NO signaling in retinal bipolar cells.

Nitric oxide (NO) is a neuromodulator involved in physiological and pathological processes in the retina. In the inner retina, a subgroup of amacrine cells have been shown to synthesize NO, but bipolar cells remain controversial as NO sources. This study correlates NO synthesis in dark-adapted retinas, through labeling with the NO marker DAF-FM, with neuronal nitric oxide synthase (nNOS) and inducible NOS expression, and presence of the NO receptor soluble guanylate cyclase in bipolar cells. NO containing bipolar cells were morphologically identified by dialysis of DAF fluorescent cells with intracellular dyes, or by DAF labeling followed by immunohistochemistry for nNOS and other cellular markers. DAF fluorescence was observed in all types of bipolar cells that could be identified, but the most intense DAF fluorescence was observed in bipolar cells with severed processes, supporting pathological NO signaling. Among nNOS expressing bipolar cells, type 9 was confirmed unequivocally, while types 2, 3a, 3b, 4, 5, 7, 8 and the rod bipolar cell were devoid of this enzyme. These results establish specific bipolar cell types as NO sources in the inner retina, and support the involvement of NO signaling in physiological and pathological processes in the inner retina.

Cigarette smoke condensate inhibits collagen gel contraction and prostaglandin E2 production in human gingival fibroblasts.

BACKGROUND: Granulation tissue remodeling and myofibroblastic differentiation are critically important events during wound healing. Tobacco smoking has a detrimental effect in gingival tissue repair. However, studies evaluating the effects of cigarette smoke on these events are lacking. MATERIAL AND METHODS: We used gingival fibroblasts cultured within free-floating and restrained collagen gels to simulate the initial and final steps of the granulation tissue phase during tissue repair. Collagen gel contraction was stimulated with serum or transforming growth factor-ß1. Cigarette smoke condensate (CSC) was used to evaluate the effects of tobacco smoke on gel contraction. Protein levels of alpha-smooth muscle actin, ß1 integrin, matrix metalloproteinase-3 and connective tissue growth factor were evaluated through Western blot. Prostaglandin E(2) (PGE(2)) levels were determined through ELISA. Actin organization was evaluated through confocal microscopy. RESULTS: CSC reduced collagen gel contraction induced by serum and transforming growth factor-ß1 in restrained collagen gels. CSC also altered the development of actin stress fibers in fibroblasts cultured within restrained collagen gels. PGE(2) levels were strongly diminished by CSC in three-dimensional cell cultures. However, other proteins involved in granulation tissue remodeling and myofibroblastic differentiation such as alpha-smooth muscle actin, ß1 integrin, matrix metalloproteinase-3 and connective tissue growth factor, were unmodified by CSC. CONCLUSIONS: CSC may alter the capacity of gingival fibroblasts to remodel and contract a collagen matrix. Inhibition of PGE(2) production and alterations of actin stress fibers in these cells may impair proper tissue maturation during wound healing in smokers.

Comparative genomics of Listeria species.

Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has also emerged as a paradigm for intracellular parasitism. We present and compare the genome sequences of L. monocytogenes (2,944,528 base pairs) and a nonpathogenic species, L. innocua (3,011,209 base pairs). We found a large number of predicted genes encoding surface and secreted proteins, transporters, and transcriptional regulators, consistent with the ability of both species to adapt to diverse environments. The presence of 270 L. monocytogenes and 149 L. innocua strain-specific genes (clustered in 100 and 63 islets, respectively) suggests that virulence in Listeria results from multiple gene acquisition and deletion events.

Schwann Cell Phenotype Changes in Aging Human Dental Pulp.

Schwann cells are glial cells that support axonal development, maintenance, defense, and regeneration in the peripheral nervous system. There is limited knowledge regarding the organization, plasticity, and aging of Schwann cells within the dental pulp in adult permanent teeth. The present study sought to relate changes in the pattern of Schwann cell phenotypes between young and old adult teeth with neuronal, immune, and vascular components of the dental pulp. Schwann cells are shown to form a prominent glial network at the dentin-pulp interface, consisting of nonmyelinating and myelinating phenotypes, forming a multicellular neuroimmune interface in association with nerve fibers and dendritic cells. Schwann cell phenotypes are recognized by the expression of S100, glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), Sox10, GAP43, and p75NTR markers. In young adult teeth, a dense population of nonmyelinating Schwann cells projects processes in close association with sensory nerve terminals through the odontoblast layer, reaching the adjacent predentin/dentin domain. While GAP43 and p75NTR are highly expressed in nonmyelinating Schwann cells from young adult teeth, the presence of these markers declines significantly in old adult teeth. Myelinated axons, identified by MBP expression, are mainly present at the Raschkow plexus and within nerve bundles in the dental pulp, but their density is significantly reduced in old adult versus young adult teeth. These data reveal age-related changes within the glial network of the dental pulp, in association with a reduction of coronal dental pulp innervation in old adult versus young adult teeth. The prominence of Schwann cells as a cellular component at the dentin-pulp interface supports the notion that their association with sensory nerve terminals and immune system components forms part of an integrated multicellular barrier for defense against pathogens and dentin repair.

