Cell kinetics of melanocytes in common and dysplastic nevi and in primary and metastatic cutaneous melanoma.

The 3H-thymidine labelling (LI) and mitotic (MI) indexes were calculated in 29 cutaneous melanocytic lesions: 6 common nevi (CN), 11 dysplastic nevi, subclassified as nevi with architectural atypia (NAA = 4) and nevi with cyto-architectural atypia (NCAA = 7), 2 melanomas in situ (MIS), 4 invasive superficial spreading melanomas (IM) and 6 metastatic melanomas (MM). The LI mean values resulted to be: CN = 0.23%, NAA = 0.98%, NCAA = 1.79%, MIS = 5.75%, IM = 5.16%, MM = 3.80%. In CN, NAA, NCAA and MIS, these values were calculated at epidermal level; in IM and MM at dermal level. At dermal level, the LI mean values of CN, NAA and NCAA were: 0.20%, 0.20%, 0.23% respectively. The MI mean value was close to 0 in CN, NAA, NCAA, MIS; 0.18% in IM, 0.16% in MM. Confirming a low proliferative activity in CN and a high activity in melanomas (MIS, IM, MM), the results showed that dysplastic nevi (NAA, NCAA) had a proliferative activity intermediate between common nevi and melanomas. The lesions with melanocytic atypia (NCAA) resulted to have a higher proliferative activity than those without this histological feature (NAA).     

Expression of the proteoglycans versican and mel-CSPG in dysplastic nevi

Nevi with architectural disorder and cytologic atypia of melanocytes (NAD) (also called dysplastic nevi) have been controversial with regard to their relationship with melanoma risk and to their gradation in 3 degrees of atypia. Versican and the melanoma-associated proteoglycan (mel-CSPG) are 2 major proteoglycans expressed by malignant melanoma, and they have a role in the regulation of cell adhesion, migration, and differentiation. We evaluated the differences in versican and mel-CSPG expression in nevi, NAD with several degrees of atypia, and primary malignant melanoma. Immunoreactivity for versican was negative in benign melanocytic nevi, positive in NAD (ranging from weakly to intensely positive), and intensely positive in malignant melanoma. Immunostaining for mel-CSPG was negative in benign melanocytic nevi and mild to moderately positive in NAD and melanoma. Our results suggest that versican expression may be of value for distinguishing NAD from benign melanocytic nevi and for distinguishing severe NAD from mild and moderate NAD.     

TIA-1 positive tumor-infiltrating lymphocytes in nevi and melanomas.

Tumor-infiltrating lymphocytes (TIL) have been shown to be an independent prognostic factor in melanomas. To better characterize the host immune response, we have classified TIL by their immunoreactivity against lymphoid markers in formalin-fixed, paraffin-embedded tissue. Monoclonal antibodies to leukocyte common antigen (LCA) and TIA-1 (a granule-associated protein of cytotoxic T cells and NK cells) were used to immunostain a series of benign nevi, nontumorigenic radial growth phase, and tumorigenic vertical growth phase melanomas and metastases. Among nine nevi, few LCA+ TIL were found, among which rare cells were positive for TIA-1 (mean, 2.0). Five nontumorigenic radial growth phase melanomas also had few total TIL and rare TIA-1+ TIL (mean, 3.4); the nontumorigenic radial growth phase component of seven tumorigenic vertical growth phase melanomas had higher numbers of TIA-1+ TIL (mean, 11). Twelve cases of tumorigenic vertical growth phase melanoma showed a variable but significantly greater number of both LCA+ TIL and TIA-1+ TIL (mean, 30.6). Nine cases of metastatic melanoma had a wide range of variation in LCA as well as in TIA-1+ TIL (mean, 46). Although the mean total number of TIA-1+ TIL increased from nontumorigenic radial growth phase to tumorigenic vertical growth phase to metastases, TIA-1+ as a percentage of TIL declined across these categories of tumor progression (42%, 31%, and 26%, respectively). Our results show that these attributes of TIA-1+ TIL, both increasing total number but decreasing percentage, appear to be a marker of tumor progression of malignant melanomas. In addition, there was significant variability in the number of TIA-1+ TIL among advanced melanomas, raising the possibility that an assessment of TIA-1+ TIL may prove a useful prognostic tool for the evaluation of primary melanomas.     

