Differentially expressed gene profile in the 6-hydroxy-dopamine-induced cell culture model of Parkinson's disease.

Parkinson's disease (PD) is a chronic neurodegenerative disorder characterized by progressive loss of dopaminergic (DA) neurons of the substantia nigra pars compacta with unknown aetiology. 6-Hydroxydopamine (6-OHDA) treatment of neuronal cells is an established in vivo model for mimicking the effect of oxidative stress found in PD brains. We examined the effects of 6-OHDA treatment on human neuroblastoma cells (SH-SY5Y) and primary mesencephalic cultures. Using a reverse arbitrarily primed polymerase chain reaction (RAP-PCR) approach we generated reproducible genetic fingerprints of differential expression levels in cell cultures treated with 6-OHDA. Of the resulting sequences, 23 showed considerable homology to known human coding sequences. The results of the RAP-PCR were validated by reverse transcription PCR, real-time PCR and, for selected genes, by Western blot analysis and immunofluorescence. In four cases, [tomoregulin-1 (TMEFF-1), collapsin response mediator protein 1 (CRMP-1), neurexin-1, and phosphoribosylaminoimidazole synthetase (GART)], a down-regulation of mRNA and protein levels was detected. Further studies will be necessary on the physiological role of the identified proteins and their impact on pathways leading to neurodegeneration in PD.

Microtubule-dependent cell morphogenesis in the fission yeast.

In many systems, microtubules contribute spatial information to cell morphogenesis, for instance in cell migration and division. In rod-shaped fission yeast cells, microtubules control cell morphogenesis by transporting polarity factors, namely the Tea1-Tea4 complex, to cell tips. This complex then recruits the DYRK kinase Pom1 to cell ends. Interestingly, recent work has shown that these proteins also provide long-range spatial cues to position the division site in the middle of the cell and temporal signals to coordinate cell length with the cell cycle. Here I review how these microtubule-associated proteins form polar morphogenesis centers that control and integrate both spatial and temporal aspects of cell morphogenesis.

Evaluation of spleen cell population and effect of splenectomy on granuloma modulation in BALB/c mice infected with Schistosoma mansoni

A kinetic study of the cells present in the spleen of BALB/c mice infected with Schistosoma mansoni was carried out. The lymphocytes were evaluated phenotypically with monoclonal antibodies and the effect of splenectomy on the modulation of periovular granuloma was also investigated. The infected mice had proportional increases in the numbers of neutophils, plasma cells, macrophages and eosinophils in the spleen. The largest number of neutrophil, plasma cells and macrophage were found between the 8th and the 12th week of infection, while the amount of eosinophils were higher later on, around the 20th week. The lymphocytes phenotipically characterized as Thy 1.2, Lyt 1.2 (CD4) increased mildly in proportional numbers. However, the percentage of lymphocytes with the Lyt 2.2 (CD8) phenotype, which is characteristic of supressor and cytotoxic T cells, increased significantly with the progress of the disease. The numbers of B lymphocytes, which comprise 50% of the mononuclear cells present in the spleen, increased significantly till the 16th week they began to decrease. The mean diameters of periovular granulomas were comparatively similar in both experimental groups (splenectomized and non-splenectomized mice). However, the evolutive types of granuloma (exudative, intermediate and fibrous) in splenectomized mice were proprtionally different from those of non splenectomized mice in the 16th and 24th week of infection. It is inferred that lymphonodes or other secondary lymphoide organs, in the abscence of the spleen, assume a modulating action on periovular granulomas, although the evolution of the granulomas is somehow delayed in splenectomized mice.

Endothelial Cell-Derived Nitric Oxide Enhances Aerobic Glycolysis in Astrocytes via HIF-1α-Mediated Target Gene Activation.

