Neuroprotective strategies in hippocampal neurodegeneration induced by the neurotoxicant trimethyltin.

The selective vulnerability of specific neuronal subpopulations to trimethyltin (TMT), an organotin compound with neurotoxicant effects selectively involving the limbic system and especially marked in the hippocampus, makes it useful to obtain in vivo models of neurodegeneration associated with behavioural alterations, such as hyperactivity and aggression, cognitive impairment as well as temporal lobe epilepsy. TMT has been widely used to study neuronal and glial factors involved in selective neuronal death, as well as the molecular mechanisms leading to hippocampal neurodegeneration (including neuroinflammation, excitotoxicity, intracellular calcium overload, mitochondrial dysfunction and oxidative stress). It also offers a valuable instrument to study the cell-cell interactions and signalling pathways that modulate injury-induced neurogenesis, including the involvement of newly generated neurons in the possible repair processes. Since TMT appears to be a useful tool to damage the brain and study the various responses to damage, this review summarises current data from in vivo and in vitro studies on neuroprotective strategies to counteract TMT-induced neuronal death, that may be useful to elucidate the role of putative candidates for translational medical research on neurodegenerative diseases.

Distribution and time-course of 4-hydroxynonenal, heat shock protein 110/105 family members and cyclooxygenase-2 expression in the hippocampus of rat during trimethyltin-induced neurodegeneration.

Trimethyltin (TMT), an organotin compound considered a useful tool to obtain an experimental model of neurodegeneration, exhibits neurotoxicant effects selectively localised in the limbic system and especially in the hippocampus, which are different in the rat and in mice. In the rat hippocampus, we investigated the expression of aldehyde 4-hydroxynonenal, a major bioactive marker of membrane lipid peroxidation, heat shock protein (HSP) 110/105 family members, markers of oxidative stress, and the neuroinflammatory marker cyclooxygenase-2 after TMT-intoxication at various time points after treatment. Our data show that TMT-induced neurodegeneration in the rat hippocampus is associated specifically with oxidative stress and lipid peroxidation, but not with HSP expression, indicating species-specific differences in the neurotoxicity of TMT between rats and mice.

Transcritpional effects of S100B on neuroblastoma cells: perturbation of cholesterol homeostasis and interference on the cell cycle.

S100B is a Ca2+ binding protein mainly secreted by astrocytes in the vertebrate brain that is considered a multifunctional cytokine and/or a damage-associated molecular pattern (DAMP) protein and a marker of brain injury and neurodegeneration when measured in different body fluids. It has been widely shown that this protein can exert diverse effects in neural cultures depending on its concentration, having detrimental effects at micromolar concentrations. The molecular mechanisms underlying this effect are still largely unknown. This study attempts to delineate the genome-wide gene expression analysis of the events associated with exposure to micromolar concentration of S100B in a human neuroblastoma cell line. In this experimental condition cells undergo a severe perturbation of lipid homeostasis along with cell cycle arrest. These mechanisms might reasonably mediate some aspects of the S100B-related detrimental effects of S100B, although obvious differences between mature neurons and neuroblastoma cells have to be considered.

S100B as a new fecal biomarker of inflammatory bowel diseases.

OBJECTIVE: S100 proteins are demonstrated to exert a protective role in the gastrointestinal tract. In the present study, we investigated whether S100B protein, that is typically expressed by enteroglial cells, is detectable in feces and could be a useful noninvasive indicator of gut chronic inflammation. PATIENTS AND METHODS: This clinical prospective study included n=48 patients suffering Crohn's disease (CD) or ulcerative colitis (UC) and non IBD-controls. The clinical disease activity was evaluated using Harvey-Bradshaw or Mayo Score Index while the diagnosis of IBD was defined based on standard endoscopic and histological criteria. S100B and calprotectin were extracted and analyzed using commercial enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: Unlike calprotectin, S100B was significantly decreased in both CD and UC compared to non IBD-patients. The strongest quantitative alterations of S100B were detected concomitantly with signs of active or quiescent disease, including high/normal expression of fecal calprotectin, mucosal damage/cryptitis, mucin depletion and inflammatory infiltrate, as defined by endoscopic evaluation and histological analysis. At the onset of disease and under no Infliximab-based therapy, the lowest was detected suggesting that S100B in feces could have a potential diagnostic value for IBD. CONCLUSIONS: Testing for S100B and calprotectin could be a useful screening tool to better predict IBD activity.

Ex vivo-transduced autologous skin fibroblasts expressing human Lim mineralization protein-3 efficiently form new bone in animal models.

