An optimized method for establishing high purity murine CD8+ T cell cultures.

Establishing CD8(+) T cell cultures has been empirical and the published methods have been largely individual laboratory based. In this study, we optimized culturing conditions and show that IL-2 concentration is the most critical factor for the success of establishing CD8(+) T cell cultures. High IL-2 concentration encouraged T cells to non-specifically proliferate, express a B cell marker, B220, and undergo apoptosis. These cells also lose typical irregular T cell morphology and are incapable of sustaining long-term cultures. Using tetramer and intracellular cytokine assessments, we further demonstrated that many antigen-specific T cells have been rendered nonfunctional when expanded under high IL-2 concentration. When IL-2 is used in the correct range, B220-mediated cell depletion greatly enhanced the success rate of such T cell cultures.

Ectodysplasin has a dual role in ectodermal organogenesis: inhibition of Bmp activity and induction of Shh expression.

Ectodermal organogenesis is regulated by inductive and reciprocal signalling cascades that involve multiple signal molecules in several conserved families. Ectodysplasin-A (Eda), a tumour necrosis factor-like signalling molecule, and its receptor Edar are required for the development of a number of ectodermal organs in vertebrates. In mice, lack of Eda leads to failure in primary hair placode formation and missing or abnormally shaped teeth, whereas mice overexpressing Eda are characterized by enlarged hair placodes and supernumerary teeth and mammary glands. Here, we report two signalling outcomes of the Eda pathway: suppression of bone morphogenetic protein (Bmp) activity and upregulation of sonic hedgehog (Shh) signalling. Recombinant Eda counteracted Bmp4 activity in developing teeth and, importantly, inhibition of BMP activity by exogenous noggin partially restored primary hair placode formation in Eda-deficient skin in vitro, indicating that suppression of Bmp activity was compromised in the absence of Eda. The downstream effects of the Eda pathway are likely to be mediated by transcription factor nuclear factor-kappaB (NF-kappaB), but the transcriptional targets of Edar have remained unknown. Using a quantitative approach, we show in cultured embryonic skin that Eda induced the expression of two Bmp inhibitors, Ccn2/Ctgf (CCN family protein 2/connective tissue growth factor) and follistatin. Moreover, our data indicate that Shh is a likely transcriptional target of Edar, but, unlike noggin, recombinant Shh was unable to rescue primary hair placode formation in Eda-deficient skin explants.

Introducció experimental a la biotecnologia amb clavellina: cultiu in vitro

Treball de recerca realitzat per una alumna d'ensenyament secundari i guardonat amb un Premi CIRIT per fomentar l'esperit científic del Jovent l'any 2009. Es tracta d’una recerca experimental en la que s’han assajat vuit tècniques de cultiu in vitro amb clavellina. El material vegetal s’ha esterilitzat per immersió en una solució diluïda de lleixiu i s’ha manipulat de manera estèril. En tots els casos el medi de cultiu utilitzat ha estat el MS amb una concentració de sacarosa i reguladors de creixement variable segons l’experiment. La incubació dels cultius s’han dut a terme en una cambra amb control de fotoperíode durant 4 setmanes. Els diferents reguladors de creixement han mostrat un clar efecte sobre les seccions de tija. Els explants cultivats en medi lliure d’hormones han crescut menys que els exposats a diverses concentracions de NAA i BA. Aquests tractaments hormonals han originat símptomes de creixement anòmals (engruiximents a la base i vitrificació). La presencia de 2,4-D ha afavorit la formació de cal•lus i d’arrels per organogènesi adventícia indirecta. L’obtenció de plàntules per germinació in vitro de llavors ha permès reduir notablement les pèrdues per contaminació, mentre que el subcultiu d’aquestes ha donat unes tases de micropropagació de 7.2 seccions/plàntula. Ha estat possible aclimatar aquestes vitroplantes per tal d’adaptar-les a les condicions de camp. No hem pogut obtenir organogènesis adventícia ni embriogènesi somàtica a partir d anteres ni hem pogut iniciar un cultiu de cèl•lules a partir dels cal•lus. Tot i la complexitat d’aquestes tècniques, és possible dur-les a terme en un laboratori escolar.

Hypoxia/hypoglycemia preconditioning prevents the loss of functional electrical activity in organotypic slice cultures.