Progression-related expression of beta3 integrin in melanomas and nevi.

The expression of the beta3 integrin subunit was investigated in 130 fixed, paraffin-embedded specimens of human melanomas and nevi using two different monoclonal antibodies. Expression was not observed in melanocytes and was absent or low in most nevi. In primary melanomas, expression was absent or low in the nontumorigenic radial growth phase, which includes the classes of in situ and microinvasive melanomas. In contrast, expression was high in the tumorigenic or vertical growth phase compartment of many primary melanomas and in most metastatic melanomas. Expression patterns were similar with the two antibodies, SSA6 and SAP, and was membrane-related as well as cytoplasmically expressed. In those nevi that reacted focally, the reactivity tended to occur in the dermal component of neurotized nevi, and in Spitz nevi, where the reactivity was stronger and more diffuse. A few dysplastic nevi showed focal reactivity of the junctional component. These results are consistent with tumor progression-related expression of the beta3 integrin, which is expressed in melanocytic tumors as the alphavbeta3 integrin, having affinity for matrix molecules, including vitronectin and fibronectin. In all melanomas, and in the subset of tumorigenic vertical growth phase melanomas, expression increased with thickness (P < .01). For this reason, and because ligation of this integrin has been shown in vitro to have several properties that may be related to the malignant phenotype, it is likely that expression of this marker may have prognostic value. However, because of its consistent and strong expression in Spitz nevi, the diagnostic utility of this marker will likely be limited.     

S100A6 protein expression is different in Spitz nevi and melanomas.

The Spitz nevus is a benign melanocytic lesion that can be identified reliably in many cases by conventional histopathological criteria. However, there are subsets of Spitz nevi and of malignant melanoma that closely resemble each other and represent diagnostic challenges. S100 proteins are of interest because of their involvement in neoplastic processes and their genes are clustered in chromosome 1q21. Chromosome 1 contains mutations in several types of tumors, including melanomas. The expression of different S100 proteins (A2, A6 and A8/A9 or A12) was examined in 42 Spitz nevi, 105 melanomas, and 73 melanocytic nevi to test the hypothesis that their expression differs among these entities and may contribute to the distinction between these entities. The results showed an up-regulation of S100A6 protein in Spitz nevi, melanomas, and melanocytic nevi but with a different percentage of positivity and pattern of immunoreactivity. The differences between these three entities were statistically significant (P <.001). All 42 Spitz nevi (100%) showed strong and diffuse S100A6 protein expression, both in junctional and in dermal components of the nevi. Thirty-three percent of melanomas expressed S100A6 (35/105). The expression was mainly weak (30/35) and patchy in the dermal component and was negative or minimal in the junctional component. Fifty-six percent of different subtypes of melanocytic nevi (41/73) expressed S100A6, almost all of them weakly (40/41) and in the dermal component. Normal intraepidermal melanocytes were negative. The melanocytic cells in these three entities did not express S100A2, S100A8/A9 or A12. However, an up-regulation of S100A2 and S100A8/A9 or A12 proteins was observed in normal keratinocytes in the epidermis overlying Spitz nevi and melanomas, without differences. In summary, a simple immunohistochemical test for S100A6 protein differentiated between Spitz nevi, melanomas, and melanocytic nevi. This marker could be used when the distinction is very difficult or controversial in routine studies, especially when there is a junctional component. Further molecular analyses of the S100A6 protein and gene should be performed to study the underlying genetic bases for such differences.     

