Capturing epidermal stemness for regenerative medicine.

The skin is privileged because several skin-derived stem cells (epithelial stem cells from epidermis and its appendages, mesenchymal stem cells from dermis and subcutis, melanocyte stem cells) can be efficiently captured for therapeutic use. Main indications remain the permanent coverage of extensive third degree burns and healing of chronic cutaneous wounds, but recent advances in gene therapy technology open the door to the treatment of disabling inherited skin diseases with genetically corrected keratinocyte stem cells. Therapeutic skin stem cells that were initially cultured in research or hospital laboratories must be produced according strict regulatory guidelines, which ensure patients and medical teams that the medicinal cell products are safe, of constant quality and manufactured according to state-of-the art technology. Nonetheless, it does not warrant clinical efficacy and permanent engraftment of autologous stem cells remains variable. There are many challenges ahead to improve efficacy among which to keep telomere-dependent senescence and telomere-independent senescence (clonal conversion) to a minimum in cell culture and to understand the cellular and molecular mechanisms implicated in engraftment. Finally, medicinal stem cells are expansive to produce and reimbursement of costs by health insurances is a major concern in many countries.

The 3' half of the mouse mammary tumor virus orf gene is not sufficient for its superantigen function in transgenic mice.

The Mouse Mammary Tumor Virus (MMTV) long terminal repeat contains an open reading frame (orf) of 960 nucleotides encoding a 36 kDa polypeptide with a putative transmembrane domain and five N-glycosylation sites in the N-terminal part of the protein. Transgenic mice bearing either the complete or the 3' terminal half of the orf sequence of MMTV-GR under the control of the SV40 promoter were raised. As shown previously by FACS analysis transgenic mice which express the complete orf gene have a significant deletion of V beta 14 expressing T cells at 6 weeks of age. Here we show that no clonal deletion of V beta 14 bearing T cells takes place in transgenic mice that contain orf sequences from the fifth ATG to the termination codon. The pattern of tissues expressing the truncated transgene was studied by the Polymerase Chain Reaction (PCR) and was very similar to the one obtained in the V beta 14 deleting animals. These data suggest that the amino-terminal portion of the ORF protein (pORF) is required for a superantigen function, while our previous data indicated that determinants from the carboxy-terminus play an important role for TCR V beta specificity.

Preferential infection of immature dendritic cells and B cells by mouse mammary tumor virus.

Until now it was thought that the retrovirus mouse mammary tumor virus preferentially infects B cells, which thereafter proliferate and differentiate due to superantigen-mediated T cell help. We describe in this study that dendritic cells are infectable at levels comparable to B cells in the first days after virus injection. Moreover, IgM knockout mice have chronically deleted superantigen-reactive T cells after MMTV injection, indicating that superantigen presentation by dendritic cells is sufficient for T cell deletion. In both subsets initially only few cells were infected, but there was an exponential increase in numbers of infected B cells due to superantigen-mediated T cell help, explaining that at the peak of the response infection is almost exclusively found in B cells. The level of infection in vivo was below 1 in 1000 dendritic cells or B cells. Infection levels in freshly isolated dendritic cells from spleen, Langerhans cells from skin, or bone marrow-derived dendritic cells were compared in an in vitro infection assay. Immature dendritic cells such as Langerhans cells or bone marrow-derived dendritic cells were infected 10- to 30-fold more efficiently than mature splenic dendritic cells. Bone marrow-derived dendritic cells carrying an endogenous mouse mammary tumor virus superantigen were highly efficient at inducing a superantigen response in vivo. These results highlight the importance of professional APC and efficient T cell priming for the establishment of a persistent infection by mouse mammary tumor virus.

Degeneracy of antigen recognition as the molecular basis for the high frequency of naive A2/Melan-a peptide multimer(+) CD8(+) T cells in humans.

In contrast with the low frequency of most single epitope reactive T cells in the preimmune repertoire, up to 1 of 1,000 naive CD8(+) T cells from A2(+) individuals specifically bind fluorescent A2/peptide multimers incorporating the A27L analogue of the immunodominant 26-35 peptide from the melanocyte differentiation and melanoma associated antigen Melan-A. This represents the only naive antigen-specific T cell repertoire accessible to direct analysis in humans up to date. To get insight into the molecular basis for the selection and maintenance of such an abundant repertoire, we analyzed the functional diversity of T cells composing this repertoire ex vivo at the clonal level. Surprisingly, we found a significant proportion of multimer(+) clonotypes that failed to recognize both Melan-A analogue and parental peptides in a functional assay but efficiently recognized peptides from proteins of self- or pathogen origin selected for their potential functional cross-reactivity with Melan-A. Consistent with these data, multimers incorporating some of the most frequently recognized peptides specifically stained a proportion of naive CD8(+) T cells similar to that observed with Melan-A multimers. Altogether these results indicate that the high frequency of Melan-A multimer(+) T cells can be explained by the existence of largely cross-reactive subsets of naive CD8(+) T cells displaying multiple specificities.

