and phenotypic characteristics of human nevus cells in culture.

Nevus cells were isolated from the three cutaneous components, epidermis, basal layer, and dermis, of nonmalignant pigmented lesions and were cultured separately in the presence or absence of the phorbol ester 12-0-tetradecanoyl phorbol-13-acetate in medium that supports the rapid proliferation of melanocytic cells. The separation procedure used provided cultures that were essentially free from normal melanocytes (dermis) or fibroblasts (epidermis). In short term culture, nevus cells of all skin compartments expressed markers associated with differentiated melanocytes, such as presence of premelanosomes and melanosomes and elevated tyrosinase levels. Nevus cells also expressed melanoma-associated antigens, such as NGF-receptor, transferrin-related p97, proteoglycan, and HLA-DR as detected with monoclonal antibodies. After several subpassages, cells showed a decreased expression of melanoma-associated antigens, decreased tyrrosinase levels, and melanosomes could no longer be detected. Morphologically, these cells were similar to fibroblasts. The disappearance of melanoma-associated cell surface antigens was concomitant with the appearance of a melanocyte-associated 145 kd protein that might serve as a marker of fibroblast-like differentiation in nevus cells and normal melanocytes. Nevus cell cultures grown in the presence of 12-0-tetradecanoyl phorbol-13-acetate maintained a stable differentiated phenotype throughout their lifespan. As reported earlier, nevus cells in culture, irrespective of the presence or absence of 12-0-tetradecanoyl phorbol-13-acetate, have a finite lifespan in vitro, grow anchorage-independent in soft agar, but do not form tumors when xenografted to nude mice. These studies demonstrate that nevus cells isolated from the epidermal, basal layer, and dermal components of lesional skin can serve as models to characterize the initial steps of tumor progression in a human cell system.

Adolescent↗
PubMed1987

Biology of tumor progression in human melanocytes.

Tumor progression in the human melanocyte system can be delineated into 6 sequential stages. The first three steps represent nonmalignant melanocyte lesions from focal proliferations of structurally normal melanocytes to lesions with architectural and cytologic atypia. Primary melanoma may be divided into radial growth phase without competence for metastasis and vertical growth phase with metastatic competence. Melanocytes isolated from normal skin, nonmalignant pigmented lesions, and melanomas and maintained in culture have properties that are characteristic for each stage of tumor progression. Cytogenetic studies revealed nonrandom chromosomal abnormalities of advanced melanomas involving chromosomes 1, 6, and 7. Recent progress in tissue culture techniques has allowed studies of growth regulation of normal and malignant cells. Six growth factor receptor-growth factor systems seem to be of biologic significance in the melanocyte system: EGF, NGF, FGF, PDGF, insulin, and beta-TGF. Monoclonal antibodies have characterized a large number of antigens on melanocytes of the various stages of tumor progression, making melanoma one of the most widely studied human tumor systems.

Antigens, Neoplasm↗
PubMed1986

Ultrastructural observations in lichen nitidus.

Lichen nitidus (LN) and lichen planus (LP) are considered by some investigators to be two variants of the same disease, and by others to be two distinct dermatoses. In order to obtain further information about the relationship between LN and LP we examined the ultrastructure of lesions from two LN patients. In the central part of the lesion, the basement membrane was absent, or was interrupted by migrating phagocytes or lymphocytes. The basal cells and the lower cells of the stratum spinosum exhibited karyolysis and appeared to be compressed and often necrotic. In the upper dermis irregular cell debris full of clumps of tonofilaments and colloid-body-like structures was observed. A dense dermal infiltrate of macrophages, lymphocytes, fibrocytes, and Sezary-like cells was present. Signs of cooperation between lymphocytes and macrophages were also evident. The periphery of the lesion showed no pathological features except for enlargement of the intercellular spaces and the presence of mononuclear cells scattered through the epidermis. Several normal Langerhans cells were noticed. These ultrastructural findings were quite similar to those reported for LP.

Adult↗
PubMed1984

[Cosmetic acne and a test of comedogenicity].

A comedogenic test was carried out on the internal ear canal of four adult, masculine, albino rabbits, using butter of cacao and linseed oil, both known to be present in various cosmetic products. Histologic observation after 14 days showed follicular hyperkeratosis conferming the validity of this test.

Acne Vulgaris↗