Astrocytes exhibit a prominent glycolytic activity, but whether such a metabolic profile is influenced by intercellular communication is unknown. Treatment of primary cultures of mouse cortical astrocytes with the nitric oxide (NO) donor DetaNONOate induced a time-dependent enhancement in the expression of genes encoding various glycolytic enzymes as well as transporters for glucose and lactate. Such an effect was shown to be dependent on the hypoxia-inducible factor HIF-1α, which is stabilized and translocated to the nucleus to exert its transcriptional regulation. NO action was dependent on both the PI3K/Akt/mTOR and MEK signaling pathways and required the activation of COX, but was independent of the soluble guanylate cyclase pathway. Furthermore, as a consequence of NO treatment, an enhanced lactate production and release by astrocytes was evidenced, which was prevented by downregulating HIF-1α. Several brain cell types represent possible sources of NO. It was found that endothelial cells, which express the endothelial NO synthase (eNOS) isoform, constitutively produced the largest amount of NO in culture. When astrocytes were cocultured with primary cultures of brain vascular endothelial cells, stabilization of HIF-1α and an enhancement in glucose transporter-1, hexokinase-2, and monocarboxylate transporter-4 expression as well as increased lactate production was found in astrocytes. This effect was inhibited by the NOS inhibitor l-NAME and was not seen when astrocytes were cocultured with primary cultures of cortical neurons. Our findings suggest that endothelial cell-derived NO participates to the maintenance of a high glycolytic activity in astrocytes mediated by astrocytic HIF-1α activation.

Cell-specific localization of monocarboxylate transporters, MCT1 and MCT2, in the adult mouse brain revealed by double immunohistochemical labeling and confocal microscopy.

Recent evidence suggests that lactate could be a preferential energy substrate transferred from astrocytes to neurons. This would imply the presence of specific transporters for lactate on both cell types. We have investigated the immunohistochemical localization of two monocarboxylate transporters, MCT1 and MCT2, in the adult mouse brain. Using specific antibodies raised against MCT1 and MCT2, we found strong immunoreactivity for each transporter in glia limitans, ependymocytes and several microvessel-like elements. In addition, small processes distributed throughout the cerebral parenchyma were immunolabeled for monocarboxylate transporters. Double immunofluorescent labeling and confocal microscopy examination of these small processes revealed no co-localization between glial fibrillary acidic protein and monocarboxylate transporters, although many glial fibrillary acidic protein-positive processes were often in close apposition to elements labeled for monocarboxylate transporters. In contrast, several elements expressing the S100beta protein, another astrocytic marker found to be located in distinct parts of the same cell when compared with glial fibrillary acidic protein, were also strongly immunoreactive for MCT1, suggesting expression of this transporter by astrocytes. In contrast, MCT2 was expressed in a small subset of microtubule-associated protein-2-positive elements, indicating a neuronal localization. In conclusion, these observations are consistent with the possibility that lactate, produced and released by astrocytes (via MCT1), could be taken up (via MCT2) and used by neurons as an energy substrate.

Compensatory β-cell mass expansion: a big role for a tiny actor.

Comment on: Jacovetti C, et al. J Clin Invest 2012; 122:3541-51.

Improved enzyme-linked immunoadsorbent assay (ELISA) for the study of Trypanosoma cruzi-host cell interaction in vitro

We herein present an improved assay for detecting the presence of Trypanosoma cruzi in infected cultures. Using chagasic human sera (CHS), we were able to detect T. cruzi infection in primary cultures of both peritoneal macrophages and heart muscle cells (MHC). To avoid elevated background levels - hitherto observed in all experiments especially in those using HMC - CHS were preincubated with uninfected cells in monolayers or suspensions prior to being used for detection of T. cruzi in infected monolayers. Preincubation with cell suspensions gave better results than with monolayers, reducing background by up to three times and increasing sensitivity by to twenty times. In addition, the continous fibroplastic cell line L929 was shown to be suitable for preadsorption of CHS. These results indicate that the high background levels observed in previous reports may be due to the presence of human autoantibodies that recognize surface and/or extracellular matrix components in cell monolayers. We therefore propose a modified procedure that increases the performance of the ELISA method, making it an useful tool even in cultures that would otherwise be expected to present low levels of infection or high levels of background

Evidence for multiple B- and T-cell epitopes in Plasmodium falciparum liver-stage antigen 3.