Tissue homing and persistence of defined antigen-specific CD8+ tumor-reactive T-cell clones in long-term melanoma survivors.

Tumor antigen-specific cytotoxic T cells (CTLs) play a major role in the adaptive immune response to cancers. This CTL response is often insufficient because of functional impairment, tumor escape mechanisms, or inhibitory tumor microenvironment. However, little is known about the fate of given tumor-specific CTL clones in cancer patients. Studies in patients with favorable outcomes may be very informative. In this longitudinal study, we tracked, quantified, and characterized functionally defined antigen-specific T-cell clones ex vivo, in peripheral blood and at tumor sites, in two long-term melanoma survivors. MAGE-A10-specific CD8+ T-cell clones with high avidity to antigenic peptide and tumor lytic capabilities persisted in peripheral blood over more than 10 years, with quantitative variations correlating with the clinical course. These clones were also found in emerging metastases, and, in one patient, circulating clonal T cells displayed a fully differentiated effector phenotype at the time of relapse. Longevity, tumor homing, differentiation phenotype, and quantitative adaptation to the disease phases suggest the contribution of the tracked tumor-reactive clones in the tumor control of these long-term metastatic survivor patients. Focusing research on patients with favorable outcomes may help to identify parameters that are crucial for an efficient antitumor response and to optimize cancer immunotherapy.

Estimativas de repetibilidade de caracteres de produção em laranjeiras-doces no Acre

O objetivo deste trabalho foi estimar o coeficiente de repetibilidade em laranjeiras-doces e o número mínimo de avaliações a serem feitas para a determinação do valor real dos indivíduos. De março a junho de 1999, foram coletadas borbulhas de laranjeiras pé-franco, em fase de produção, em nove municípios do Acre. As borbulhas foram enxertadas sobre porta-enxertos de limoeiro 'Cravo', o que resultou em 54 clones. Esses clones foram avaliados em conjunto com a cultivar Aquiri, recomendada para o Estado. O delineamento experimental foi o de blocos ao acaso, com 55 tratamentos (55 clones), três repetições, com uma planta por parcela. O número total de frutos por planta, a produção de frutos por planta e o peso médio de fruto foram avaliados em 2002, 2003, 2004, 2005, 2006 e 2008. Foram realizadas as análises de variância, de componentes principais e estrutural. A estimativa de repetibilidade para peso médio do fruto demonstrou regularidade na classificação dos clones, de um ciclo para outro, e foram necessários cinco ciclos de avaliação para a predição do valor real dos indivíduos, com acurácia de 90%, pelo método dos componentes principais (matriz de co-variância). Apesar de as características número de frutos total por planta e produção de frutos por planta serem influenciadas pelo ambiente, oito e nove medições, respectivamente, permitem obter coeficientes de determinação de 95%.

Repetibilidade da produção de sementes em clones de guaraná

O objetivo deste trabalho foi avaliar a repetibilidade da produção de sementes 27 clones guaraná (Paullinia cupana). Os clones foram avaliados em dez ensaios instalados em blocos ao acaso com duas repetições e três plantas por parcela, com espaçamento de 5x5 m. A produção de sementes foi avaliada por quatro anos (1998, 1999, 2000 e 2001) a partir do início da fase produtiva, após dois anos do plantio. O coeficiente de repetibilidade foi estimado pelos métodos: análise de variância (ANOVA), componentes principais com base em matriz de correlações (CPCOR), componentes principais com base em matriz de variâncias e covariâncias fenotípicas (CPCOV) e análise estrutural com base na matriz de correlações (AECOR). Foram estimados também o número mínimo de observações e o coeficiente de determinação. As estimativas dos coeficientes de repetibilidade com base nos quatro anos, nos dez ambientes, foram relativamente baixas - 0,2618 (AECOR), 0,3064 (CPCOR), 0,3183 (ANOVA) e 0,5308 (CPCOV) - e variaram de acordo com o método e o ambiente. São necessários, no mínimo, quatro anos de avaliação para a obtenção de valores genotípicos dos clones com coeficiente de determinação de 80%.

In vitro negative selection of viral superantigen-reactive thymocytes by thymic dendritic cells.

