Differential expression of disialic acids in the cerebellum of senile mice.

It is known that disialic acids (diSia) are present in the mammalian brain. However, the precise anatomical distribution and the chronology of its expression along life are not well studied yet. It is accepted that the transfer of diSia in the brain is mediated mainly by the enzyme ST8Sia III (α2,8-sialyltransferase III). We studied the expression of diSia glycoepitopes and of the ST8Sia III gene in different structures of the mouse brain at different postnatal stages by immunohistochemistry and real-time polymerase chain reaction, respectively. C57BL/6 mice of different stages were used. Samples of hippocampus, olfactory bulb, cortex and cerebellum were processed for studies of molecular biology and immunohistochemistry. Histological analysis revealed an important decrease in diSia labeling in the senile cerebellum compared with other structures and stages (P â‰ª 0.001). In concordance with these results, a significant decrease in ST8Sia III gene expression was found in the cerebellum of senile animals (P < 0.001). These results suggest that diSia are constantly expressed but with differential expression in various areas of the mouse central nervous system. On the other hand, the concordance in the decreased expression of ST8Sia III and the diSia epitope in the cerebellum of senile animals suggest a role of diSia in this structure or, inversely, an influence of aging on the expression of diSia in the cerebellum. Further research in that direction could elucidate the roles of diSia in brain function in health and disease.

Episodic-like memory: new perspectives from a behavioral test in rats.

In order to have a tool to empirically test the ideas derived from a theoretical model, we extended a protocol for evaluation of episodic-like memory in rats, based on the triad "what, where, context" for definition of memories. As with the computational model, our intention was for the animal being tested to store a specific number of object-place-context configurations as different memories, which would then be retrievable from cues. The aim of this work was to evaluate the influence of the number of configurations to be memorized on the performance of the task. Sixty-five Wistar male rats were evaluated. In accordance with previous work, for two configurations, the recognition index was indicative of recognition of the element mismatching the original memory (mean = 0.28; SEM = 0.12). The recognition index for three configurations was lower (mean = 0.15; SEM = 0.10), evidencing less recall with increasing requirements. The results also showed a trend toward recognition of novelty for the first and the last memory when evaluating three configurations (a "U" shape in the exploratory preference's curve), showing the primacy and recency effects typical of memory both in humans and animals. Nonetheless, the data presented a high inter-subject variability which makes the test non-robust for small groups. However, if used before and after a treatment for a same subject, we suggest that the protocol presented in this work can be a useful behavioral test for the evaluation of episodic-like memory in rats in terms of a variable task demand.

Influence of CYP3A5 polymorphism on tacrolimus maintenance doses and serum levels after renal transplantation: age dependency and pharmacological interaction with steroids.

TAC, MMF and MP are used in pediatric kidney tx. The cytochrome P450 (CYP)3A5 enzyme appears to play a role in TAC metabolism. The aims of this study were to investigate CYP3A5 polymorphism's effect on TAC dosing and the age dependency of TAC dosing by testing blood concentrations, and the interaction between steroids and TAC during the first year after tx. Genomic DNA was extracted and amplified with specific primers. CYP3A5 alleles were confirmed by direct sequencing of PCR products on an automated AB13100 capillary sequencer. We studied 48 renal transplant patients (age at tx 12±0.5yr, 22 boys) receiving TAC, MMF, MP. Of these, 79% were CYP3A5*3/*3 (non-expressers homozygotes) and 21% were CYP3A5*1/*3 (expressers). TAC trough levels were 7.1±0.4ng/mL in CYP3A5*3/*3 patients and 6.5±0.7ng/mL in CYP3A5*1/*3 group (p=0.03). CYP3A5*1/*3 patients had lower levels of dose-adjusted TAC (36.7±5.8ng/mL/mg/kg/day) to achieve target blood concentration and required higher daily dose per weight (0.21±0.03mg/kg/day) than CYP3A5*3/*3 patients, 72.4±8.0ng/mL/mg/kg/day and 0.13±0.01mg/kg/day (p<0.001). Prepubertal patients with different CYP3A5 polymorphisms required significant higher TAC doses and achieved lower dose-normalized concentration compared with pubertal patients. Both TAC dose and adjusted-dose correlated with daily MP dose in CYP3A5*1*3 (r: 0.4, p<0.03 and r: 0.4, p<0.03) and in CYP3A5*3*3 (r: 0.6, p<0.01 and r: 0.47, p<0.001) patients. CYP3A5 polymorphism performed before tx could contribute to a better individualization of TAC therapy. The higher TAC dose in prepubertal patients and the pharmacological interactions between MP and TAC may not be fully explained by different CYP3A5 polymorphisms.

