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1.
Cell Rep ; 32(3): 107910, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32697991

RESUMO

Cell competition in the thymus is a homeostatic process that drives turnover. If the process is impaired, thymopoiesis can be autonomously maintained for several weeks, but this causes leukemia. We aimed to understand the effect of cell competition on thymopoiesis, identify the cells involved, and determine how the process is regulated. Using thymus transplantation experiments, we found that cell competition occurs within the double-negative 2 (DN2) and 3 early (DN3e) thymocytes and inhibits thymus autonomy. Furthermore, the expansion of DN2b is regulated by a negative feedback loop that is imposed by double-positive thymocytes and determines the kinetics of thymopoiesis. This feedback loop affects the cell cycle duration of DN2b, in a response controlled by interleukin 7 availability. Altogether, we show that thymocytes do not merely follow a pre-determined path if provided with the correct signals. Instead, thymopoiesis dynamically integrates cell-autonomous and non-cell-autonomous aspects that fine-tune normal thymus function.


Assuntos
Competição entre as Células , Timócitos/citologia , Timo/citologia , Animais , Contagem de Células , Ciclo Celular , Diferenciação Celular , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Interleucina-7/metabolismo , Cinética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Timo/transplante
2.
J Pharm Sci ; 109(1): 316-322, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31255685

RESUMO

Optimization of protein formulations at subzero temperatures is required for many applications such as storage, transport, and lyophilization. Using isochoric cooling (constant volume) is possible to reach subzero temperatures without freezing aqueous solutions. This accelerates protein damage as protein may unfold by cold denaturation and diffusional and conformational freedom is still present. The use of isochoric cooling to faster protein formulations was first demonstrated for the biomedical relevant protein disulfide isomerase A1. Three osmolytes, sucrose, glycerol, and l-arginine, significantly increased the stability of protein disulfide isomerase A1 at -20°C with all tested under isochoric cooling within the short time frame of 700 h. The redox green fluorescent protein 2 was used to evaluate the applicability of isochoric cooling for stability analysis of highly stable proteins. This derivative of GFP is 2.6-fold more stable than the highly stable GFP ß-barrel structure. Nevertheless, it was possible to denature a fraction of roGFP2 at -20°C and to assign a stabilizing effect to sucrose. Isochoric cooling was further applied to insulin. Protein damage was evaluated through a signaling event elicited on human hepatocyte carcinoma cells. Insulin at -20°C under isochoric cooling lost 22% of its function after 15 days and 0.6M sucrose prevented insulin deactivation.


Assuntos
Proteínas de Fluorescência Verde/química , Proteínas Recombinantes/química , Temperatura Baixa , Composição de Medicamentos , Estabilidade de Medicamentos , Congelamento , Hemoglobinas/química , Células Hep G2 , Humanos , Insulina/química , Insulina/farmacologia , Transição de Fase , Pró-Colágeno-Prolina Dioxigenase/química , Desnaturação Proteica , Isomerases de Dissulfetos de Proteínas/química , Redobramento de Proteína , Estabilidade Proteica , Termodinâmica
3.
J Immunol ; 202(4): 1137-1144, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30651344

RESUMO

Thymus autonomy is the capacity of the thymus to maintain T lymphocyte development and export independently of bone marrow contribution. Prolonging thymus autonomy was shown to be permissive to the development of T cell acute lymphoblastic leukemia (T-ALL), similar to the human disease. In this study, performing thymus transplantation experiments in mice, we report that thymus autonomy can occur in several experimental conditions, and all are permissive to T-ALL. We show that wild type thymi maintain their function of T lymphocyte production upon transplantation into recipients with several genotypes (and corresponding phenotypic differences), i.e., Rag2 - / - γc - / -, γc - / -, Rag2 - / - IL-7rα - / -, and IL-7rα - / - We found that the cellularity of the thymus grafts is influenced exclusively by the genotype of the host, i.e., IL-7rα-/- versus γc -/- Nonetheless, the difference in cellularity detected in thymus autonomy bore no impact on onset, incidence, immunophenotype, or pathologic condition of T-ALL. In all tested conditions, T-ALL reached an incidence of 80%, demonstrating that thymus autonomy bears a high risk of leukemia. We also analyzed the microbiota composition of the recipients and their genetic background, but none of the differences found influenced the development of T-ALL. Taken together, our data support that IL-7 drives cellular turnover non-cell autonomously, which is required for prevention of T-ALL. We found no influence for T-ALL in the specific combination of the genotypic mutations tested (including the developmental block caused by Rag deficiency), in microbiota composition, or minor differences in the genetic background of the strains.


