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.

Increased expression of Aquaporin 4 in the rat hippocampus and cortex during trimethyltin-induced neurodegeneration.

Trimethyltin chloride (TMT) is a neurotoxicant producing neuronal degeneration and reactive astrogliosis in the mammalian central nervous system, especially the hippocampus. A previous magnetic resonance imaging investigation in TMT-treated rats evidenced dilation of lateral ventricles, also suggesting alterations in blood-brain barrier permeability and brain edema. Aquaporin 4 (AQP4), a glial water channel protein expressed mainly in the nervous system, is considered a specific marker of vascular permeability and thought to play an important role in brain edema (conditions). We studied AQP4 expression in the hippocampus and cerebral cortex of TMT-treated rats in order to explore the molecular mechanisms involved in brain edema occurring in these experimental conditions. Real-time PCR and western blotting data showed significant up-regulation of both AQP4 mRNA and protein levels starting 14 days after TMT treatment in the hippocampus and cortex. Parallel immunofluorescence studies indicated intense astrogliosis and AQP4 immunoreactivity diffusely pronounced in the hippocampal and cortex areas starting 14 days after TMT intoxication. In order to study the effects of TMT on vascular integrity, double-label immunofluorescence experiments for rat immunoglobulin G (IgG) and rat endothelial cell antigen-1 (RECA-1) or neuronal nuclei (NeuN) (endothelial and neuronal markers respectively) were performed. The results indicated, at 21 and 35 days after treatment, the presence of rat IgG in paravasal parenchyma and in some neuronal cells of the hippocampus and cortex. The extravasated IgG staining was temporally correlated with over-expression of neuronal vascular endothelial growth factor (VEGF) and the active phosphorylated form of its neuronal receptor (VEGFR-2P), suggesting that these factors may cooperate in mediating vascular leakage.

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.

Cathepsin D plays a crucial role in the trimethyltin-induced hippocampal neurodegeneration process.

Trimethyltin chloride (TMT) is known to produce neuronal damage in the rat hippocampus, especially in the CA(1)/CA(3) subfields, together with reactive astrogliosis. Previous studies indicate that in cultured rat hippocampal neurons the Ca(2+) cytosolic increase induced by TMT is correlated with apoptotic cell death, although some molecular aspects of the hippocampal neurodegeneration induced by this neurotoxicant still remain to be clarified. Cathepsin D (Cat D) is a lysosomal aspartic protease involved in some neurodegenerative processes and also seems to play an important role in the processes that regulate apoptosis. We investigated the specific activity and cellular expression of Cat D in the rat hippocampus in vivo and in cultured organotypic rat hippocampal slices. The role of Cat D in cell death processes and the mechanisms controlling Cat D were also investigated. Cat D activity was assayed in hippocampus homogenates of control and TMT-treated rats. In order to visualize the distribution of Cat D immunoreactivity in the hippocampus, double-label immunofluorescence for Cat D and Neu N, GFAP, OX42 was performed. In addition, in order to clarify the possible relationship between Cat D activity, neuronal calcium overload and neuronal death processes, organotypic hippocampal cultures were also treated with a Cat D inhibitor (Pepstatin A) or Calpain inhibitor (Calpeptin) or an intracellular Ca(2+) chelator (BAPTA-AM) in the presence of TMT. TMT treatment in rat hippocampus induced high levels of Cat D activity both in vivo and in vitro, in glial cells and in CA(3) neurons, where a marked TMT-induced neuronal loss also occurred. Cat D is actively involved in CA3 neuronal death and the protease increase is a calcium-Calpain dependent phenomenon.

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.

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.

A human umbilical cord stem cell rescue therapy in a murine model of toxic liver injury.