Local gene transfer of the human Lim mineralization protein (LMP), a novel intracellular positive regulator of the osteoblast differentiation program, can induce efficient bone formation in rodents. To develop a clinically relevant gene therapy approach to facilitate bone healing, we have used primary dermal fibroblasts transduced ex vivo with Ad.LMP-3 and seeded on a hydroxyapatite/collagen matrix prior to autologous implantation. Here, we demonstrate that genetically modified autologous dermal fibroblasts expressing Ad.LMP-3 are able to induce ectopic bone formation following implantation of the matrix into mouse triceps and paravertebral muscles. Moreover, implantation of the Ad.LMP-3-modified dermal fibroblasts into a rat mandibular bone critical size defect model results in efficient healing, as determined by X-rays, histology and three-dimensional microcomputed tomography (3DmuCT). These results demonstrate the effectiveness of the non-secreted intracellular osteogenic factor LMP-3 in inducing bone formation in vivo. Moreover, the utilization of autologous dermal fibroblasts implanted on a biomaterial represents a promising approach for possible future clinical applications aimed at inducing new bone formation.

How does human stem cell therapy influence gene expression after liver injury? Microarray evaluation on a rat model.

BACKGROUND: Tissue homeostasis is guaranteed by stem proliferating reserve, depending on dynamic changes in gene expression. A high plasticity is shown by the haematopoietic stem cells, potential source for liver regeneration. AIM: We aimed to evaluate the gene expression modifications induced by human haematopoietic stem cell therapy after liver injury in rats. SUBJECTS: Rats were sorted as follows: (A) human-haematopoietic stem cell injection after allyl alcohol liver damage; (B) only haematopoietic stem cell injection; (C) only allyl alcohol injection; and (D) sacrifice without any treatment. METHODS: Livers, spleens and bone marrows were analysed with flow-cytometry. Livers were also studied by reverse-transcription PCR, histology, immunohistochemistry and microarray analysis; selected genes were confirmed by real-time PCR. RESULTS: In subset A, haematopoietic stem cells were selectively recruited by liver, with respect to the group B, and they improved the liver regeneration process compared to group C. As regards microarrays, haematopoietic stem cell infusion upregulates 265 genes and downregulates 149 genes. Differentially regulated genes belong to a broad range of functional pathways, including proliferation, differentiation, adhesion/migration and transcripts related to oval-cell activation. Real-time PCR validated array results. CONCLUSIONS: Our study confirmed the capacity of haematopoietic stem cells to contribute to liver regeneration. Moreover, microarray analysis led to the identification of genes whose regulation strongly correlates with a more efficient process of liver repair after haematopoietic stem cell injection.

Improvement of mortality rate and decrease in histologic hepatic injury after human cord blood stem cell infusion in a murine model of hepatotoxicity.

BACKGROUND AND AIMS: Because of their plasticity potential local and systemic application of cord blood stem cells may represent excellent candidates for cell-based therapeutic strategies in toxic liver injuries. It is already known that intraperitoneal administration of hematopoietic stem cells provides rapid liver homing in animal models of hepatic injury. We sought to assess the efficacy of a hematopoietic stem cell infusion to decrease the histologic damage and the mortality rate of animals previously damaged by allyl alcohol. MATERIAL AND METHODS: NOD/SCID mice were divided into two groups. (1) animals treated by intraperitoneal administration of allyl alcohol and (2) animals treated with allyl alcohol and 24 hours later with an intraperitoneal infusion of human cord blood cells. Flow cytometry, histology, immunohistochemistry, and RT-PCR were performed to monitor human cell engraftment by evidences of human hepatic markers. RESULTS: Human stem cells were able to transdifferentiate into hepatocytes, improve liver regeneration after damage, and reduce the mortality rate even when requiring qualitative and quantitative differences in the transdifferentiation processes. The mortality rate decreased from 70% to 20%, with a significant improvement in the histologic findings. CONCLUSION: We demonstrated that the infusion of hematopoietic stem cells into the liver in the early stage of damage might initiate endogenous hepatic tissue regeneration that oppose the injury inflicted by toxicants.

Human cordonal stem cell intraperitoneal injection can represent a rescue therapy after an acute hepatic damage in immunocompetent rats.