Axonal Degeneration in Dental Pulp Precedes Human Primary Teeth Exfoliation.

The dental pulp in human primary teeth is densely innervated by a plethora of nerve endings at the coronal pulp-dentin interface. This study analyzed how the physiological root resorption (PRR) process affects dental pulp innervation before exfoliation of primary teeth. Forty-four primary canine teeth, classified into 3 defined PRR stages (early, middle, and advanced) were fixed and demineralized. Longitudinal cryosections of each tooth were stained for immunohistochemical and quantitative analysis of dental pulp nerve fibers and associated components with confocal and electron microscopy. During PRR, axonal degeneration was prominent and progressive in a Wallerian-like scheme, comprising nerve fiber bundles and nerve endings within the coronal and root pulp. Neurofilament fragmentation increased significantly during PRR progression and was accompanied by myelin degradation and a progressive loss of myelinated axons. Myelin sheath degradation involved activation of autophagic activity by Schwann cells to remove myelin debris. These cells expressed a sequence of responses comprising dedifferentiation, proliferative activity, GAP-43 overexpression, and Büngner band formation. During the advanced PRR stage, increased immune cell recruitment within the dental pulp and major histocompatibility complex (MHC) class II upregulation by Schwann cells characterized an inflammatory condition associated with the denervation process in preexfoliative primary teeth. The ensuing loss of dental pulp axons is likely to be responsible for the progressive reduction of sensory function of the dental pulp during preexfoliative stages.

Ultrastructural changes during the life cycle of human odontoblasts.

In developing premolars there are four successive stages of odontoblasts related to their location within the tooth, namely pre-odontoblasts, secretory, transitional and aged odontoblasts. Pre-odontoblasts are at the growing tip of the root; they are immature and slightly polarized. They become fully differentiated to give rise to the most active, secretory stage when primary dentine is produced. After completion of primary dentine formation in the coronal region, the aged odontoblasts have a reduced number of organelles which are relocated within the infranuclear region. This final stage is regressive and results from an autophagic process that is prominent in the transitional stage.

Reactionary Dentinogenesis and Neuroimmune Response in Dental Caries.

Reactionary dentin formation is an adaptive secretory response mediated by odontoblasts to moderate dentin injury. The implications of this process for neuroimmune interactions operating to contain pathogens have not been fully appreciated. The purpose of the present study was to describe the relationship between reactionary dentinogenesis, the neurogenic changes of dental pulp innervation, and dendritic cell recruitment to caries progression, using a comparative immunohistochemical approach in human teeth from young adult individuals. Reactionary dentin formation during dentin caries progression is associated with changes in the integrity of junctional complexes within the odontoblast layer. Diminished coexpression of Cx43 and zonula occludens 1 implies a reduced level of intercellular connectivity between odontoblasts. Dentin caries also causes overexpression of growth-associated protein 43, a modulator of neural plasticity that promotes extensive sprouting of nerve endings into the reactionary dentin matrix. At the same time, an elevated number of HLA-DR-positive dendritic cells infiltrate the odontoblast layer and subsequently invade reactionary dentin formed underneath the early caries-affected regions. Simultaneous odontoblast layer remodeling, nerve fiber sprouting, and activation of dendritic cells during caries progression suggest a coordinated neuroimmune response to fight caries pathogen invasion and to promote dentin-pulp healing. We propose that reactionary dentin formation hinders pathogen invasion and supports defensive neuroimmune interactions against infection. The eventual understanding of this complex scenario may contribute to the development of novel approaches to dental caries treatment.

The amazing odontoblast: activity, autophagy, and aging.

