Cecilia Malentacchi

Transforming growth factor-β isoform and receptor expression in chondrosarcoma of bone

Abstract. It has been shown that transforming growth factor-β (TGF-β) has a potent stimulatory effect on the growth of chondrosarcoma cells in vitro. In order to examine the production of this family of growth factors and their receptors in vivo, we studied the expression of TGF-β isoforms 1, 2, and 3 and of TGF-β receptor types I and II (TGF-βRI and TGF-βRII) in a series of 24 chondrosarcomas of bone using immunohistochemistry and reverse-transcription polymerase chain reaction analysis. For comparison, five enchondromas and five osteochondromas were also analyzed. TGF-β1 was expressed in 3 benign lesions (30%) and 18 chondrosarcomas (75%), with a significantly higher expression in grade-2 and -3 tumors than in grade-1 tumors (P=0.002). TGF-β2 was identified in 8 benign lesions (89%) and 21 chondrosarcomas (87.5%), with increased expression in grade-2 and -3 chondrosarcomas in comparison with grade-1 tumors (P=0.05). TGF-β3 was detected in 6 benign lesions (60%) and 17 chondrosarcomas (70.8%), with no significant differences between chondrosarcomas of different histologic grade (P=0.6). Twenty-three chondrosarcomas (95.8%) expressed both TGF-β receptor types I and II. Reverse-transcription polymerase chain reaction analysis performed on ten chondrosarcomas confirmed the presence of low or absent levels of TGF-β1 and -β2 mRNA in grade-1 chondrosarcomas, while grade-2 chondrosarcomas presented high levels of transcript of both cytokines. High levels of TGF-βRI and RII mRNA were also detected. Chondrosarcomas with TGF-β1 and TGF-β2 overexpression (>20% of tumor cells) had a significantly higher expression of the cell proliferation marker MIB-1 (P=0.006 and P=0.0003, respectively), while no significant correlation was found between TGF-β3 expression and proliferative activity (P=0.5). When TGF-β isoform and receptor expression were examined with respect to disease-free survival, TGF-β1 overexpression was significantly associated with a shorter disease-free survival (P=0.004, log-rank test). Our data indicate that TGF-β isoforms are produced by neoplastic cells of chondrosarcomas and could have a potential role as autocrine growth stimulators in these neoplasms.

Serous Cutaneous Glands in Phyllobates bicolor (Anura:Dendrobatidae): an Ontogenetic, Ultrastructural Study on Secretory Product Biosynthesis and Maturation

Abstract Secretory activity of serous (poison) cutaneous glands was studied with light and transmission electron microscopy over the ontogenetic cycle of the Black-legged Dart Frog Phyllobates bicolor. Serous gland activity includes two steps: biosynthesis and maturation. Biosynthesis is performed by the rough endoplasmic reticulum, smooth endoplasmic reticulum, and Golgi apparatus, and follows the usual pathways of exocrine secretory processes. Maturation is typical of anuran serous glands, which accumulate their products in syncytial secretory units. Maturation involves merging processes between secretory deposits (vesicles and/or granules) distally from the Golgi stacks, as well as an increase in the surface area between the syncytial cytoplasm and the secretory vesicles/granules. In P. bicolor, this interface area increases through the formation of peculiar cytoplasmic outgrowths. These outgrowths allow transport processes involved in maturational changes of secretory product. During maturational storage, the secretory products of P. bicolor acquire a repeating substructure of spherical to short tubular subunits. This recurrent pattern is seen in cutaneous poisons of several anuran species, regardless of their phylogenetic relationships. Among anurans, condensing serous cutaneous products into repeating aggregations of subunits appears to be a successful strategy, which allows intracytoplasmic accumulation of pharmacologically active and/or toxic molecules through various post-Golgian processes. The present findings provide evidence of a novel repertory of cytological activities that stress the large diversity of the maturational pathways leading to the peculiar repeating arrangement of secretory subunits.