In cerebral ischemic preconditioning (IPC), a first sublethal ischemia increases the resistance of neurons to a subsequent severe ischemia. Despite numerous studies, the mechanisms are not yet fully understood. Our goal is to develop an in vitro model of IPC on hippocampal organotypic slice cultures. Instead of anoxia, we chose to apply varying degrees of hypoxia that allows us various levels of insult graded from mild to severe. Cultures are exposed to combined oxygen and glucose deprivation (OGD) of varying intensities, ranging from mild to severe, assessing both the electrical activity and cell death. IPC was accomplished by exposure to the mildest ischemia condition (10% of O2 for 15 min) 24 h before the severe deprivation (5% of O2 for 30 min). Interestingly, IPC not only prevented delayed ischemic cell death 6 days after insult but also the transient loss of evoked potential response. The major interest and advantage of this system over both the acute slice preparation and primary cell cultures is the ability to simultaneously measure the delayed neuronal damage and neuronal function.

Optimum in vitro expansion of human antigen-specific CD8 T cells for adoptive transfer therapy.

Increasing evidence suggests that adoptive transfer of antigen-specific CD8(+) T cells could represent an effective strategy in the fight against chronic viral infections and malignancies such as melanoma. None the less, a major limitation in the implementation of such therapy resides in the difficulties associated with achieving rapid and efficient expansion of functional T cells in culture necessary to obtain the large numbers required for intravenous infusion. Recently, the critical role of the cytokines interleukin (IL)-2, IL-7 and IL-15 in driving T cell proliferation has been emphasized, thus suggesting their use in the optimization of expansion protocols. We have used major histocompatibility complex (MHC) class I/peptide multimers to monitor the expansion of antigen-specific CD8 T lymphocytes from whole blood, exploring the effect of antigenic peptide dose, IL-2, IL-7 and IL-15 concentrations on the magnitude and functional characteristics of the antigen-specific CD8(+) T cells generated. We show here that significant expansions of antigen-specific T cells, up to 50% of the CD8(+) T cell population, can be obtained after a single round of antigen/cytokine (IL-2 or IL-15) stimulation, and that these cells display good cytolytic and interferon (IFN)-gamma secretion capabilities. Our results provide an important basis for the rapid in vitro expansion of autologous T cells from the circulating lymphocyte pool using a simple procedure, which is necessary for the development of adoptive transfer therapies.

Substituts épidermiques et dermo-épidermiques vivants pour le traitement des grands brûlés [Living epidermal and dermal substitutes for treatment of severely burned patients]

Epidermal and dermal cells can be multiplied in vitro using different techniques. Under particular conditions, the structure and the function of the original tissues are partly recreated. Autologous epidermal substitutes for wound coverage in deep burns are prepared in less than three weeks. Bilayered skin equivalents containing a dermal component are obtained by growing epidermal cells on a reconstructed dermal substitute or by juxtaposing stratified cultures of keratinocytes and fibroblasts. New technologies are required to optimise the nutrition of three-dimensional cultures of skin cells, which should lead to further progress in the area of skin reconstruction.

A role for CD147 in thymic development.

We have previously identified a mAb that binds to a molecule expressed preferentially on the surface of cycling thymocytes. In this study the molecule recognized by this mAb has been identified in the mouse as CD147 (basigin) by expression cloning. We show that CD147 expression correlates with cycling of immature thymocytes even in the absence of TCRbeta selection and that ligation of this molecule on immature fetal thymocytes inhibits their further development into mature T cells.

Involvement of nuclear factor-kappaB in macrophage migration inhibitory factor gene transcription up-regulation induced by interleukin- 1 beta in ectopic endometrial cells.

OBJECTIVE: To investigate the involvement of the nuclear factor (NF)-kappaB in the interleukin (IL)-1 beta-mediated macrophage migration inhibitory factor (MIF) gene activation. DESIGN: Prospective study. SETTING: Human reproduction research laboratory. PATIENT(S): Nine women with endometriotic lesions. INTERVENTION(S): Endometriotic lesions were obtained during laparoscopic surgery. MAIN OUTCOME MEASURE(S): The MIF protein secretion was analyzed by ELISA, MIF mRNA expression by quantitative real-time polymerase chain reaction (PCR), NF-kappaB translocation into the nucleus by electrophoresis mobility shift assay, I kappaB phosphorylation and degradation by Western blot, and human MIF promoter activity by transient cell transfection. RESULT(S): This study showed a significant dose-dependent increase of MIF protein secretion and mRNA expression, the NF-kappaB translocation into the nucleus, I kappaB phosphorylation, I kappaB degradation, and human MIF promoter activity in endometriotic stromal cells in response to IL-1 beta. Curcumin (NF-kappaB inhibitor) significantly inhibited all these IL-1 beta-mediated effects. Analysis of the activity of deletion constructs of the human MIF promoter and a computer search localized two putative regulatory elements corresponding to NF-kappaB binding sites at positions -2538/-2528 bp and -1389/-1380 bp. CONCLUSION(S): This study suggests the involvement of the nuclear transcription factor NF-kappaB in MIF gene activation in ectopic endometrial cells in response to IL-1 beta and identifies a possible pathway of endometriosis-associated inflammation and ectopic cell growth.