BACKGROUND AND AIM: Tissue homeostasis and turnover require reserve stem proliferating cells. Several studies performed on immunodeficient animals have suggested a degree of plasticity by the hematopoietic stem cell compartment that may represent source for liver regeneration. We sought to explore the hepatic differentiation potential of hematopoietic stem cells from human cord blood, after toxic liver damage induced by allyl-alcohol in immunocompetent rats. MATERIALS AND METHODS: Wistar rats were divided into groups (A) allyl-alcohol intraperitoneal injection with hematopoietic stem cell intraperitoneal infusion at 1 day and sacrifice 3 days later; (B) stem cell injection and sacrifice 3 days later; (C) allyl-alcohol infusion and sacrifice 4 days later; and (D) sacrifice without any treatment. Livers, spleens, and bone marrows were analysed for human stem cells using flow-cytometry; livers were also tested by histology and immunohistochemistry to study the pattern of hepatic regeneration after damage and human stem cell conversion into hepatocyte-like cells, respectively. RESULTS: Flow-cytometry revealed selective recruitment of human hematopoietic stem cells by damaged livers (group A) compared with control group B. In addition, liver damage was reduced in animals treated with stem cells. Immunohistochemistry demonstrated that human stem cells could convert hepatic cells. CONCLUSIONS: Our study demonstrated that hematopoietic stem cells selectively recruited by injured livers can contribute to hepatic regeneration after acute toxic damage in immunocompetent recipients.

Localization of the loosely bound nuclear proteins of rat brain: an immunocytochemical ultrastructural study.

Antibodies against the loosely bound subnuclear protein fraction (0.35 M NaCl-extractable subnuclear fraction) of rat brain were raised in rabbits, and the ultrastructural distribution of the antigenic determinants in rat cerebellum was studied using the unlabeled antibody peroxidase-antiperoxidase (PAP) method. A localization restricted to the nucleus was observed. Both neuronal and neuroglial nuclei exhibited antigens, whereas nuclei of pericytes and endothelial cells did not. The immunoreaction product was homogeneously distributed in dispersed chromatin and was absent from condensed chromatin, suggesting that the antigens were confined to the active regions of the genoma. The outer nuclear membrane and the nucleolus appeared to be free of the antigens, while a perinucleolar ring of immunoreaction was detectable. Liver preparations showed a nuclear reaction markedly weaker than the one for brain nuclei. Adsorption of the serum with isolated liver nuclei nullified the reactivity in liver tissue, whereas a sharp reaction was still observed in the cerebellum, indicating the subcellular reaction under examination to contain antigens specifically concentrated in the nervous system or unique to the brain.

Immunochemical and immunohistochemical detection of S-100-like immunoreactivity in spinach tissues.

S-100 proteins represent a group of closely related acidic, calcium binding proteins originally isolated from the mammalian nervous system and later detected in non-neural cell types and in a wide variety of vertebrate and invertebrate species. The present study used immunochemical and immunohistochemical methods to extend the investigation of S-100 during phylogenesis to plant tissues. The presence of S-100-like immunoreactive material was detected in extracts of spinach (Spinacia oleracea L.) terminal buds and young leaves by the ELISA method and by Western blotting using different anti-S-100 rabbit antisera. Using the PAP method, serial sections of young spinach leaves treated with the same antisera exhibited an immunoreaction product that was confined to the cytoplasm and nucleus (but absent from the vacuoles) in meristematic, epidermal, and parenchymal cells. The present data enlarge the field of investigation of S-100 proteins in the search of the function(s) of S-100 in biological organisms.

Localization of S-100 protein in Müller cells of the retina--2. Electron microscopical immunocytochemistry.

The cellular and subcellular distribution of S-100 protein was investigated at the ultrastructural level in the rat retina by the immunocytochemical PAP method. S-100 appeared to be localized in the cytoplasm and nucleus of Müller cells, offering conclusive evidence that in the mammalian retina, the protein is confined to glial cells. S-100, as a marker for Müller cells, may be a useful tool in order to study the cytoarchitecture of the retina in normal as well as in pathologic conditions. In addition, the retina may represent a suitable model for further investigation on the biologic role of S-100.

Localization of S-100 protein in Müller cells of the retina--1. Light microscopical immunocytochemistry.

S-100 is an acidic brain protein previously found to be present in glial cells of the brain and the nervous system of gut and respiratory tract. Immunocytochemistry at the light microscopical level localized immunoreactivity for S-100 in the Müller cells in the retina of rat, guinea pig, and Chinese hamster. The Müller cells represent the main glial component of the retina, with a structural role in the support and insulation of neurons and sensory elements. The use of S-100 protein as an immunocytochemical marker of Müller cells may be useful in the study of pathologic conditions of the retina where glial cell proliferation could reflect the index of neuronal injury.