[Cell therapy for diabetes mellitus: beyond stem cells]. / Terapia celular para el tratamiento de la diabetes: Más allá de las células madre.

Type 1 diabetes is an autoimmune disease of unknown etiology characterized by destruction of pancreatic beta cells, leading to absolute insulin deficiency. Standard therapy includes the use of exogenous insulin. However, due to the difficulty to achieve a tight metabolic control, a number of patients will present severe complications, including vascular, renal and ophthalmologic disease. On the other hand, a more strict metabolic control is often associated with episodes of life threatening hypoglycemia. This motivated the research and development of new alternative treatments, such as the transplantation of insulin producing beta cells, obtained from cadaveric pancreatic islets. Best results with this therapy were observed with consecutive islet injection from more than one donor and immunosuppressive therapy without steroids. However, the scarcity of organs as well as an increased immune reaction derived from the use of pancreas from different donors have limited this therapy to markedly selected patients and highly experienced centers. Furthermore, lifelong administration of immunosuppressive drugs may produce undesired secondary effects. Regenerative medicine opens the possibility of using stem cells capable of differentiating into insulin-producing cells after stimulation by diverse trophic factors that may act over stem cells located within a specific tissue.

A simple approach for mouse embryonic stem cells isolation and differentiation inducing embryoid body formation.

Stem cells were derived from hatched blastocyst-stage mouse embryos of the C57BL/6 strain employing a knockout serum replacement instead of the traditional fetal calf serum, thereby avoiding the use of immunosurgery. Although fetal calf serum was not good for isolation of stem cells, a combination of this serum plus knockout serum increased the expansion rate of the cell culture. The derived cells were capable of maintaining an undifferentiated state during several passages, as demonstrated by the presence of alkaline phosphatase activity, stage-specific embryonic antigen 1 (SSEA-1), and octamer binding protein 4 (Oct-4). Suspension culture in bacteriological dishes gave better results than the hanging drop method for differentiation by means of embryoid body formation. Mouse embryonic stem cells showed spontaneous differentiation into derivatives of the 3 germ layers in culture media supplemented with fetal calf serum but not with knockout serum.

Reconsolidation of declarative memory in humans.

The reconsolidation hypothesis states that a consolidated memory could again become unstable and susceptible to facilitation or impairment for a discrete period of time after a reminder presentation. The phenomenon has been demonstrated in very diverse species and types of memory, including the human procedural memory of a motor skill task but not the human declarative one. Here we provide evidence for both consolidation and reconsolidation in a paired-associate learning (i.e., learning an association between a cue syllable and the respective response syllable). Subjects were given two training sessions with a 24-h interval on distinct verbal material, and afterward, they received at testing two successive retrievals corresponding to the first and second learning, respectively. Two main results are noted. First, the first acquired memory was impaired when a reminder was presented 5 min before the second training (reconsolidation), and also when the second training was given 5 min instead of 24 h after the first one (consolidation). Second, the first retrieval proved to influence negatively on the later one (the retrieval-induced forgetting [RIF] effect), and we used the absence of this RIF effect as a very indicator of the target memory impairment. We consider the demonstration of reconsolidation in human declarative memory as backing the universality of this phenomenon and having potential clinical relevance. On the other hand, we discuss the possibility of using the human declarative memory as a model to address several key topics of the reconsolidation hypothesis.

[Structural and functional polarity of porcine hepatocyte cultured spheroids]. / Análisis de la polaridad estructural y funcional de estructuras tridimensionales (esferoides) de células hepáticas porcinas cultivadas in vitro.

