Morphofunctional Investigation in a Transgenic Mouse Model of Alzheimer's Disease: Non-Reactive Astrocytes Are Involved in Aß Load and Reactive Astrocytes in Plaque Build-Up.

The term neuroinflammation defines the reactions of astrocytes and microglia to alterations in homeostasis in the diseased central nervous system (CNS), the exacerbation of which contributes to the neurodegenerative effects of Alzheimer's disease (AD). Local environmental conditions, such as the presence of proinflammatory molecules, mechanical properties of the extracellular matrix (ECM), and local cell-cell interactions, are determinants of glial cell phenotypes. In AD, the load of the cytotoxic/proinflammatory amyloid ß (Aß) peptide is a microenvironmental component increasingly growing in the CNS, imposing time-evolving challenges on resident cells. This study aimed to investigate the temporal and spatial variations of the effects produced by this process on astrocytes and microglia, either directly or by interfering in their interactions. Ex vivo confocal analyses of hippocampal sections from the mouse model TgCRND8 at different ages have shown that overproduction of Aß peptide induced early and time-persistent disassembly of functional astroglial syncytium and promoted a senile phenotype of reactive microglia, hindering Aß clearance. In the late stages of the disease, these patterns were altered in the presence of Aß-plaques, surrounded by typically reactive astrocytes and microglia. Morphofunctional characterization of peri-plaque gliosis revealed a direct contribution of astrocytes in plaque buildup that might result in shielding Aß-peptide cytotoxicity and, as a side effect, in exacerbating neuroinflammation.

Correlation between Sialylation Status and Cell Susceptibility to Amyloid Toxicity.

The interaction between the cell membrane and misfolded protein species plays a crucial role in the development of neurodegeneration. This study was designed to clarify the relationship between plasma membrane composition in terms of the differently linked sialic acid (Sia) content and cell susceptibility to toxic and misfolded Aß-42 peptides. The sialylation status in different cell lines was investigated by lectin histochemistry and confocal immunofluorescence and then correlated with the different propensities to bind amyloid fibrils and with the relative cell susceptibility to amyloid damage. This study reveals that expressions of Sias α2,3 and α2,6 linked to galactose/N-acetyl-galactosamine, and PolySia are positively correlated with Aß-42-induced cell toxicity. PolySia shows an early strong interaction with amyloid fibrils, favoring their binding to GM1 ganglioside containing α2,3 galactose-linked Sia and a loss of cell viability. Our findings demonstrate that cell lines with a prevailing plastic neuron-like phenotype and high monoSia and PolySia contents are highly susceptible to amyloid Aß-42 toxicity. This toxicity may involve a change in neuron metabolism and promote a compensative/protective increase in PolySia, which, in turn, could favor amyloid binding to GM1, thus exacerbating cell dysmetabolism and further amyloid aggregation.

Neuroinflammation: Integrated Nervous Tissue Response through Intercellular Interactions at the "Whole System" Scale.

Different cell populations in the nervous tissue establish numerous, heterotypic interactions and perform specific, frequently intersecting activities devoted to the maintenance of homeostasis. Microglia and astrocytes, respectively the immune and the "housekeeper" cells of nervous tissue, play a key role in neurodegenerative diseases. Alterations of tissue homeostasis trigger neuroinflammation, a collective dynamic response of glial cells. Reactive astrocytes and microglia express various functional phenotypes, ranging from anti-inflammatory to pro-inflammatory. Chronic neuroinflammation is characterized by a gradual shift of astroglial and microglial phenotypes from anti-inflammatory to pro-inflammatory, switching their activities from cytoprotective to cytotoxic. In this scenario, the different cell populations reciprocally modulate their phenotypes through intense, reverberating signaling. Current evidence suggests that heterotypic interactions are links in an intricate network of mutual influences and interdependencies connecting all cell types in the nervous system. In this view, activation, modulation, as well as outcomes of neuroinflammation, should be ascribed to the nervous tissue as a whole. While the need remains of identifying further links in this network, a step back to rethink our view of neuroinflammation in the light of the "whole system" scale, could help us to understand some of its most controversial and puzzling features.

Multiplex gene expression profile in inflamed mucosa of patients with Crohn's disease ileal localization: A pilot study.