BACKGROUND: Several studies have demonstrated that bone marrow contains a subpopulation of stem cells capable of participating in the hepatic regenerative process, even if some reports indicate quite a low level of liver repopulation by human stem cells in the normal and transiently injured liver. AIMS: In order to overcome the low engraftment levels seen in previous models, we tried the direct intraperitoneal administration of human cord blood stem cells, using a model of hepatic damage induced by allyl alcohol in NOD/SCID mice. METHODS: We designed a protocol based on stem cell infusion following liver damage in the absence of irradiation. Flow cytometry, histology, immunohistochemistry and RT-PCR for human hepatic markers were performed to monitor human cell engraftment. RESULTS: Human stem cells were able to transdifferentiate into hepatocytes, to improve liver regeneration after damage and to reduce the mortality rate both in both protocols, even if with qualitative and quantitative differences in the transdifferentiation process. CONCLUSIONS: We demonstrated for the first time that the intraperitoneal administration of stem cells can guarantee a rapid liver engraftment. Moreover, the new protocol based on stem cell infusion following liver damage in the absence of irradiation may represent a step forward for the clinical application of stem cell transplantation.

Gamma-aminobutyric acidergic interneuron vulnerability to aging in canine prefrontal cortex.

The aged dog is considered a promising model for examining molecular and cellular processes involved in a variety of human neurological disorders. By using the canine counterpart of senile dementia of the Alzheimer's type (ccSDAT), we investigated the specific vulnerability of the gamma-aminobutyric acid (GABA) cortical subset of interneurons, characterized by their calcium-binding protein content, to neuronal death. Dogs representing a large variety of breeds were classified into three groups: young control, aged control, and ccSDAT. In all dogs, the general distribution and cell typology of parvalbumin-, calretinin-, and calbindin-positive neurons were found to be similar to those in the human. As in Alzheimer's disease patients, neurons displaying parvalbumin or calretinin immunoreactivity were resistant and the calbindin-positive ones depleted. Together with aging, amyloid deposition in its early phase (stage II) participates in this specific neuronal death, but with a lower potency. In conclusion, our data provide evidence that preservation of GABAergic cortical interneurons has to be focused on the early stage of beta-amyloid deposition. We also demonstrate the usefulness of dogs of all breeds for investigating the early phases of human brain aging and Alzheimer's disease.

Phentolamine administration increases blood S100B protein levels in pediatric open-heart surgery patients.

AIM: Phentolamine administration during open-heart surgery shortens the cooling and rewarming phases of cardiopulmonary bypass (CPB) and hastens weaning from mechanical ventilation and extubation. Data on the effects of phentolamine on cerebral circulation and function in this setting are lacking. This study reports the cerebral effects of phentolamine using blood S100B protein levels and the middle cerebral artery pulsatility index (MCA PI). METHODS: Sixty pediatric patients undergoing congenital heart disease repair were randomly assigned to receive either phentolamine 0.2 mg kg(-1) i.v. (n = 30) or placebo (n = 30) before the cooling and rewarming phases of CPB. Samples for S100B measurement were collected at seven predetermined time-points before, during and after surgery. MCA PI values were recorded at the same times as sampling. RESULTS: S100B blood levels were higher in the phentolamine-treated group than in controls after rewarming (3.53 +/- 1.88 vs 1.58 +/- 0.53 microg l(-1); p < 0.001), remained persistently higher at the end of surgery (2.95 +/- 0.91 vs 0.79 +/- 0.21 microg l(-1); p < 0.001) and returned to normal ranges 12 h later than in the placebo group (p > 0.05). MCA PI values were also significantly higher at the end of surgery in the phentolamine-treated group (1.83 +/- 0.50 vs 1.22 +/- 0.34; p < 0.01). Cooling and rewarming times were shorter in the phentolamine-treated group (p < 0.01, for all). CONCLUSION: Despite improved peripheral vasodilatation and perfusion, phentolamine administration in pediatric open-heart surgery is correlated with increased cerebrovascular resistance and brain damage.

S100B protein cord blood levels and development of fetal behavioral states: a study in normal and small-for-dates fetuses.