Hepatocytes are epithelial cells that show a complex polarity in vivo. However, hepatocytes isolated and cultured in vitro normally lose both their differentiated properties and polarity. Culturing hepatocyte spheroids seems to be the accurate approach to maintain tissue level of organization. The structural and functionalpolarities of pig liver spheroids were analyzed in this work. Swine liver cells were isolated and cultured as spheroids. Their metabolic activity was proved through the metabolism of diazepam, ammonium and synthesis of albumin. Several structural features show the presence of polarity in the cells inside the spheroids. Reticular and collagen fibers, as well as Ck19(+) cells forming duct-like structures were found. _eta and _-catenins and pancadherins were positive in different regions of the spheroids, mainly in the outer cell layers, which have cuboidal epithelia features. The scanning electron microscopy showed a tightly compacted architecture, with smooth surface. The transmission electron microscopy analysis showed bile canaliculi with microvilli, tight junctions, zonula adherens and desmosome-like junctions. Well-maintained cellular organelles, as mitochondria, nucleus, nucleolus, peroxisomes, endoplasmic reticulum, were seen in the spheroids. A complex inner bile canaliculi network was shown by using a fluorescent bile acid analogue incorporated and excreted by the spheroids. Furthermore, excretion of a normal pattern of bile acids was demonstrated. The morphology and functionality of the spheroids may provide an appropriate model for applications where the maintenance of liver-specific functions is crucial, as a bioartificial liver device.

Association between GABA(A) receptor subunit polymorphisms and autism spectrum disorder (ASD).

ASD might be associated with alterations in excitation/inhibition ratio and GABA(A) has been implicated since it mediates synaptic inhibition. Polymorphisms in GABA receptor (GABAR) were studied: significant differences in allele and genotype frequencies observed between cases and controls (rs1912960, GABRA4). Haplotype analysis: rs1912960 (GABRA4) and rs211037 (GABRG2) overrepresented in cases. Rs1912960 has been associated with ASD and rs211037 with epilepsy. GABRA4 is associated with autism in the Argentinean dataset independently or in combination with GABRG2.

Effect of the embolization of completely unpurified islets on portal vein pressure and hepatic biochemistry in clinical practice.

Here we report on the impact of completely unpurified islet transplantation on the portal vein pressure (PVP) and the hepatic biochemistry in the peritransplant period and on follow-up. Type I diabetic patients underwent simultaneous kidney and islet transplantation. Islets were not purified from the acinar tissue to prevent loss of endocrine mass. Each patient received a mean 521,846 +/- 201,539.4 islet equivalents (7812.1 islet equivalents/kg/recipient). Immunosuppression and peritransplant medication were given according to the Giessen protocol. The islets were injected into the left hepatic lobe through the umbilical vein. PVP was recorded at time 0 and every 5 min throughout cell infusion. Liver function was assessed daily for the first 10 days, and on follow-up. Basal, peak, and final PVP were 12 +/- 3.8, 25.1 +/- 7.9, and 19.5 +/- 6.2 mmHg, respectively (basal vs. final, p < 0.05). Bilirubin, alkaline phosphatase, prothrombin time, and APTT stayed within normal range. Peak aspartate aminotransferase (AST), alanine aminotransferase (ALT), and serum amylase were 109.4 +/- 61.2 IU/L (basal vs. peak, not significant), 79.5 +/- 56.9 IU/L (basal vs. peak, not significant), and 887.5 +/- 153.6 IU/L (basal vs. peak, p = 0.02), respectively. In all cases AST, ALT, and amylase normalized within 6 days posttransplant and remained so on follow-up (longest control, 33 months posttransplant). Although the intrahepatic infusion of unpurified pancreatic islets affects both the portal vein pressure and the hepatic biochemical profile, this effect is transient and does not compromise the safety of the procedure.

Evidence for interaction between markers in GABA(A) receptor subunit genes in an Argentinean autism spectrum disorder population.

Autism spectrum disorders (ASD) can be conceptualized as a genetic dysfunction that disrupts development and function of brain circuits mediating social cognition and language. At least some forms of ASD may be associated with high level of excitation in neural circuits, and gamma-aminobutyric acid (GABA) has been implicated in its etiology. Single-nucleotide polymorphisms (SNP) located within the GABA receptor (GABAR) subunit genes GABRA1, GABRG2, GABRB3, and GABRD were screened. A hundred and thirty-six Argentinean ASD patients and 150 controls were studied, and the contribution of the SNPs in the etiology of ASD was evaluated independently and/or through gene-gene interaction using multifactor dimensionality reduction (MDR) method. From the 18 SNP studied, 11 were not present in our Argentinean population (patients and controls) and 1 SNP had minor allele frequency < 0.1%. For the remaining six SNPs, none provided statistical significant association with ASD when considering allelic or genotypic frequencies. Non-significant association with ASD was found for the haplotype analysis. MDR identified evidence for synergy between markers in GABRB3 (chromosome 15) and GABRD (chromosome 1), suggesting potential gene-gene interaction across chromosomes associated with increased risk for autism (testing balanced accuracy: 0.6081 and cross-validation consistency: 10/10, P < 0.001). Considering our Argentinean ASD sample, it can be inferred that GABRB3 would be involved in the etiology of autism through interaction with GABRD. These results support the hypothesis that GABAR subunit genes are involved in autism, most likely via complex gene-gene interactions.