Measuring the inflammasome.

Inflammasomes are multiprotein complexes whose activity has been implicated in physiological and pathological inflammation. The hallmarks of inflammasome activation are the secretion of the mature forms of Caspase-1 and IL-1β from cells of the innate immune system. This protocol covers the methods required to study inflammasome activation using mouse bone marrow-derived dendritic cells (BMDCs) as a model system. The protocol includes the generation and handling of BMDCs, the stimulation of BMDCs with established Nlrp3 inflammasome activators, and the measurement of activation by both ELISA and western blot. These methods can be useful for the study of potential inflammasome activators, and of the signaling pathways involved in inflammasome activation. General considerations are provided that may help in the design and optimization of modified methods for the study of other types of inflammasomes and in other cell types.

Autophagy promotes survival of retinal ganglion cells after optic nerve axotomy in mice.

Autophagy is an essential recycling pathway implicated in neurodegeneration either as a pro-survival or a pro-death mechanism. Its role after axonal injury is still uncertain. Axotomy of the optic nerve is a classical model of neurodegeneration. It induces retinal ganglion cell death, a process also occurring in glaucoma and other optic neuropathies. We analyzed autophagy induction and cell survival following optic nerve transection (ONT) in mice. Our results demonstrate activation of autophagy shortly after axotomy with autophagosome formation, upregulation of the autophagy regulator Atg5 and apoptotic death of 50% of the retinal ganglion cells (RGCs) after 5 days. Genetic downregulation of autophagy using knockout mice for Atg4B (another regulator of autophagy) or with specific deletion of Atg5 in retinal ganglion cells, using the Atg5(flox/flox) mice reduces cell survival after ONT, whereas pharmacological induction of autophagy in vivo increases the number of surviving cells. In conclusion, our data support that autophagy has a cytoprotective role in RGCs after traumatic injury and may provide a new therapeutic strategy to ameliorate retinal diseases.

Vasopressin-stimulated CFTR Cl- currents are increased in the renal collecting duct cells of a mouse model of Liddle's syndrome.

Liddle's syndrome is a genetic form of hypertension linked to Na(+) retention caused by activating mutations in the COOH terminus of the beta or gamma subunit of the epithelial sodium channel (ENaC). In this study, we used the short-circuit current (I(sc)) method to investigate the effects of deamino-8-d-arginine vasopressin (dDAVP) on Na(+) and Cl(-) fluxes in primary cultures of cortical collecting ducts (CCDs) microdissected from the kidneys of mice with Liddle's syndrome carrying a stop codon mutation, corresponding to the beta-ENaC R(566) stop mutation (L) found in the original pedigree. Compared to wild-type (+/+) CCD cells, untreated L/+ and L/L CCD cells exhibited 2.7- and 4.2-fold increases, respectively, in amiloride-sensitive (Ams) I(sc), reflecting ENaC-dependent Na(+) absorption. Short-term incubation with dDAVP caused a rapid and significant increase (approximately 2-fold) in Ams I(sc) in +/+, but not in L/+ or L/L CCD cells. In sharp contrast, dDAVP induced a greater increase in 5-nitro-2-(3-phenylpropamino)benzoate (NPPB)-inhibited apical Cl(-) currents in amiloride-treated L/L and L/+ cells than in their +/+ counterparts. I(sc) recordings performed under apical ion substituted conditions revealed that the dDAVP-stimulated apical secretion of Cl(-), which was absent in cultured CCDs lacking CFTR, was 1.8-fold greater in L/+ and 3.7-fold greater in L/L CCD cells than in their +/+ CCD counterparts. After the basal membrane had been permeabilized with nystatin and a basal-to-apical Cl(-) gradient had been imposed, dDAVP also stimulated larger Cl(-) currents across L/L and L/+ CCD layers than +/+ CCD layers. These findings demonstrate that vasopressin stimulates greater apical CFTR Cl(-) conductance in the renal CCD cells of mice with Liddle's syndrome than in wild-type mice. This effect could contribute to the enhanced NaCl reabsorption observed in the distal nephron of patients with Liddle's syndrome.

Preparation and analysis of fetal liver extracts.