Odontoblasts are dentin-secreting cells that survive for the whole life of a healthy tooth. Once teeth are completely erupted, odontoblasts transform into a mature stage that allows for their functional conservation for decades, while maintaining the capacity for secondary and reactionary dentin secretion. Odontoblasts are also critically involved in the transmission of sensory stimuli from the dentin-pulp complex and in the cellular defense against pathogens. Their longevity is sustained by an elaborate autophagic-lysosomal system that ensures organelle and protein renewal. However, progressive dysfunction of this system, in part caused by lipofuscin accumulation, reduces the fitness of odontoblasts and eventually impairs their dentin maintenance capacity. Here we review the functional activities assumed by mature odontoblasts throughout life. Understanding the biological basis of age-related changes in human odontoblasts is crucial to improving tooth preservation in the elderly.

Mitochondrial autophagy and lipofuscin accumulation in aging odontoblasts.

Aging of long-lived post-mitotic cells is characterized as a progressive and irreversible reduction of functional activity. In such cells, mitochondria are organelles critical for bioenergetic supply, whose turnover is mediated by an autophagic-lysosomal pathway. In human teeth, odontoblasts are post-mitotic cells responsible for sensory function and dentin preservation. Here, human odontoblasts were processed for immunohistochemistry with antibodies against mitochondrial (MTCO2) and lysosomal (LAMP2) markers, and comparatively analyzed in two age groups (young-adult and adult) with light and electron microscopy. Selective engulfment of mitochondrial profiles into autophagic vacuoles is common in young-adult odontoblasts, suggesting a microautophagic pathway. With age, the odontoblast layer is reduced in width, and mitochondrial elements converge around large clusters of autofluorescent lipofuscin deposits. Age-related changes in odontoblasts are observed as a long-term process in which the progressive accumulation of intralysosomal debris limits the autophagic turnover of mitochondrial components, causing an eventual decline in physiological cell functions, which leads to increased vulnerability under stress conditions.

Autophagic activity and aging in human odontoblasts.

Odontoblasts are long-lived post-mitotic cells in the dental pulp, whose function is to form and maintain dentin. The survival mechanisms that preserve the viability of terminally differentiated odontoblasts during the life of a healthy tooth have not been described. In the present study, we characterized the autophagic-lysosomal system of human odontoblasts with transmission electron microscopy and immunocytochemistry, to analyze the mechanisms that maintain the functional viability of these dentinogenic cells. Odontoblasts were found to develop an autophagic-lysosomal system organized mainly by large autophagic vacuoles that are acid-phosphatase-positive to various degrees. These vacuoles expressed the autophagosomal and lysosomal markers LC3 and LAMP2, respectively, in an age-related pattern indicating the organization of a dynamic autophagic machinery. Progressive accumulation of lipofuscin within lysosomes indicates reduced lysosomal activity as a function of odontoblast aging. Our results suggest that autophagic activity in odontoblasts is a fundamental mechanism to ensure turnover and degradation of subcellular components. A reduction in the efficacy of this system might compromise cell viability and dentinogenic secretory capacity. In adult teeth, this condition is described as an 'old odontoblast' stage.

Changes in the predentin thickness and mineralization front configuration in developing human premolars.

The thickness of the predentin layer was studied at three different levels of developing human premolars. The results demonstrate that at the growing end next to the apex, where dentinogenesis is most active, predentin exhibits its greatest thickness (mean value 40.4 micron). However, at the coronal region, where primary dentin has been completely formed, predentin width is reduced to a mean value of 14.8 micron. Changes in the calcospheritic configuration of the mineralization front were established for each of the predentin levels studied. A comparative analysis of these calcospheritic changes and the morphology of fluorescent tetracycline lines detected in ground sections of premolars was established. Fluorescent lines observed at the coronal circumpulpal dentin showed large calcospheritic forms beneath the mantle dentin. However, lines found near the dentin-pulp border showed small calcospherites. It is concluded that the thickness of the predentin layer and the mineralization front configuration vary as a function of dentinogenic activity during development of human premolars.

Analysis of pore-rich areas on the nuclear envelope of spermatocytes.

Serial sections analysis of spermatocytes of the rodent Phyllotis darwini, reveal a persistent positional relationship between nucleolar material and centrioles. Pore rich areas interposed between these nucleocytoplasmic components, are represented graphically. Centriole replication takes place during the pachytene stage. Pore rich areas could be the result of a preferential aggregational process which insures the exchange of information between nuclear and cytoplasmic regions.

Morphometric analysis of the nucleolus during the life cycle of human odontoblasts.