Intrathymic expression of endogenous mouse mammary tumor virus (MMTV)-encoded superantigens (SAg) induces the clonal deletion of T cells bearing SAg-reactive T-cell receptor (TCR) Vbeta elements. However, the identity of the thymic antigen-presenting cells (APC) involved in the induction of SAg tolerance remains to be defined. We have analyzed the potential of dendritic cells (DC) to mediate the clonal deletion of Mtv-7-reactive TCR alphabeta P14 transgenic thymocytes in an in vitro assay. Our results show that both thymic and splenic DC induced the deletion of TCR transgenic double positive (DP) thymocytes. DC appear to be more efficient than splenic B cells as negatively selecting APC in this experimental system. Interestingly, thymic and splenic DC display a differential ability to induce CD4+ SP thymocyte proliferation. These observations suggest that thymic DC may have an important role in the induction of SAg tolerance in vivo.

Stability of Citrus tristeza virus protective isolates in field conditions

The objective of this work was to monitor the maintenance of Citrus tristeza virus (CTV) protective isolates stability in selected clones of 'Pêra' sweet orange (Citrus sinensis), preimmunized or naturally infected by the virus, after successive clonal propagations. The work was carried out in field conditions in the north of Paraná State, Brazil. Coat protein gene (CPG) analysis of 33 isolates collected from 16 clones of 'Pêra' sweet orange was performed using single strand conformational polymorphism (SSCP). Initially, the isolates were characterized by symptoms of stem pitting observed in clones. Then viral genome was extracted and used as template for the amplification of CPG by reverse transcription polimerase chain reaction (RTPCR). RTPCR products electrophoretic profiles were analyzed using the Jaccard coefficient and the UPGMA method. The majority of the clones had weak to moderate stem pitting symptoms and its CTV isolates showed alterations in the SSCP profiles. However, the stability of the protective complex has been maintained, except for isolates from two analised clones. Low genetic variability was observed within the isolates during the studied years.

Induction of CD8 T-cell responses restricted to multiple HLA class I alleles in a cancer patient by immunization with a 20-mer NY-ESO-1f (NY-ESO-1 91-110) peptide.

Immunogenicity of a long 20-mer NY-ESO-1f peptide vaccine was evaluated in a lung cancer patient TK-f01, immunized with the peptide with Picibanil OK-432 and Montanide ISA-51. We showed that internalization of the peptide was necessary to present CD8 T-cell epitopes on APC, contrasting with the direct presentation of the short epitope. CD8 T-cell responses restricted to all five HLA class I alleles were induced in the patient after the peptide vaccination. Clonal analysis showed that B*35:01 and B*52:01-restricted CD8 T-cell responses were the two dominant responses. The minimal epitopes recognized by A*24:02, B*35:01, B*52:01 and C*12:02-restricted CD8 T-cell clones were defined and peptide/HLA tetramers were produced. NY-ESO-1 91-101 on A*24:02, NY-ESO-1 92-102 on B*35:01, NY-ESO-1 96-104 on B*52:01 and NY-ESO-1 96-104 on C*12:02 were new epitopes first defined in this study. Identification of the A*24:02 epitope is highly relevant for studying the Japanese population because of its high expression frequency (60%). High affinity CD8 T-cells recognizing tumor cells naturally expressing the epitopes and matched HLA were induced at a significant level. The findings suggest the usefulness of a long 20-mer NY-ESO-1f peptide harboring multiple CD8 T-cell epitopes as an NY-ESO-1 vaccine. Characterization of CD8 T-cell responses in immunomonitoring using peptide/HLA tetramers revealed that multiple CD8 T-cell responses comprised the dominant response.

Cyanogenesis and the onset of tapping panel dryness in rubber tree

The objective of this work was to study the influence of cyanogenesis on the onset of irreversible tapping panel dryness (TPD) and the physiological and histological aspects of secondary phloem in the trunk (tapping panel) of rubber trees (Hevea spp.). Two cyanogenic compounds, linamarin and KCN, were applied separately on the trunk bark of healthy mature trees belonging to two Brazilian clones (Fx 4098 and Fx 3899). Changes in histology, latex pressure potential (ΨP) and cyanogenic potential (HCNp) were followed in the trunk inner barks. In addition, the HCNp levels were determined in TPD-affected plants of both clones. The applications of linamarin or KCN in healthy plants decreased latex ΨP, and formed tylosoids associated with in situ coagulation of latex. The clone Fx 4098 had the higher HCNp and showed the quicker and stronger responses to the cyanogenic compounds. Plants with TPD syntoms had a higher HCNp than the untreated healthy ones. Since histological changes are also structural markers of early TPD, it can be inferred that excessive release of cyanide can induce it in sensitive rubber clones

Transcriptome of a mouse kidney cortical collecting duct cell line: effects of aldosterone and vasopressin.