BACKGROUND: Crohn's disease (CD) is a complex disorder resulting from the interaction of genetic, environmental, and microbial factors. The pathogenic process may potentially affect any segment of the gastrointestinal tract, but a selective location in the terminal ileum was reported in 50% of patients. AIM: To characterize clinical sub-phenotypes (colonic and/or ileal) within the same disease, in order to identify new therapeutic targets. METHODS: 14 consecutive patients undergoing surgery for ileal CD were recruited for this study. Peripheral blood samples from each patient were collected and the main polymorphisms of the gene Card15/Nod2 (R702W, G908R, and 1007fs) were analyzed in each sample. In addition, tissue samples were taken from both the tract affected by CD and from the apparently healthy and disease-free margins (internal controls). We used a multiplex gene assay in specimens obtained from patients with ileal localization of CD to evaluate the simultaneous expression of 24 genes involved in the pathogenesis of the disease. We also processed surgery gut samples with routine light microscopy (LM) and transmission electron microscopy (TEM) techniques to evaluate their structural and ultrastructural features. RESULTS: We found a significant increase of Th17 (IL17A and IL17F, IL 23R and CCR6) and Th1 (IFN-γ) gene expression in inflamed mucosa compared to non-inflamed sites of 14 CD patients. DEFB4 and HAMP, two genes coding for antimicrobial peptides, were also strongly activated in inflamed ileal mucosa, suggesting the overwhelming stimulation of epithelial cells by commensal microbiota. IFN-γ and CCR6 were more expressed in inflamed mucosa of CD patients with ileal localization compared with patients with colonic localization suggesting a more aggressive inflammation process in this site. Morphological analysis of the epithelial lining of Lieberkün crypts disclosed enhanced release activity from goblet mucocytes, whereas the lamina propria contained numerous cells pertaining to various lines. CONCLUSION: We observed that the expression of ileal genes related to Th1 and Th17 activity is strongly activated as well as the expression of genes involved in microbiota regulation.

Skin of the Red Eye Tree Frog Agalychnis Callidryas (Hylidae, Phyllomedusinae) Contains Lipid Glands of the Type Described in the Genus Phyllomedusa.

Several anuran species of the genus Phyllomedusa are known to possess specialized cutaneous glands producing lipids and exhibit a peculiar wiping behavior. This behavior is a stereotyped repertory of fore and hind limb movements distributing hydrophobic molecules onto the body surface and reducing evaporative water loss. No reports are presently available on the occurrence of lipid glands in other phyllomedusine genera, and data on the structure of the secretory units specialized for the production of cutaneous lipids are still unclear. The present report is aimed to answer both questions: it describes lipid glands of the Phyllomedusa type in Agalychnis callidryas and provides light and transmission electron microscope evidence of the syncytial structure of their secretory units, a typical feature of serous glands in anuran skin. This morphological trait supports the hypothesis that lipid glands are a specialized subset of the anuran serous glands, and underlines their flexible role in the skin adaption to sub-aerial environments. Anat Rec, 300:503-506, 2017. © 2016 Wiley Periodicals, Inc.

Clasmatodendrosis and ß-amyloidosis in aging hippocampus.

Alterations of the tightly interwoven neuron/astrocyte interactions are frequent traits of aging, but also favor neurodegenerative diseases, such as Alzheimer disease (AD). These alterations reflect impairments of the innate responses to inflammation-related processes, such as ß-amyloid (Aß) burdening. Multidisciplinary studies, spanning from the tissue to the molecular level, are needed to assess how neuron/astrocyte interactions are influenced by aging. Our study addressed this requirement by joining fluorescence-lifetime imaging microscopy/phasor multiphoton analysis with confocal microscopy, implemented with a novel method to separate spectrally overlapped immunofluorescence and Aß autofluorescence. By comparing data from young control rats, chronically inflamed rats, and old rats, we identified age-specific alterations of neuron/astrocyte interactions in the hippocampus. We found a correlation between Aß aggregation (+300 and +800% of aggregated Aß peptide in chronically inflamed and oldvs.control rats, respectively) and fragmentation (clasmatodendrosis) of astrocyte projections (APJs) (+250 and +1300% of APJ fragments in chronically inflamed and oldvs.control rats, respectively). Clasmatodendrosis, in aged rats, associates with impairment of astrocyte-mediated Aß clearance (-45% of Aß deposits on APJs, and +33% of Aß deposits on neurons in oldvs.chronically inflamed rats). Furthermore, APJ fragments colocalize with Aß deposits and are involved in novel Aß-mediated adhesions between neurons. These data define the effects of Aß deposition on astrocyte/neuron interactions as a key topic in AD biology.-Mercatelli, R., Lana, D., Bucciantini, M., Giovannini, M. G., Cerbai, F., Quercioli, F., Zecchi-Orlandini, S., Delfino, G., Wenk, G. L., Nos, D. Clasmatodendrosis and ß-amyloidosis in aging hippocampus.

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.

Histology of the suprapubic and anogenital cutaneous glands in male cotton top tamarins (Saguinus oedipus).