Developing first premolars were used as a model system to obtain information on the nucleolar structure of human odontoblasts at several stages of their life cycle. Four stages were defined by their location within the tooth: a) preodontoblasts were located at the growing tip of the root; b) secretory odontoblasts in the apical region; c) transitional odontoblasts in the middle region; and d) aged odontoblasts in the coronal region. Preodontoblasts have a small nucleolus (0.55 micron 2) with few strands of dense fibrillar material radiating from the fibrillar center. Secretory odontoblasts are characterized by a large (1.24 micron 2), irregular, and reticulated nucleolus. The fibrillogranular material, the largest component in all nucleoli, reaches maximal size at this stage (0.88 micron 2). Fibrillar centers occupy about the same area (0.1 micron 2) throughout the odontoblast's life cycle. As the formation of primary dentin is completed, the nucleolus of transitional odontoblasts is reduced in size (0.54 micron 2). Finally, the aged odontoblasts have a small, compact nucleolus (0.39 micron 2), with segregated components. Morphologic analysis and quantification of size and component areas of nucleoli obtained with an image analyzer indicated that secretory odontoblasts had the most active, and aged odontoblasts the least active, nucleolus.

Dysmyelination, demyelination and reactive astrogliosis in the optic nerve of the taiep rat.

Taiep is an autosomal recessive mutant rat that shows a highly hypomyelinated central nervous system (CNS). Oligodendrocytes accumulate microtubules (MTs) in association with endoplasmic reticulum (ER) membranes forming MT-ER complexes. The microtubular defect in oligodendrocytes, the abnormal formation of CNS myelin and the astrocytic reaction were characterized by immunocytochemical and ultrastructural methods during the first year of life. Optic nerves of both control and taiep rats were processed by the immunoperoxidase method using antibodies against tubulin, myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP). Taiep oligodendrocytes are strongly immunoreactive against tubulin, indicative of a significant accumulation of microtubules. Early differentiated oligodendrocytes observed with electron microscopy show that MT-ER complexes are mainly present in the cell body. This defect increases during the first year of life; oligodendrocytes show large MT-ER complexes projected within oligodendrocyte processes. Using anti-MBP, there was a progressive reduction of immunolabeling in the myelin sheaths as taiep rats grew older. Ultrastructural analysis revealed severely dysmyelinated axons with a frequently collapsed periaxonal collar. However, through age the myelin sheath became gradually infiltrated by MTs, suggesting their contribution to premature loss of myelin in the taiep rat. Axons of one-year-old taiep rats were severely demyelinated. Modifications in astrocytes revealed by the GFAP antibody showed a strong hypertrophy with increased immunostaining in their processes. As demyelination of axons progressed, taiep rats developed a strong astrogliosis. The present findings suggest that in taiep rats the early abnormal myelination of axons affects the adequate maintenance of myelin, leading to a progressive loss of myelin components and severe astrogliosis, features that should be considered in the pathogenesis of dysmyelinating diseases.

Infectious pancreatic necrosis virus internalization and endocytic organelles in CHSE-214 cells.

The early events that take place during the internalization of infectious pancreatic necrosis virus (IPNV) into Chinook salmon embryo cells (CHSE-214) were analyzed ultrastructurally. Endocytic tracers were employed in order to characterize the organization of endocytic organelles in CHSE-214 cells, as well its relation to the IPNV penetration. Results demonstrate that IPNV appear internalized within vesicular compartments which are located peripherally in CHSE-214 cells. Despite the high rate of infectious multiplicity few virus particles were detected inside the cells. Endocytic tracer labelling of tubulovesicular elements and endosomes of host cells showed a well developed endocytic apparatus. Results suggest that endocytosis may be involved during the initiating events in the productive IPNV infection.

Cell polarity changes and migration during early development of the avian peripheral auditory system.

The intracellular location of organelles was studied in avian embryos immediately before and during the initial detachment of cells from the dorsoanterolateral wall of the otocyst, using light and electron microscopy. The Golgi apparatus was silver-impregnated, and its location within the otic epithelium was determined quantitatively. The present study demonstrates that a sub-population of cells of the dorsoanterolateral otic epithelium changes the intracellular location of its organelles, particularly the Golgi apparatus and the centrosome, from an apical to a basal position. Concomitantly, cells lose specializations characteristically present at apical (tight junctions, microvilli) and basal (basal lamina) surfaces. At basolateral cell surfaces, filopodia form ahead of the Golgi apparatus and centrosome and penetrate the previously continuous underlying basal lamina. Thereafter, cells detach from the otocyst and migrate medially toward the hind-brain. Thus, concomitant with changes in surface polarity, the cells that comprise the dorsoanterolateral wall of the otocyst undergo profound changes in the intracellular location of their organelles, especially the Golgi apparatus and the centrosome, so that by the time cells detach from the otic epithelium a reversal in their "normal" internal polarity has occurred. We suggest that the change in cell polarity may be related to the mechanisms that allow cells to leave the otocyst.