Mesothelial cells can detach from the mesentery and differentiate into macrophage-like cells

Peritoneal cell suspension is composed of heterogeneous cell population. Macrophages are the most numerous cells among them. They can originate from different sources and can be resident, exudate and elicited. When we used Freund's adjuvant to elicit peritoneal macrophages, cells having large amount of caveolae on their plasma membrane appeared in the peritoneal wash. The number of these caveolae-rich cells increased by the time of the Freund's adjuvant treatment. Although their morphology was different form from the common macrophages, they were labelled with pan-macrophage antibodies. As the origin of these cells is unknown in this work, we tried to find out where they can originate from. Our interest turned towards the mesothelial cells. We found that the adjuvant treatment resulted in significant morphological changes in these cells and stimulate them to leave the surface of the mesentery. By the time of the adjuvant treatment, the macrophage markers expression increased in the mesothelial cells and more cells were found to detach from the mesentery. These results strongly suggest that under special stimuli mesothelial cells can leave the mesentery and differentiate into phagocytotic (macrophage-like) cells. These data raises the idea that mesothelial cells might not entirely differentiated and represent a multipotential cell lineage. To study whether this is the case we used anti-nestin antibody, which is a specific marker for multifunctional, multi-lineage progenitor cells. Mesothelial cells showed strong labelling with this antibody indicating that these cells really represent a 'young', not entirely differentiated cell population.     

In vivo assessment of melanocytic nests in nevi and melanomas by reflectance confocal microscopy.

In vivo reflectance confocal microscopy is a novel technique for the noninvasive study and diagnosis of the skin. The aim of this study was to describe and characterize the cytological and architectural aspects of cell clusters in melanocytic lesions observed by confocal microscopy, and to correlate them with routine histopathology. A total of 55 melanocytic lesions comprising 20 melanomas, 25 acquired nevi and 10 Spitz nevi were studied by means of reflectance confocal microscopy, dermoscopy and routine histopathology. Three different types of cell clusters at confocal microscopy observation (dense, sparse cell and cerebriform clusters) were identified and correlated with histopathology. Dense clusters appeared characteristic for benign lesions, although present in 13 out of 20 melanomas. Sparse cell clusters were more frequently observable in melanomas, but also sporadically present in one Spitz nevus. Moreover, cerebriform clusters were exclusively observed in five out of 20 melanomas. Confocal microscopy allowed the in vivo characterization of aspects of melanocytic nests and their exact correlation with histopathology.     

Genetic changes in neoplasms arising in congenital melanocytic nevi: differences between nodular proliferations and melanomas

Large congenital melanocytic nevi (CMN) are at an increased risk of developing melanoma. Several forms of secondary proliferations can arise in congenital nevi on rare occasions. Although some of these closely resemble melanoma both clinically and histologically, metastasis is rare. We used comparative genomic hybridization to analyze chromosomal aberrations in different types of proliferations arising in CMN and compared them to typical congenital nevi, clear-cut melanomas arising in congenital nevi, as well as primary cutaneous melanomas that were not associated with a CMN. Cases of CMN and CMN with secondary proliferations were assigned to six groups according to the predominant histological pattern: group I, bland congenital nevi (n = 6); group II, congenital nevi with foci of increased cellularity (n = 4); group III, CMN with a proliferation simulating superficial spreading melanoma in situ (n = 3); group IV, CMN with a proliferation simulating nodular melanoma (n = 9); group V, proliferating neurocristic hamartoma (n = 1); and group VI, melanoma arising in congenital nevus (n = 6). No aberrations were found in groups I to III, whereas seven of nine cases of group IV, and one of one case of group V, showed aberrations. In group IV six of seven cases with aberrations (86%) showed numerical aberrations of whole chromosomes exclusively. This pattern differed significantly from the findings in melanoma that arose within CMN (n = 6), group VI, or independent of CMN (n = 122) in which only 5% showed numerical changes only. The single case in group V showed aberrations similar to melanoma. The finding of frequent numerical chromosomal aberrations in atypical nodular proliferations arising in CMN identifies these as clonal neoplasms with a genomic instability consistent with a mitotic spindle checkpoint defect. This difference compared to the aberration pattern found in melanoma might explain their more benign clinical behavior and may be of diagnostic value in ambiguous cases.     