Skin fibroblasts from patients with type 1 diabetes (T1D) can be chemically transdifferentiated into insulin-expressing clusters: a transgene-free approach.

The conversion of differentiated cells into insulin-producing cells is a promising approach for the autologous replacement of pancreatic cells in patients with type 1 diabetes (T1D). At present, cellular reprogramming strategies encompass ethical problems, epigenetic failure or teratoma formation, which has prompted the development of new approaches. Here, we report a novel technique for the conversion of skin fibroblasts from T1D patients into insulin-expressing clusters using only drug-based induction. Our results demonstrate that skin fibroblasts from diabetic patients have pancreatic differentiation capacities and avoid the necessity of using transgenic strategies, stem cell sources or global demethylation steps. These findings open new possibilities for studying diabetes mechanisms, drug screenings and ultimately autologous transgenic-free regenerative medicine therapies in patients with T1D.

Inhibition of brain ST8SiaIII sialyltransferase leads to impairment of procedural memory in mice.

Several glycoproteins in mammalian brains contain α2,8-linked disialic acid residues. We previously showed a constant expression of disialic acid (DiSia) in the hippocampus, olfactory bulb and cortex, and a gradual decrease of expression in the cerebellum from neonatal to senile mice. Previous publications indicate that neurite extension of neuroblastoma-derived Neuro2A cells is inhibited in the presence of DiSia antibody. Based on this, we treated Neuro2A cell cultures with RNA interference for ST8SiaIII mRNA, the enzyme responsible for DiSia formation. We observed that neurite extension was inhibited by this treatment. Taking this evidence into consideration and the relationship of the cerebellum with learning and memory, we studied the role of DiSia expression in a learning task. Through delivery of pST8SiaIII into the brains of C57BL/6 neonatal mice, we inhibited the expression of ST8SiaIII. ST8SiaIII mRNA and protein expressions were analyzed by real-time PCR and western blot, respectively. In this work, we showed that pST8SiaIII-treated mice presented a significantly reduced level of ST8SiaIII mRNA in the cerebellum (p<0.01) in comparison to control mice at 8 days after treatment. It is also noted that these levels returned to baseline values in the adulthood. Then, we evaluated behavioural performance in the T-Maze, a learning task that estimates procedural memory. At all ages, pST8SiaIII-treated mice showed a lower performance in the test session, being most evident at older ages (p<0.001). Taken all together, we conclude that gene expression of ST8SiaIII is necessary for some cognitive tasks at early postnatal ages, since reduced levels impaired procedural memory in adult mice.

Neurogenesis interferes with the retrieval of remote memories: forgetting in neurocomputational terms.

In contrast to models and theories that relate adult neurogenesis with the processes of learning and memory, almost no solid hypotheses have been formulated that involve a possible neurocomputational influence of adult neurogenesis on forgetting. Based on data from a previous study that implemented a simple but complete model of the main hippocampal circuitry (Weisz & Argibay, 2009), we now test this model under different situations to better study the case of remote memories. The results of this work show that following neurogenesis, the new, ongoing memories in the hippocampus are better retained than when no neurogenesis occurs at all, while the older memories are affected (to a lesser extent) by a special type of interference that is different from interference that occurs with an increasing number of memories per se. This work adds a new point of analysis in support of the interference view that might lead to the forgetting of memories in the hippocampus as they are transferred to neocortex for long-term storage, consistent with the Complementary Learning Systems models of system-level consolidation. Attention should be directed to the specific causes of interference; the results of this work signal a type of distortion of remote memories that is produced by the birth and the growth of new processing units, which results in a subtly impoverished retrieval as new neurons become active. The proposals of this model fit well with some empirical findings that are related to the issue. In the future, as new evidence emerges, we believe that this biological process, which is largely related to learning and memory, will also help to shape our ideas about normal forgetting and its possible contributions to system consolidation.