Aldosterone and vasopressin are responsible for the final adjustment of sodium and water reabsorption in the kidney. In principal cells of the kidney cortical collecting duct (CCD), the integral response to aldosterone and the long-term functional effects of vasopressin depend on transcription. In this study, we analyzed the transcriptome of a highly differentiated mouse clonal CCD principal cell line (mpkCCD(cl4)) and the changes in the transcriptome induced by aldosterone and vasopressin. Serial analysis of gene expression (SAGE) was performed on untreated cells and on cells treated with either aldosterone or vasopressin for 4 h. The transcriptomes in these three experimental conditions were determined by sequencing 169,721 transcript tags from the corresponding SAGE libraries. Limiting the analysis to tags that occurred twice or more in the data set, 14,654 different transcripts were identified, 3,642 of which do not match known mouse sequences. Statistical comparison (at P < 0.05 level) of the three SAGE libraries revealed 34 AITs (aldosterone-induced transcripts), 29 ARTs (aldosterone-repressed transcripts), 48 VITs (vasopressin-induced transcripts) and 11 VRTs (vasopressin-repressed transcripts). A selection of the differentially-expressed, hormone-specific transcripts (5 VITs, 2 AITs and 1 ART) has been validated in the mpkCCD(cl4) cell line either by Northern blot hybridization or reverse transcription-PCR. The hepatocyte nuclear transcription factor HNF-3-alpha (VIT39), the receptor activity modifying protein RAMP3 (VIT48), and the glucocorticoid-induced leucine zipper protein (GILZ) (AIT28) are candidate proteins playing a role in physiological responses of this cell line to vasopressin and aldosterone.

Desempenho silvicultural e seleção precoce de clones de híbridos de eucalipto

O objetivo deste trabalho foi avaliar a variabilidade genética e o desempenho silvicultural de clones de híbridos interespecíficos de Eucalyptus urophylla, E. globulus, E. maidenii, E. saligna, E. grandis, E. pellita, E. resinifera, E. kirtoniana e E. dunnii, e determinar a viabilidade da seleção precoce em selecionar clones superiores. Os diâmetros do tronco à altura do peito aos três (DAP3) e sete (DAP7) anos de idade e a altura total das árvores aos sete anos de idade foram mensurados. Utilizou-se o delineamento experimental de blocos ao acaso, com 138 clones, 10 repetições e seis plantas por parcela. Os dados foram submetidos à análise de variância e agrupados pelo método das k-médias, tendo-se realizado a comparação de médias e determinado a correlação de Pearson (r) entre as variáveis avaliadas. A validação do agrupamento foi realizada por meio de análise de variância e do teste de Tukey. Os clones de híbridos interespecíficos de Eucalyptus apresentaram variabilidade genética. Foram estabelecidos cinco grupos de clones com base no desempenho silvicultural (DAP3, DAP7 e altura). O DAP3 é altamente correlacionado ao DAP7 e à altura aos sete anos de idade. A seleção com base no DAP3 pode ser utilizada para identificar clones superiores de Eucalyptus com grande vigor de crescimento.

The effect of recombination in Aeromonas

Although several approaches have been attempted, the estimation of recombination frequencies in natural populations ofbacteria remains challenging. Previous studies have demonstrated awide variety of situations among bacterial species, ranging from theclonal diversification of Salmonella or Escherichia coli, which aremainly due to mutation, to the frequent recombination found inNeisseria gonorrhoeae or Helicobacter pylori. Most of the populationstudies done with bacterial species suggest that recombination occursin nature but that it is infrequent compared to mutation. Consequently,bacterial populations consist largely of independent clonal lineages.Our research suggests little or null influence of recombination in thegenetic structure of "Aeromonas hydrophila Species Complex", despite the presence of some strains with recombinant gene fragments.

Unexpected genomic variability in clinical and environmental strains of the pathogenic yeast Candida parapsilosis

Invasive candidiasis is the most commonly reported invasive fungal infection worldwide. Although Candida albicans remains the main cause, the incidence of emerging Candida species, such as C. parapsilosis is increasing. It has been postulated that C. parapsilosis clinical isolates result from a recent global expansion of a virulent clone. However, the availability of a single genome for this species has so far prevented testing this hypothesis at genomic scales. We present here the sequence of three additional strains from clinical and environmental samples. Our analyses reveal unexpected patterns of genomic variation, shared among distant strains, that argue against the clonal expansion hypothesis. All strains carry independent expansions involving an arsenite transporter homolog, pointing to the existence of directional selection in the environment, and independent origins of the two clinical isolates. Furthermore, we report the first evidence for the existence of recombination in this species. Altogether, our results shed new light onto the dynamics of genome evolution in C. parapsilosis.