A comparative study of proliferation indices and ploidy in dysplastic naevi and malignant melanomas using flow cytometry

Cell proliferation indices and DNA content have been determined in 18 intradermal naevi, 40 dysplastic naevi and 16 superficial malignant melanomas (less than 0.76 mm depth of invasion) using flow cytometry. In this study, proliferation indices of intradermal naevi and dysplastic naevi were not significantly different from each other. Abnormalities of DNA ploidy were not identified in the intradermal naevi or dysplastic naevi; whereas three of the malignant melanomas were aneuploid. In addition, cellular proliferation was increased within the group of malignant melanomas, in comparison with the naevi. This study has found no evidence to indicate that sporadic dysplastic naevi were more likely than intradermal naevi to transform to malignant melanoma, when objective criteria were employed. However, dysplastic naevi could be distinguished from some early malignant melanomas by absence of aneuploidy and by low cell proliferation indices.     

Correlation of histologic architectural and cytoplasmic features with nuclear atypia in atypical (dysplastic) nevomelanocytic nevi

The dysplastic nevus in nonfamilial melanoma is a clinicopathologic entity consistently demonstrating an eightfold or greater association with malignant melanoma. The present report quantifies the relationship between nuclear atypia and 16 architectural and cytoplasmic features in 153 pigmented nevi removed from a similar number of patients with newly diagnosed nonfamilial melanoma. All lesions were evaluated by one dermatopathologist, and most lesions were reviewed by a second dermatopathologist. Nuclear atypia of nevomelanocytes was defined as at least three of the following: nuclear enlargement, pleomorphism, hyperchromatism, and prominent nucleoli easily observed throughout each lesion. Seventeen percent of the total nevi had such atypia. On univariate analysis, 11 parameters (lentiginous hyperplasia of the epidermis, basal melanocytic hyperplasia, junctional nest disarray, fusion [bridging] of theques, suprabasal melanocytes, lymphoid response, prominent vascularity, fibroplasia, abundant cytoplasm, "dusty" cytoplasm, and large melanin granules) showed an association with nuclear atypia (P less than .05). However, on multivariate analysis only five parameters continued to be important: basal melanocytic hyperplasia, junctional nest disarray, melanophages (inverse correlation), prominent vascularity, and large melanin granules. These data support the idea that multiple histopathologic characteristics, correlating objectively with nuclear atypia, are important for the diagnosis of dysplastic nevi. In our view, the minimal essential histologic criteria for dysplastic nevi based on these findings include nuclear atypia and abnormal patterns of intraepidermal nevomelanocytic proliferation (ie, basal melanocytic hyperplasia and/or junctional nest disarray).     

Subcutaneous adipocytes can differentiate into bone-forming cells in vitro and in vivo

Interconversion of bone marrow osteoblasts and adipocytes has been reported previously. However, the osteogenic potential of extramedullary adipocytes is not known. Thus, we incubated a pure culture of human subcutaneous adipocytes in control medium for 1-2 weeks. Afterward, the cells were incubated in either osteoblast medium (OB medium) containing various combinations of calcitriol, dexamethasone, ascorbic acid, and beta-glycerophosphate or in adipocyte medium (AD medium) containing HEPES, biotin, pantothenate, insulin, triiodothyronine, dexamethasone, and isobutylmethylxanthine for 4 weeks. Expression of osteoblastic and adipocytic phenotypes was examined by determination of lineage-specific mRNA markers and in vitro adipocyte and osteoblast formation. Cells were also implanted, mixed with hydroxyapatite-tricalcium phosphate powder, in the subcutaneous tissue of immunodeficient mice in order to assess in vivo bone formation potential. One week after incubation in control medium, cells formed fusiform elongated fibroblast-like cells. In OB medium, cells stained positive for alkaline phosphatase (AP) and expressed mRNAs encoding Cbfa1/Runx2, AP, and osteocalcin. In AD medium cells reacquired adipocyte morphology with multilocular lipid-filled cells. Also, the cells expressed adipocyte-specific mRNA markers: lipoprotein lipase and peroxisome proliferator-activated receptor gamma2. Bone was formed only in the in vivo implants of cells incubated in OB medium. In conclusion, extramedullary adipocytes can transdifferentiate to bone-forming cells. Because of their ease of isolation, adipocytes may be good candidates for tissue-engineering protocols aimed at creating bone tissue for the repair of nonunion fractures and large bone defects.     