Evaluation of the serum-free medium MDSS2 for the production of poliovirus on vero cells in bioreactors.

The serum-free medium MDSS2 (Merten et al., 1994), was used for cultivating Vero cells as well as for producing poliovirus (Sabin type 1) in static and in perfused micro-carrier cultures. At slightly different growth rates of 0.0120/h and 0.0106/h, respectively, static cultures in serum-containing (SCM) and serum-free (SFM) medium produced titers of (106.75) and 10(6.67) TCID50 per 50 µl; signifying a specific productivity of 0.89 and 1.07 TCID50/c.Serum-free bioreactor cultures of Vero cells on DEAE-dextran microcarriers at 6.25 g/l produced cell densities of about 1.5×10(6)c/ml. After infection with virus (multiplicity of infection (MOI) 0.1-0.3) titers of about 6.3×10(8) TCID50/ml were obtained, signifying an average specific productivity of 7.1 TCID50/c.h. Although these values were 4 and 2 fold, respectively, higher than in classical resum-based production processes (Montagnon et al. Dev. biol. Stand. 1981, 47, 55), a reference culture, for which cell growth was done in SCM and only virus production was done in SFM, produced 2×10(9) TCID/ml with an average specific virus production rate of 18.9 TCID50/c.h. The differences between the fully serum-free and our reference process were mainly due to physiological differences of cells grown in SCM and SFM and also due to strongly modified consumption kinetics after virus infection leading to limitations of one or several essential medium compounds, like glucose and amino acids. Avoiding these limitations by increasing the residual concentration of glucose, glutamine, histidine, and SH-amino acids, led to specific virus production rates (of about 17.9 TCID59/c.h.) comparable to those found in the reference virus production process. The optimisation of the production of the poliovirus (Sabin 1) will be described with respect to the modification of the medium composition.

Binding of microtubules to transitional elements in oligodendrocytes of the myelin mutant taiep rat.

The presence of microtubules physically bound to smooth endoplasmic reticulum profiles of oligodendrocytes constitutes the most conspicuous feature observed in the myelin mutant taiep rat. The endoplasmic reticulum membranes associated with microtubules were morphologically characterized as transitional elements that constitute the intermediate compartment according to their topographic location close to the cis-Golgi apparatus. The development of this surprising microtubular defect appears to be associated with the early events of myelination. Transitional elements associated with microtubules operate in protein transport from endoplasmic reticulum to cis-Golgi. This microtubular defect could explain the dysmyelination and neurologic alterations observed in taiep rats. Moreover, these findings allow us to propose that there is a blockage of protein traffic at the intermediate compartment of taiep oligodendrocytes, a situation that could explain the hypomyelinated axons observed in this myelin mutant. The binding of microtubules to membranous organelles promotes the stabilization of microtubules, a feature that has important implications regarding its accumulation within the cytoplasm of oligodendrocytes during the temporal evolution of this neurologic disorder. The taiep rat is a myelin mutant with a long survival and could be a useful model for understanding dysmyelinating diseases in which the intracellular transport of myelin components is affected.

A simple serum-free freezing medium for serum-free cultured cells.

Because the presence of serum in cell culture raises safety problems for the production of biologicals, we have developed a serum-free medium (SFM) for the cryopreservation of animal cells. This medium is based of the SFM MDSS2, to which 10% dimethylsulfoxide (DMSO) and 0.1% methylcellulose or 3% polyvinyl pyrrolidone or no other additive than DMSO were added. Both, Vero and BHK-21 cells regularly cultivated in MDSS2, could be cryopreserved in the three serum-free freezing media and could be thawed without any cell loss. No differences could be found between the cells in the standard freezing medium (DMEM containing 10% fetal calf serum and 10% DMSO) and those frozen in SFM, with respect to cell growth and viability. In the case of the Vero cells no differences were observed with respect to their attachment. This medium represents the last step in fulfilling a complete serum-free animal cell based bioprocess.