Adaptive evolution of secretory cell lines in vertebrate skin

Abstract The embryonic horny layer of vertebrates contains distinctive cell lines that account for the remarkable, diverse secretory performances of their cutaneous apparatuses. Aquatic classes (jawless, cartilaginous and bony fishes) possess single gland cells, scattered among ordinary epidermal cells, that manufacture proteinaceous or mucous substances. This dichotomy is retained in adult amphibians, although the secretory cells in anamnionic tetrapode begin to be arranged in complex glands located in the loose dermis. In the Amniota, intensive keratinisation involving the epidermis results in the loss of the ancestral secretory lines, accompanied by the onset of a novel, lipid- producing gland type, which in mammals is flanked by the exclusive sweat gland apparatus. In this concise up-to-date review, we attempt to phylogenetically narrow the large variety of secretory structures in vertebrate skin into a few basic schemes, in the light of the close relationships between environmental challenges and ecological, ethological as well as physiological roles of the cutaneous apparatus.

Sequence diversity within the HA-1 gene as detected by melting temperature assay without oligonucleotide probes

BackgroundThe minor histocompatibility antigens (mHags) are self-peptides derived from common cellular proteins and presented by MHC class I and II molecules. Disparities in mHags are a potential risk for the development of graft-versus-host disease (GvHD) in the recipients of bone marrow from HLA-identical donors. Two alleles have been identified in the mHag HA-1. The correlation between mismatches of the mHag HA-1 and GvHD has been suggested and methods to facilitate large-scale testing were afterwards developed.MethodsWe used sequence specific primer (SSP) PCR and direct sequencing to detect HA-1 gene polymorphisms in a sample of 131 unrelated Italian subjects. We then set up a novel melting temperature (Tm) assay that may help identification of HA-1 alleles without oligonucleotide probes.ResultsWe report the frequencies of HA-1 alleles in the Italian population and the presence of an intronic 5 base-pair deletion associated with the immunogeneic allele HA-1H. We also detected novel variable sites with respect to the consensus sequence of HA-1 locus. Even though recombination/gene conversion events are documented, there is considerable linkage disequilibrium in the data. The gametic associations between HA-1R/H alleles and the intronic 5-bp ins/del polymorphism prompted us to try the Tm analysis with SYBR® Green I. We show that the addition of dimethylsulfoxide (DMSO) during the assay yields distinct patterns when amplicons from HA-1H homozygotes, HA-1R homozygotes, and heterozygotes are analysed.ConclusionThe possibility to use SYBR® Green I to detect Tm differences between allelic variants is attractive but requires great caution. We succeeded in allele discrimination of the HA-1 locus using a relatively short (101 bp) amplicon, only in the presence of DMSO. We believe that, at least in certain assets, Tm assays may benefit by the addition of DMSO or other agents affecting DNA strand conformation and stability.

Chemical skin defence in the Eastern fire-bellied toadBombina orientalis : an ultrastructural approach to the mechanism of poison gland rehabilitation after discharge

Type I serous glands in the skin of the Eastern yellow-bellied toad Bombina orientalis released their product massively after 10 -3 M nor-adrenalin (NA) stimulation, mimicking orthosympathetic control on poison emission in chemical skin defence. Features of cutaneous glands involved in this bulk discharge were observed under light and electron microscopes. Furthermore, restoration of depleted glands was followed after 1, 2 and 3 weeks, and compared with serous biosynthesis during larval gland development. Bulk discharge was caused by contraction of myoepithelial cells (mecs) encircling the secretory units. Mec compression dramatically affected the secretory unit, but parts of this syncytial cytoplasm were saved from degeneration and cooperated in gland renewal with stem cells from the gland neck. These adenoblasts underwent proliferation and secretory cytodifferentiation, until merging with the syncytium. Cytoplasm that had resumed secretory activity showed the features typical of larval gland development: the endoplasmic reticulum (rer) cisterns were aligned in close parallel arrangement and Golgi stacks released minute type I granules. Secretory rehabilitation led to increasing amounts of granule content. In the meantime, rough cisterns decreased in number and assumed the less ordered pattern described in control specimens. Data collected in the present study revealed that chemical skin defence in anurans is a multi-factorial mechanism involving specific activities: mechanical from mecs, biosynthetic from secretory syncytium and proliferative from intercalated stem cells.