Calcium and magnesium binding to thin and thick filaments in skinned muscle fibres: electron probe analysis

Summary Electron probe analysis of ultrathin cryosections with high spatial resolution was used to determinein situ the concentrations of Ca²⁺ and Mg²⁺ bound in the absence of ATP to myofilaments in the I and A-bands of skinned frog skeletal muscle. At 2.2×10^–11 m Ca²⁺ and 2.7×10^–9 m Mg²⁺, the inexchangeably bound Mg²⁺ in the I-band was equivalent to the amount of divalent cations known to be inexchangeably bound to F-actin, while the Ca²⁺ bound to the I-band was not significantly above zero. The bound Mg²⁺ in the I-band was not exchangeable with Ca²⁺ even when the skinned fibres were exposed to 10mm Ca²⁺ solution. These results clearly indicate that Mg²⁺, rather than Ca²⁺, is the divalent cation bound to F-actin in the thin filamentsin situ. In the presence of 1mm Mg²⁺, the exchangeable Ca²⁺ bound to the I-band was increased as a function of the free Ca²⁺, while that in the A-band was not significantly changed with [Ca²⁺] up to 2 × 10^–5 m, and increased to approximately 0.8 mol Ca²⁺ per mol myosin at 10^–4 m Ca²⁺. At a saturating free Ca²⁺ in Tris-Cl solution, the bound Ca²⁺ content (2–3 mol Ca²⁺ per mol troponin) of the nonoverlapping I-band was unexpectedly low; the replacement of Tris with Na⁺ enhanced Ca²⁺ binding to the level equivalent to 3–4 mol Ca²⁺ per mol troponin. The depressant effect of Tris on Ca²⁺ binding was greater in the absence of Mg²⁺. High concentrations of Tris also reduced the maximum tension induced by 10^–4 m Ca²⁺ buffered with 10mm EGTA. At 1.3×10^–7 m Ca²⁺, thought to be close to the cytoplasmic free Ca²⁺ in resting muscle, the I-band bound a significant amount of Ca²⁺: equivalent to about 1 mol Ca²⁺ per mol troponin. In rabbit myofibrils there was a significant amount (approximately 1.5 mol/mol myosin) of Ca²⁺ bound by the A-band at a free Ca²⁺ of 10^–4 m.     

Cell cycle perturbations induced by human herpesvirus 6 infection and their effect on virus replication

In this study, we demonstrate that infection of HSB-2 cells with human herpesvirus 6 (HHV-6) resulted in the accumulation of infected cells in the G2/M phase of the cell cycle. Analysis of various cell-cycle-regulatory proteins indicated that the levels of cyclins A2, B1, and E1 were increased in HHV-6-infected cells, but there was no difference in cyclin D1 levels between mock-infected and HHV-6-infected cells. Our data also showed that inducing G2/M phase arrest in cells infected by HHV-6 provided favorable conditions for viral replication.     

Cell cycle components and their potential impact on the development of continuous in vitro penaeid cell replication

In vitro prawn cell culture has yet to produce an established cell line. In an effort to establish some understanding of the cellular blockage that prohibits their division in vitro we conducted several studies to characterize the cytoskeletal components of hemocytes and found no cells undergoing mitosis. Following this discovery, a molecular analysis of cell division regulatory proteins was performed. Cell cycle regulatory proteins (cyclins) have been identified as essential components in the progression of all eukaryotic cells through the cell cycle. We report here the identification of cyclin A and cyclin B proteins and their cofactor (p34^cdc2) in making up the mitosis promoting factor (MPF) in protein extracts from egg and muscle tissues of Penaeus vannamei. Molecular weight analysis confirmed the size of the target proteins to be similar to the same proteins identified in the Atlantic surf clam (Spisula solidissima).