Liver-stage antigen 3 (LSA-3) is a new vaccine candidate that can induce protection against Plasmodium falciparum sporozoite challenge. Using a series of long synthetic peptides (LSP) encompassing most of the 210-kDa LSA-3 protein, a study of the antigenicity of this protein was carried out in 203 inhabitants from the villages of Dielmo (n = 143) and Ndiop (n = 60) in Senegal (the level of malaria transmission differs in these two villages). Lymphocyte responses to each individual LSA-3 peptide were recorded, some at high prevalences (up to 43%). Antibodies were also detected to each of the 20 peptides, many at high prevalence (up to 84% of responders), and were directed to both nonrepeat and repeat regions. Immune responses to LSA-3 were detectable even in individuals of less than 5 years of age and increased with age and hence exposure to malaria, although they were not directly related to the level of malaria transmission. Thus, several valuable T- and B-cell epitopes were characterized all along the LSA-3 protein, supporting the antigenicity of this P. falciparum vaccine candidate. Finally, antibodies specific for peptide LSP10 located in a nonrepeat region of LSA-3 were found significantly associated with a lower risk of malaria attack over 1 year of daily clinical follow-up in children between the ages of 7 and 15 years, but not in older individuals.

Manipulating keratinocyte stem cell proliferation and differentiation using RNA interference

ABSTRACT : The epidermis, the outermost compartment of the skin, is a stratified and squamous epithelium that constantly self-renews. Keratinocytes, which represent the main epidermal population, are responsible for its cohesion and barrier function. Epidermal renewal necessitates a fine equilibrium between keratinocyte proliferation and differentiation. The keratinocyte stem cell, located in the basal cell layer, is responsible for epidermal homeostasis and regeneration during the wound healing process. The transcription factor p63 structurally belongs to the p53 superfamily. It is expressed in the basal and supra-basal cell layers of stratified epithelia and is thought to be important for the renewal or the differentiation of keratinocyte stem cells (Yang et al., 1999; Mills et al., 1999). In order to better understand its function, we established an in vitro model of p63 deficient human keratinocyte stem cells using a shp63 mediated RNA interference. Knockdown of endogenous p63 induces downregulation of cell-adhesion genes as previously described (Carroll et al., 2006). Interestingly, the replating of attached p63-knockdown keratinocytes on a feeder layer results in a loss of attachment and proliferation. They are no longer clonogenic. However, if the same population are replated in a fibrin matrix, extended fibrinolysis is reported, a common process in wound healing, suggesting that p63 regulates the fibrinolytic pathway. This result was confirmed by Q-PCR and shows that the urokinase pathway, which mediates fibrinolysis, is upregulated. Altogether, these findings suggest a mechanism in which the fine tuning of p63 expression promotes attachment or release of the keratinocyte stem cell from the basement membrane by inducing genes of adhesion and/or of fibrinolysis. This mechanism may be important for epidermal self-renewal, differentiation as well as wound healing. Its misregulation may be partly responsible for the p63 knockout phenotype. The downregulation of p63 also induces a decrease in LEKTI expression. LEKTI (lymphoepithelial Kazal-type serine protease inhibitor) is a serine protease inhibitor encoded by the Spink5 gene. It is expressed and secreted in the uppermost differentiated layers of stratified epithelia and plays a role in the desquamation process. When this gene is disrupted, humans develop the Netherton syndrome (Chavanas et al., 2000b). It is a dermatosis characterized by hair dysplasias, ichtyosiform erythroderma and impairment in epidermal barrier function promoting inflammation similarly as in psoriasis with inflammatory infiltrate in excess. TNFα (tumor necrosis factor alpha) and EDA1 (ectodysplasin A1) are two transmembraneprecursors that belong to the TNF superfamily, which is involved in immune and inflammation regulation (Smahi et al., 2002). We suggest that the secreted serine protease inhibitor LEKTI plays a role in the regulation of TNFα and EDA1 precursor cleavage and absence of LEKTI induces excess of inflammation. To investigate this hypothesis, we induced downregulation of Spink5 expression in rat keratinocyte stem cells by using a shSpink5 mediated RNA interference approach. Interestingly, expression of TNFα and EDA1 is modified after knockdown of Spink5 by Q-PCR. Moreover, downregulation of Spink5 induces loss of cohesiveness between keratinocytes and colonies adopt a scattered phenotype. Altogether, these preliminary data suggest that downregulation of LEKTI may play a role in the inflammatory response in Netherton syndrome patients, by regulating TNFα expression.