Assuntos
Leucemia-Linfoma Linfoblástico de Células T Precursoras/imunologia , Receptores de Interleucina-7/imunologia , Timo/imunologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Receptores de Interleucina-7/deficiência , Receptores de Interleucina-7/genética
4.
J Immunol ; 197(10): 4042-4052, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27798165

RESUMO

The ability of HIV-1 to replicate and to establish long-term reservoirs is strongly influenced by T cell activation. Through the use of membrane-tethered, genetically encoded calcium (Ca2+) indicators, we were able to detect for the first time, to our knowledge, the formation of Ca2+ territories and determine their role in coordinating the functional signaling nanostructure of the synaptic membrane. Consequently, we report a previously unknown immune subversion mechanism involving HIV-1 exploitation, through its Nef accessory protein, of the interconnectivity among three evolutionarily conserved cellular processes: vesicle traffic, signaling compartmentalization, and the second messenger Ca2+ We found that HIV-1 Nef specifically associates with the traffic regulators MAL and Rab11b compelling the vesicular accumulation of Lck. Through its association with MAL and Rab11b, Nef co-opts Lck switchlike function driving the formation Ca2+ membrane territories, which, in turn, control the fusion of LAT-transporting Rab27 and Rab37 vesicles and the formation of LAT nanoclusters at the immunological synapse. Consequently, HIV-1 Nef disengages TCR triggering from the generation of p-LAT and p-SLP nanoclusters driving TCR signal amplification and diversification. Altogether our results indicate that HIV-1 exploits the interconnectivity among vesicle traffic, Ca2+ membrane territories, and signaling nanoclusters to modulate T cell signaling and function.


Assuntos
Cálcio/metabolismo , HIV-1/fisiologia , Sinapses Imunológicas/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Células HEK293 , HIV-1/imunologia , Humanos , Sinapses Imunológicas/ultraestrutura , Células Jurkat , Ativação Linfocitária , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Glicoproteínas de Membrana/metabolismo , Fosforilação , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Interleucina-1/metabolismo , Linfócitos T/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/química , Produtos do Gene nef do Vírus da Imunodeficiência Humana/imunologia , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab27 de Ligação ao GTP
5.
Clin Endocrinol (Oxf) ; 77(6): 926-33, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22882326

RESUMO

OBJECTIVE: FOXE1 is a transcription factor required for thyroid differentiation and function. FOXE1 locus polymorphisms (chromosome 9q22.33) were recently associated with increased sporadic thyroid cancer risk. In this study, we aimed to investigate the association of FOXE1 variants with nonmedullary thyroid cancer (NMTC), in both sporadic and familial (FNMTC) cases from the Portuguese population. DESIGN AND METHODS: Nine variants located at the FOXE1 locus were sequenced in genomic DNA from 60 FNMTC probands and 80 patients with sporadic NMTC. Alleles were tested for association with thyroid cancer, against 130 healthy matched Portuguese controls. RESULTS: All variants were significantly associated with increased thyroid cancer risk when combining familial and sporadic cases (OR range = 1·62-2·58). In particular, two reported risk variants were associated with the disease: rs965513 (allele A) with familial (OR = 2·30, 95% CI = 1·48-3·59, P = 0·0002) and sporadic (OR = 2·81, 95% CI = 1·87-4·22, P < 0·0001) NMTC and rs1867277 (allele A) with the sporadic (OR = 1·76, 95% CI = 1·18-2·62, P = 0·0052) and combined NMTC cases (OR = 1·70, 95% CI = 1·21-2·40, P = 0·0022). Interestingly, we also identified association of FOXE1 polyalanine tract expansions (>14 alanines) with thyroid cancer risk, in both familial (OR = 2·56, 95% CI = 1·64-4·01, P < 0·0001) and sporadic (OR = 2·44, 95% CI = 1·61-3·68, P < 0·0001) cases. CONCLUSIONS: We found compelling evidence of association between FOXE1 variants and thyroid cancer risk in the Portuguese population. To our knowledge, this is the first study supporting the association of this locus with both sporadic and familial NMTC susceptibility.


Assuntos
Fatores de Transcrição Forkhead/genética , Predisposição Genética para Doença , Polimorfismo Genético/genética , Neoplasias da Glândula Tireoide/genética , Adenocarcinoma Folicular/epidemiologia , Adenocarcinoma Folicular/genética , Idoso , Idoso de 80 Anos ou mais , Frequência do Gene , Humanos , Desequilíbrio de Ligação , Pessoa de Meia-Idade , Portugal/epidemiologia , Análise de Sequência de DNA , Neoplasias da Glândula Tireoide/epidemiologia
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