In cotton top tamarins (Saguinus oedipus), scent glands have been mostly studied in females from museum collections. This work aims to extend the investigation to male specimens, introducing a novel source of skin samples. Two adult males from zoo populations, one intact and one castrated, were immediately frozen after natural death. Skin samples were later collected at the thawing onset, soaked with cold fixative and processed for light microscopy. Sebaceous units of scent glands showed phasic secretory activity in the intact male and marked fibrosis in the castrated male. It appears, therefore, that light microscopy samples from frozen tissues provide detailed features that can disclose distinctive traits in specimens characterized by different hormonal balances.

The venom apparatus in stenogastrine wasps: subcellular features of the convoluted gland.

In the wasp venom apparatus, the convoluted gland is the tract of the thin secretory unit, i.e. filament, contained in the muscular reservoir. Previous transmission electron microscope investigation on Stenogastrinae disclosed that the free filaments consist of distal and proximal tracts, from/to the venom reservoir, characterized by class 3 and 2 gland patterns, respectively. This study aims to extend the ultrastructural analysis to the convoluted tract, in order to provide a thorough, subcellular representation of the venom gland in these Asian wasps. Our findings showed that the convoluted gland is a continuation of the proximal tract, with secretory cells provided with a peculiar apical invagination, the extracellular cavity, collecting their products. This compartment holds a simple end-apparatus lined by large and ramified microvilli that contribute to the processing of the secretory product. A comparison between previous and present findings reveals a noticeable regionalization of the stenogastrine venom filaments and suggests that the secretory product acquires its ultimate composition in the convoluted tract.

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.

Cadmium modulates proliferation and differentiation of human neuroblasts.

Cadmium is an environmental pollutant inducing numerous pathological effects, including neurological disorders and brain diseases. However, little is known about the molecular mechanisms of cadmium in affecting neurons and in inducing neurotoxicity in the development of the human brain. We have recently established, cloned, and propagated in vitro a primary long-term cell culture (FNC-B4) obtained from the human fetal olfactory neuroepithelium. In the present study, we show that different concentrations of cadmium chloride (CdCl(2)) induced dose-dependent biological effects in FNC-B4 cells. A low concentration (10 microM) of CdCl(2) stimulated neuroblast growth, whereas a high concentration (100 microM) inhibited the growth and the viability of neuroblasts inducing morphological and cytoskeletal alterations as well as apoptotic cell death. We also observed that CdCl(2) affected, in a dose-dependent manner, the differentiation of FNC-B4 neuroblasts, with increased mRNA and protein levels of differentiation markers and decreased expression levels of neuronal stem markers. Furthermore, differentiated cells co-expressed glial and neuronal markers. We suggest that CdCl(2) in FNC-B4 neuroblasts might represent a selective cue by which, in a heterogeneous primary culture, the more differentiated mature cells die, whereas the undifferentiated cells, at the same time glial and neuronal progenitors, are forced to access a state of differentiation.

Bombesin promotes vasculogenesis and angiogenesis in chick chorio-allantoic membrane: A morphometric, structural, and ultrastructural study.

Experiments were performed on the chorio-allantoic membrane (CAM) of the chick to evaluate the effects of bombesin (BN) on vascular neoformation. In morphometrical assays, 10(-13)-10(-4) M BN promoted dose-dependent vascular development. Newly formed vessels converged toward the BN release site in a spoked wheel arrangement, suggesting a diffusion gradient mechanism. Structural and ultrastructural analysis of CAM specimens collected near the BN release site showed that both vasculogenetic and angiogenetic processes cooperated in vascular neoformation that involved committed cells from the mesenchyme (angioblasts) as well as endothelial cells. No pattern of vascular development was detected away from the BN release site. Findings from the present study emphasize the role of BN in vascular net development of respiratory organs.

Serous cutaneous glands in the South American horned frog Ceratophrys ornata (Leptodactylformes, Chthonobatrachia, Ceratophrydae): ultrastructural expression of poison biosynthesis and maturation.

Serous cutaneous glands are described in newly metamorphosed and juvenile specimens of the horned frog Ceratophrys ornata using light and transmission electron microscopy. We report patterns of biosynthesis and maturation of the specific product of the gland secretory unit. The syncytial, secretory compartment possesses a complex of endoplasmic reticulum (predominantly smooth endoplasmic reticulum after metamorphosis) and Golgi stacks. The serous product is weak in density and is contained in vesicles involved in repeating merging processes. During this maturation activity, secondary lysosomes are observed, which derive from autophagic processes (crinophagy) involving the secretory materials. Ceratophrys ornata, a species representative of the type genus of the family Ceratophrydae, belongs to the heterogeneous group of anurans that, possibly as the result of convergence, all produce cutaneous poisons consisting of vesicles or faint density granules.

Ultrastructure of poison glands of South American frogs: a comparison between Physalaemus albonotatus and Leptodactylus chaquensis (Anura: Leptodactylidae).