HTLV-I associated cutaneous T-cell lymphoma: report of a case with atypical clinical presentation

A case of a 20-years-old black man from Salvador, Bahia with HTLV-I associated T cell lymphoma is presented. In spite of the absence of splenomegaly and leukemia, the patient had a marked cephalic tumoral infiltrationassociated with axillary tumors in a pattern not yet described in adult T cell lymphoma. Peripheral blood involvement was observed later on in the course of thedisease. The patient underwent chemotherapy but died seven monts after diagnosis.

Sialoglycoconjugates in Trypanosoma cruzi-host cell interaction: possible biological model - a review

A number of glycoconjugates, including glycolipids and glycoproteins, participate in the process of host-cell invasion by Trypanosoma cruzi and one of the most important carbohydrates involved on this interaction is sialic acid. It is known that parasite trans-sialidase participates with sialic acid in a coordinated fashion in the initial stages of invasion. Given the importance of these sialogycoconjugates, this review sets out various possible biological models for the interaction between the parasite and mammalian cells that possess a sialylated receptor/ligand system.

Emerging roles of non-coding RNAs in pancreatic β-cell function and dysfunction.

Pancreatic β-cells play a central role in glucose homeostasis by tightly regulating insulin release according to the organism's demand. Impairment of β-cell function due to hostile environment, such as hyperglycaemia and hyperlipidaemia, or due to autoimmune destruction of β-cells, results in diabetes onset. Both environmental factors and genetic predisposition are known to be involved in the development of the disease, but the exact mechanisms leading to β-cell dysfunction and death remain to be characterized. Non-coding RNA molecules, such as microRNAs (miRNAs), have been suggested to be necessary for proper β-cell development and function. The present review aims at summarizing the most recent findings about the role of non-coding RNAs in the control of β-cell functions and their involvement in diabetes. We will also provide a perspective view of the future research directions in the field of non-coding RNAs. In particular, we will discuss the implications for diabetes research of the discovery of a new communication mechanism based on cell-to-cell miRNA transfer. Moreover, we will highlight the emerging interconnections between miRNAs and epigenetics and the possible role of long non-coding RNAs in the control of β-cell activities.

Targeting B-cell lymphomas with inhibitors of the MALT1 paracaspase.

The paracaspase MALT1 is an Arg-specific protease that cleaves multiple substrates to promote lymphocyte proliferation and survival. The catalytic activity of MALT1 is normally tightly regulated by antigen receptor triggering, which promotes MALT1 activation by its inducible monoubiquitination-dependent dimerization. Constitutive MALT1 activity is a hallmark of specific subsets of B-cell lymphomas, which are characterized by chromosomal translocations or point mutations that activate MALT1 or its upstream regulators. Recent findings suggest that such lymphomas may be sensitive to treatment with MALT1 inhibitors. Here we review recent progress in the understanding of MALT1 function and regulation, and the development of small molecule MALT1 inhibitors for therapeutic applications.