Increase of S-100 immunoreactivity in the urinary bladder from patients with multiple sclerosis, an indication of peripheral neuronal lesion.

The Schwann cells in urinary bladder biopsies from multiple sclerosis patients and controls were examined by immunocytochemistry with an antiserum to S-100. S-100 immunoreactivity was found to be markedly increased in these tissues as compared with the controls, indicating a Schwann cell hyperplasia in the urinary bladder in multiple sclerosis. This finding suggests that local neuronal damage exists in the urinary bladder of patients with multiple sclerosis. Therefore, the concept of multiple sclerosis as a disease wholly of the central nervous system should be reexamined.

Detection of S-100 labelled cells in nasopharyngeal carcinoma.

S-100 antigen containing cells with dendritic features, recognisable by morphological and immunohistochemical criteria as belonging to the Langerhans' or interdigitating reticulum cell type, have been found in undifferentiated nasopharyngeal carcinoma. The presence of these cells, which have a special function of antigen presentation in immune responses, may be involved in a possible modulation of nasopharyngeal carcinoma associated Epstein-Barr virus infection and host-tumour interactions.

Soluble and membrane-bound S-100 protein in cerebral cortex synaptosomes. Properties of the S-100 receptor.

Within cerebral cortex synaptosomes, S-100 protein can be recovered in two forms: soluble and membrane-bound. Synaptosomal S-100 is mainly a soluble protein (85 percent). The membrane-bound S-100 is differently distributed in the synaptosomal membranes, intraterminal mitochondria, and synaptic vesicles. S-100 binds to a specific receptor. The binding is time-dependent, reversible and saturable with respect to S-100. The number of receptors is calculated to be about 9 times 10(12)/mg protein, since saturation is achieved at 31 ng [125I]S-100/0.1 mg protein of disrupted synaptosomes. The rate constant for association of S-100 with its receptor at 37 degrees C, k1, is 4.74 times 10(4) M(-1) sec(-1), and the rate constant for dissociation, k-1, 9.24 times 10(-4) sec(-1).

S-100 protein in the brain of hypothyroid adult rats: an immunochemical and immunocytochemical study.

The levels and the distribution of S-100 protein were studied in the brain of hypothyroid adult rats. The concentration of the protein was significantly increased in the soluble fraction of hypothyroid rat brain as compared with controls (P less than 0.001). This finding probably reflects the increased number of astroglial cells, which has been widely reported in hypothyroid adult rats. The immunocytochemical ultrastructural distribution of the protein in the cerebellar cortex was not affected by thyroid deficiency. The protein was thus confined to the cytoplasm and the nucleus of the astroglial cells both in treated and untreated rats.

Glial-like cells in sympathetic neural crest derivatives during human embryogenesis. Detection by S-100 immunohistochemistry.

The present study investigates at the light and electron microscopic levels the possible presence and distribution during human development of glial-like satellite cells in sympathetic neural crest derivatives by S-100 immunohistochemistry. From the earliest stages investigated, immunostained cells were detected inside sympathetic migrating masses and at their periphery, where they constituted a continuous layer isolating sympathetic elements from mesenchymal cells. The detection and peculiar distribution of these glial-like cells in developing sympathetic tissue could open new perspectives in the study of events linked to the migration and differentiation of some neural crest derivatives.

Comparative study by S-100 and GFAP immunohistochemistry of glial cell populations in the early stages of human spinal cord development.

The present study reports the presence and distribution of S-100-containing cells in the developing human spinal cord. From the earliest stages investigated, S-100-immunostained cells bordered the ventral third of the central canal or appeared dispersed in the basal lamina. On the other hand, radial glia processes were clearly depicted after GFAP immunolabelling. The presence and peculiar distribution of S-100-immunoreactive cells could be significant in the study of the early events of glial differentiation and migration.

Immunochemical and immunocytochemical study of S-100 protein in rat adipocytes.

S-100 is a protein originally believed to be unique to the nervous system. We report here on the presence of S-100 in rat adipocytes, using immunohistochemical and immunochemical methods. We demonstrate that the protein in adipose tissue is present at a concentration comparable to that measured in the nervous tissue and is immunologically identical to brain S-100, indicating that the protein can no longer be regarded as being specific to the nervous system.

Immunochemical detection of S-100 protein in non-nervous structures of the rabbit eye.

S-100 is a protein originally believed to be unique to the nervous system. We report here on its immunochemical detection in non-nervous structures of the rabbit eye, namely in the lens, cornea and iris, at a markedly higher concentration in the latter.