Serous (poison) cutaneous glands of the leptodactylid species Physalaemus albonotatus and Leptodactylus chaquensis were compared using light and transmission electron microscopy. Glands in the two species share structural traits common in anurans, including the peripheral contractile sheath (myoepithelium) and the syncytial secretory unit that produces, stores, and modifies the poison. At the ultrastructural level, early steps of poison production are also similar and fit the usual path of proteosynthesis, involving rough endoplasmic reticulum (RER) and Golgi stacks (dictyosomes) in the peripheral syncytial cytoplasm. However, several differences are obvious during the maturational processes that lead post-Golgian products to their ultimate ultrastructural traits. In P. albonotatus, the dense product released from the dictyosomes acquires a thick repeating substructure, which, however, becomes looser in the inner portion of the syncytium. In L. chaquensis, serous maturation involves gradual condensation, and opaque, somewhat "vacuolized" granules are formed. These different maturational paths expressed during poison manufacturing in the two species agree with the polyphyletic origin of the family Leptodactylidae. On the other hand, data collected for P. albonotatus fit previous findings from P. biligonigerus and stress the view that poisons produced by congeneric species share similar (or identical) ultrastructural features.

Specialized mucous glands and their possible adaptive role in the males of some species of Rana (Amphibia, Anura).

A structural and ultrastructural study was carried out on the cutaneous glands in some species of Rana (R. dalmatina, R. iberica, R. italica, R. "esculenta," and R. perezi), giving particular attention to the mucous secretory units. Two different types of mucous glands occur in R. dalmatina, R. iberica, and R. italica. Besides the ordinary mucous units, which are randomly distributed over the body surface in both males and females, a further population of mucous glands was observed on the male dorsal skin. The latter is recognizable by the peculiar morphology of the epithelial cells and some characteristics of the secretory product. Specialized mucous glands are absent in both sexes of R. "esculenta" and R. perezi. The possible adaptive role of the specialized mucous glands is discussed in light of the absence of vocal sacs in males of R. dalmatina, R. iberica, and R. italica. Chemosignals released by sexually dimorphic mucous units may replace vocal communication during the breeding season and so play an important role in female attraction and/or territorial announcement. The morphology and possible function of the specialized mucous glands in the three species of Rana are compared with the breeding glands of other frogs and with the hedonic glands of some urodeles (Salamandridae and Plethodontidae), which are known to produce pheromonal substances during courtship.

Serous cutaneous glands in new world hylid frogs: an ultrastructural study on skin poisons confirms phylogenetic relationships between Osteopilus septentrionalis and Phrynohyas venulosa.

Transmission electron microscope investigations of the serous (poison) skin glands in the New World tree frogs Osteopilus septentrionalis and Phrynohyas venulosa revealed that they produce granules with closely similar substructures, namely, a dense cortex and pale medulla. In both species these features, that contrast the complex, sometimes repeating patterns described in other hylid frogs, derive from similar secretory and maturational processes starting from the Golgi phase of poison biosynthesis. Observations on secretory discharge showed that the two species share common release mechanisms, based on bulk discharge (holocrine) processes. Our data provide novel evidence of the extensive ultrastructural polymorphism of serous skin products in Hylidae and agree with phylogenies that regard this family as polyphyletic in origin. Assuming that ultrastructural features of cutaneous poison biosynthesis and maturation are adequate clues for tracking anuran phylogeny, the present findings also support a close relationship between Osteopilus and Phrynohyas taxa as previously suggested by osteological evidence.

Spermathecae of Salamandrina terdigitata (Amphibia: Salamandridae): Patterns of sperm storage and degradation.

The spermathecae of female Salamandrina terdigitata were observed using light and transmission electron microscopy during the fallspring period of sperm storage and secretory activity and during the summer stasis. When sperm are stored inside the spermathecae, the product synthesized by spermathecal epithelial cells is exported into the lumen, where it bathes the sperm. During sperm storage some spermatozoa undergo degradation by the spermathecal epithelium. This process, which includes sperm capture by the apical microvilli, formation of endocytic vacuoles and production of lysosomes, becomes prominent shortly after oviposition. In many instances, cells filled with vacuolized spermatozoa and/or residual bodies undergo desquamation from the spermathecal epithelium and enter the lumen together with residual sperm. Desquamated cells, together with residual sperm, are a common feature in the spermathecal lumina at the end of the egg-laying season. Concomitant to the activity of the spermathecal epithelium, macrophages move into the spermathecae from the stroma and contribute to the degradation of both the residual sperm and desquamated epithelial cells. As a result of this degradation activity, spermathecae observed during the short summer stasis appear devoid of secretory product and sperm. By late summer, however, the spermathecae already show early signs of an imminent resumption of biosynthetic activity. © 1995 Wiley-Liss, Inc.