The computational influence of neurogenesis in the processing of spatial information in the dentate gyrus.

This study was designed to analyze the effect of hippocampal neurogenesis on the spatial maps of granule cells. Accordingly, we developed and improved an artificial neural network that was originally proposed by Aimone. Many biological processes were included in this revised model to improve the biological relevance of the results. We proposed a novel learning-testing protocol to analyze the activation of encoding place cells across contexts and over time in the dentate gyrus. We observed that, regardless of the presence of neurogenesis, the quantity and morphology of the place fields were represented in the same manner by granule cells. Additionally, we observed that neurogenesis was an effective mechanism for reducing the degree of rate remapping that occurred in the place fields of the granule cells.

[Non-symbolic addition in preschool children from Argentina]. / Adición no simbólica en niños preescolares de la Argentina.

INTRODUCTION: Children take years to learn symbolic arithmetic. Nevertheless, such as animals and human adults, infants and children can represent approximate number in arrays of objects and sequences of events, and use these capacities to perform approximate addition and subtraction. OBJECTIVE: To evaluate whether preschool children without formal education could perform non-symbolic additions, through abstract representations. METHODS: We evaluated 17 preschoolers from a private kindergarten recruited from the city of Buenos Aires. They had to add to groups of blue dots, and then compare their addition with a third group of red dots, determining if the blue or the red dots were more numerous. We measured accuracy of responses of each child. RESULTS: Across all the problems, children performed well above chance (67.89%, chance= 50%, t(16)= 6.89, p <0.001), showing the characteristic ratio effect [F(1, 16)= 8.45, p <0.01, ANOVA], and without resort to non-arithmetic strategies. CONCLUSIONS: This study provides further evidence regarding the non-symbolic arithmetic skill present before formal education, and together with recent research, raises important contributions in education, trying to understand how children learn mathematics and to establish new methods of teaching.

[Hepatic cell transplantation in childhood. State of the art]. / Estado actual del trasplante de células hepáticas en Pediatría.

To date, children who suffer from a certain type of illness such as hepatic failure, could benefit with a non conventional functional replacement as an alternative to liver transplantation. Deterioration and death of patients on waiting list encourage the search for alternatives methods within transplantation. Liver cell transplantation has become a potential alternative treatment whose validation as an alternative or as a bridge until the donor appears, will probably contribute to improve the quality of life and survival of the patients. The aim of this review is to describe the state of the art of hepatocyte transplantation in pediatric patients.

Xenotrasplante de islotes pancreáticos en el tratamiento de la diabetes insulino dependiente / Xenotransplantation of pancreatic islets in the treatment of insulin-dependent diabetes

La diabetes es una alteración metabólica de elevada incidencia: Su frecuencia aumenta en asociación con la edad. Desatendida genera notable morbilidad. El envejecimiento poblacional otorga interés cardinal al tratamiento de esta patología y una de las propuestas terapéuticas es el trasplante. El homotrasplante brinda resultados promisorios. La parvedad de donantes y la complejidad del procedimiento disminuyen su factibilidad. Se han propuesto diversos xenotrasplantes. Sucinta revisión del estado actual del asunto, en opinión de un experto

A putative role for neurogenesis in neuro-computational terms: inferences from a hippocampal model.

New neurons are generated daily in the hippocampus during adult life. They are integrated into the existing neuronal circuits according to several factors such as age, physical exercise and hormonal status. At present, the role of these new neurons is debated. Computational simulations of hippocampal function allow the effects of neurogenesis to be explored, at least from a computational perspective. The present work implements a model of neurogenesis in the hippocampus with artificial neural networks, based on a standard theoretical model of biologically plausible hippocampal circuits. The performance of the model in retrieval of a variable number of patterns or memories was evaluated (episodic memory evaluation). The model increased, in a phase subsequent to initial learning, the number of granular cells by 30% relative to their initial number. In contrast to a model without neurogenesis, the retrieval of recent memories was very significantly improved, although remote memories were only slightly affected by neurogenesis. This increase in the quality of retrieval of new memories represents a clear advantage that we attribute to the neurogenesis process. This advantage becomes more significant for higher storage loads. The model presented here suggests an important functional role of neurogenesis on learning and memory.