OBJECTIVE: We aimed to determine whether S100B protein levels in cord blood and the development of fetal behavioral states were altered and interrelated in small-for-dates (SFD) fetuses. METHODS: Umbilical cord blood samples were collected from 12 SFD fetuses with normal umbilical artery (UA) Doppler findings, from six SFD fetuses with abnormal Doppler waveform patterns and from 36 controls matched for gestational age. S100B protein levels were measured by means of a specific radioimmunoassay. Fetal behavioral state recordings were made before delivery by Cesarean section and data were expressed as percentage of quiet sleep coincidence (C1F), of activity state coincidence (C2-4F) and of no coincidence (NOC). Flow velocimetry waveforms were recorded from the uterine artery, UA and fetal middle cerebral artery (MCA). RESULTS: Mean S100B protein levels in umbilical plasma were significantly higher in the six SFD infants with abnormal prenatal Doppler findings (3.31 +/- 0.65 microg/l) than in SFD infants with normal Doppler findings (1.56 +/- 0.35 microg/l) and in controls (1.23 +/- 0.43 microg/l). Similarly in these fetuses NOC was higher and C2F significantly lower (p < 0.05), but there was no significant difference in C1F. S100B concentrations were correlated with the UA pulsatility index (PI) (r = 0.78, p < 0.01), with the MCA PI (r = -0.78, p < 0.01) and with the UA PI/MCA PI ratio (r = 0.80, p < 0.01). Also, NOC and C2F percentages were correlated with the UA PI (r = 0.61, p < 0.01 and r = -0.61, p < 0.01, respectively), with the MCAPI (r = -0.72, p < 0.001 and r = 0.66, p < 0.01, respectively), and with the UA PI/MCA PI ratio (r = 0.60, p < 0.01 and r = -0.54, p < 0.05, respectively). NOC was also correlated with S100B protein (r = 0.48, p < 0.05); the correlation of S100B protein and C2F almost reached significance (r = -0.47, p < 0.05). CONCLUSIONS: This study provides evidence of a relationship between a biochemical marker of brain development and/or integrity and the development of fetal behavioral states, offering additional information on brain maturation in normal and high-risk pregnancies.

Elevated S100B protein as an early indicator of intracranial haemorrhage in infants subjected to extracorporeal membrane oxygenation.

UNLABELLED: The aim of this investigation was to verify whether plasma S100B could be a useful tool in identifying which infants subjected to extracorporeal membrane oxygenation (ECMO) might develop intracranial haemorrhage (ICH). A case-control study of eight infants who developed ICH during ECMO was conducted. Plasma samples collected daily after ECMO insertion were assessed for S100B and compared with those obtained from eight infants supported by ECMO who did not develop ICH. Cerebral ultrasound and Doppler velocimetry waveform patterns in the middle cerebral artery (MCA PI) were also recorded at the same time as blood sampling. S100B blood concentrations were significantly higher in the group of infants with ICH 72 h before any signs of haemorrhage could be detected by ultrasound (ICH: 2.91 +/- 0.91 microg/L vs. control: 0.53 +/- 0.15 microg/L), reaching their peak at day 6, when cerebral ultrasound scan patterns were suggestive of intracranial haemorrhage (ICH: 3.50 +/- 1.03 microg/L vs. control: 0.66 +/- 0.27 microg/L) (p < 0.05, for both). The highest S100B levels were observed in the three ICH infants who expired during the ECMO procedure (3.43 microg/L, 4.0 microg/L, 4.12 microg/L, respectively). MCA PI values in the ICH group were also significantly higher, but only 24 h before any ultrasound pattern of bleeding was detected (ICH: 2.31 +/- 0.22 vs control: 1.81 +/- 0.24) (p < 0.05). CONCLUSION: This study suggests that blood S100B measurement could be a promising tool for the identification of infants at risk of ICH when imaging assessment and clinical symptoms of haemorrhage might still be silent.

Prognostic significance of the Ca(2+) binding protein S100A2 in laryngeal squamous-cell carcinoma.

We investigated by immunocytochemistry the expression of the Ca(2+) binding protein S100A2 in 62 cases of laryngeal squamous-cell carcinoma (SCC). S100A2 was detected in 18/19 (95%) low-grade tumors and in 22/43 (51%) high-grade tumors, which were partially keratinizing. The remaining 21/43 (49%) high-grade tumors were non-keratinizing, anaplastic tumors and clearly S100A2-negative. In normal laryngeal squamous epithelium and in laryngeal SCC, S100A2 expression was strictly associated with that of cytokeratins 14 (P = 0.0002) and 17 (P = 0.0021), suggesting an association of S100A2 expression and cell commitment to squamous differentiation. A correlation was found between S100A2 tumor positivity and longer relapse-free (P = 0.0005) and overall (P = 0.0095) survival.