The aim of this work is to describe the techniques that have been used for preparation and analysis of whole fetal liver extracts destined for in utero transplantation. Nine fetal livers between 12 and 17 weeks of gestation were prepared: cell counts and assessment of the hematopoietic cell viability were performed on cell suspensions. Hepatocytes represented 40 to 80% of the whole cell population. The remaining cells were constituted by hematopoietic cells (mainly erythroblasts), as well as by endothelial cells. The latter expressed CD34 on their surface, interfering with the assessment of CD34+ hematopoietic cells by flow cytometry. Direct visual morphologic control using alkaline phosphatase anti-alkaline phosphatase techniques was needed to differentiate hematopoietic from extra-hematopoietic CD34+ cells. Between 3.0 and 34.6 x 10(6) CD34+ viable hematopoietic cells were collected per fetal liver. Adequate differentiation of these cells into burst-forming units erythroid (BFU-E), colony-forming units granulocyte-macrophage (CFU-GM), and colony-forming units granulocyte erythroid macrophage megakaryocyte (CFU-GEMM) has been shown for each sample in clonogeneic cultures. In conclusion, fetal liver is a potential source of hematopoietic stem cells. Their numeration, based on the presence of CD34, is hampered by the expression of this antigen on other cells contained in the liver cell extract, in particular endothelial cells.

Echovirus 30 associated with cases of aseptic meningitis in state of Pará, Northern Brazil

Investigation of the aetiology of viral meningitis in Brazil is most often restricted to cases that occur in the Southern and Southeastern Regions; therefore, the purpose of this study is to describe the viral meningitis cases that occurred in state of Pará, Northern Brazil, from January 2005-December 2006. The detection of enterovirus (EV) in cerebrospinal fluid was performed using cell culture techniques, RT-PCR, nested PCR and nucleotide sequencing. The ages of the 91 patients ranged from < one year old to > 60 years old (median age 15.90 years). Fever (87.1%), headache (77.0%), vomiting (61.5%) and stiffness (61.5%) were the most frequent symptoms. Of 91 samples analyzed, 18 (19.8%) were positive for EV. Twelve were detected only by RT- PCR followed by nested PCR, whereas six were found by both cell culture and RT-PCR. From the last group, five were sequenced and classified as echovirus 30 (Echo 30). Phylogenetic analyses revealed that Echo 30 detected in Northern Brazil clustered within a unique group with a bootstrap value of 100% and could constitute a new subgroup (4c) according to the phylogenetic tree described by Oberste et al. (1999). This study described the first molecular characterization of Echo 30 in Brazil and this will certainly contribute to future molecular analyses involving strains detected in other regions of Brazil.

Histological and histochemical evaluation of human oral mucosa constructs developed by tissue engineering

Reconstruction of large oral mucosa defects is often challenging, since the shortage of healthy oral mucosa to replace the excised tissues is very common. In this context, tissue engineering techniques may provide a source of autologous tissues available for transplant in these patients. In this work, we developed a new model of artificial oral mucosa generated by tissue engineering using a fibrin-agarose scaffold. For that purpose, we generated primary cultures of human oral mucosa fibroblasts and keratinocytes from small biopsies of normal oral mucosa using enzymatic treatments. Then we determined the viability of the cultured cells by electron probe quantitative X-ray microanalysis, and we demonstrated that most of the cells in the primary cultures were alive and had high K/Na ratios. Once cell viability was determined, we used the cultured fibroblasts and keratinocytes to develop an artificial oral mucosa construct by using a fibrin-agarose extracellular matrix and a sequential culture technique using porous culture inserts. Histological analysis of the artificial tissues showed high similarities with normal oral mucosa controls. The epithelium of the oral substitutes had several layers, with desmosomes and apical microvilli and microplicae. Both the controls and the oral mucosa substitutes showed high suprabasal expression of cytokeratin 13 and low expression of cytokeratin 10. All these results suggest that our model of oral mucosa using fibrin-agarose scaffolds show several similarities with native human oral mucosa.

Functionalized nanostructures with application in regenerative medicine

In the last decade, both regenerative medicine and nanotechnology have been broadly developed leading important advances in biomedical research as well as in clinical practice. The manipulation on the molecular level and the use of several functionalized nanoscaled materials has application in various fields of regenerative medicine including tissue engineering, cell therapy, diagnosis and drug and gene delivery. The themes covered in this review include nanoparticle systems for tracking transplanted stem cells, self-assembling peptides, nanoparticles for gene delivery into stem cells and biomimetic scaffolds useful for 2D and 3D tissue cell cultures, transplantation and clinical application.