Ultrastructural study on host-guest relationships between an-uran serous cutaneous glands and nematodes

Abstract We described guest nematodes in serous and lipid skin glands in two anuran species from the Old and New Worlds: Rana camerani and Phyllomedusa hypochondrialis, respectively. These glands are involved in three types of response: a) invaginations from the periluminal plasma membrane which accommodate the “round worms”; b) secretory release into such hosting chambers; c) migration of macrophages from the periglandular stroma to the secretory unit. There are no obvious signs suggesting the glands are harmed by the nematodes, nor are the latter affected by defence reactions, either secretory or cell-mediated, of their hosts. Nematodes are natural tracers which allow analysis of the inner space system (lumina as well as interstitia) in the glands. Furthermore, they provide the opportunity for functional characterisation of anuran serous glands under intrusion stress, revealing peculiar reactive traits, namely a pliable periluminal plasma membrane and inducible processes of secretory release.

Ultrastructural Evidence of Serous Gland Polymorphism in the Skin of the Tungara Frog Engystomops pustulosus (Anura Leptodactylidae)

Three types of serous products were detected in the syncytial cutaneous glands of the leptodactylid tungara frog, Engystomops pustulosus: type Ia, granules with wide halos and variable density cores; type Ib, high density granules without halos; and type II, vesicles containing a finely dispersed product. Ultrastructural evidence revealed that these products were manufactured by different serous gland types and excluded that they represented different steps in the secretory cycle of a single gland type. Indeed, secretory maturation affecting the products released by the Golgi apparatus proceeded through different mechanisms: confluence (vesicles), interactions between syncytium and secretory product (type Ib granules), and a combination of both processes (type Ia granules). In conclusion, this investigation of secretory maturation was shown to be a suitable approach for the identification of serous gland polymorphism and demonstrated that the tungara frog belongs to the minority of anuran species characterized by this peculiar morpho‐functional trait. Anat Rec, 298:1659–1667, 2015.

Larval Epidermis of the Red Eye Tree Frog Agalychnis callidryas (Anura, Hylidae): Ultrastructural Investigation on the Kugelzellen, Specialized Forms of the Constitutive Skein Cell Line

An ultrastructural study was carried out on the epidermis of Agalychnis callidryas tadpoles during limb development. Larval epidermis consisted of four cell layers: basal, lower intermediate, upper intermediate, and surface or apical layers. Basal cells represented the stem compartment of intermediate cells: both belong to the skein cell (SC) lineage, described in several anuran species, on account of the conspicuous intracytoplasmic tonofilament bundles. Apical cells were secretory in nature and released mucus on the body surface. Intermediate SCs exhibited a hydrated central cytoplasm and peripheral tonofilament bundles. They closely resembled the epidermal ball‐like cells, Kugelzellen (KZn) of Xenopus laevis tadpoles, and possibly shared their turgor‐stiffness properties. In A. callidryas, the stratification of intermediated SCs on their stem cell layer provided the chance to study their cytodifferentiation in a suitable sequence, until basal cell differentiation shifted toward the keratinocyte lineage in premetamorphic stages. Present data assign A. callidryas to the anuran species with a constitutive SC population in larval epidermis, and demonstrate that KZn express the ultimate specialization of such cell line. SCs were arranged in the fashion of a random‐rubble stone groundwork, and possessed long processes. These cytoplasmic outgrowths contained a tonofilament axial rod and held together contiguous cells. Ultrastructural findings suggest that this complex structure may impart compressive as well as sliding strengths to the larval epidermis, representing a possible adaption to the fresh water environment. Anat Rec, 2011.