Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Neste estudo investigamos as consequências da restrição protéica materna durante a lactação sobre a resposta de timócitos da prole jovem de ratos Wistar (grupo D), identificando o papel da leptina nas alterações encontradas. Observamos que, quando comparados ao grupo controle, os animais do grupo D apresentaram, aos 30 dias de vida, uma diminuição significativa tanto do peso corporal quanto do timo. Contudo, não observamos alterações no número de timócitos, no perfil de células CD4/CD8 ou na resposta proliferativa destas células. Sistemicamente, o grupo D não apresentou alterações nos níveis séricos de corticosterona ou no conteúdo nuclear do seu receptor (GR) em timócitos. Apesar dos animais D não apresentarem alterações nos níveis circulantes de leptina, a expressão do seu receptor, ObRb, estava aumentada nos timócitos. Esta alteração foi acompanhada pela amplificação da resposta de sinalização da leptina nestas células, observada por um aumento na ativação de JAK2 e STAT3 após a incubação com leptina. Os timócitos isolados do grupo D apresentaram uma diminuição significativa na taxa de apoptose espontânea quando comparados ao grupo controle. Corroborando estes resultados, demonstramos que os timócitos dos animais D apresentam um aumento na expressão da proteína antiapoptótica Bcl-2 e uma redução da expressão da proteína próapoptótica Bax, além de um maior conteúdo de Pró-caspase-3, entretanto, não encontramos alterações no conteúdo de Bad. Além disso, timócitos do grupo D apresentaram um maior conteúdo da subunidade p65 do NFĸB no núcleo, associado a uma menor expressão de IĸBα no citoplasma. Finalmente, observamos um aumento na expressão do RNAm para o gene ob (leptina) mas não para o gene db (receptor) no microambiente tímico dos animais D. Em conjunto, nossos dados mostram que a restrição protéica durante a lactação afeta a homeostase tímica, induzindo uma maior atividade de leptina, que protege os timócitos da apoptose na prole jovem, sugerindo que esses animais poderiam ser mais suscetíveis a alterações na resposta imune na vida aduta. / In this study, we investigated the consequences of maternal protein deprivation during lactation on thymocyte responses of the offspring Wistar rats (PD group) and the role of leptin in these alterations. Compared to group C, PD animals showed at 30 days of age, lower body and thymus weights, with no alteration in the thymocyte number or any difference in the profile of T cell subsets, or in their proliferative response. Similar circulating levels of corticosterone and GR nuclear contents were detected in thymic cells of PD or control groups. In contrast, despite the rats from PD group did not present alterations in leptin circulating levels, the expression of leptin receptor ObRb was enhanced in their thymocytes. This change was accompanied by an amplification in leptin signaling response of thymocytes from PD rats, which showed an increase in JAK2 and STAT3 phosphorylation after stimulation with leptin. Moreover, the thymic cells from PD rats presented a decreased rate of spontaneous apoptosis when compared to controls. Accordingly, higher expression of both the anti-apoptotic protein Bcl-2 and Procaspase-3 and lower expression of pro-apoptotic protein Bax were detected in PD thymocytes, however, the pro-apoptotic protein bad expression was similar between the groups. Thymocytes from PD group also exhibited a constitutive higher nuclear content of p65 NFkB associated to a lower IkB content in the cytoplasm. Finally, although there was no change in db genic (leptin receptor) expression in PD thymocytes, a higher expression of mRNA for ob gene (leptin) was observed in the thymic microenvironment from PD animals. Taken together, the results show that maternal protein deprivation during lactation affects thymic homeostasis, inducing leptin activity, which protect thymocytes from apoptosis in young progeny and, perhaps,may prone these animals for alterations in immune response in adult life.

Abstract Trojan is a novel leukocyte-specific protein cloned from chicken (Gallus gallus) embryonic thymocytes. The molecule is a type I transmembrane protein with an extracellular CCP domain followed by two FN3 domains. Its cytoplasmic tail is predicted to possess a MAPK docking and a PKA phosphorylation site. Trojan displays differential expression on developing thymocyte subpopulations. It is high on CD4 and CD8 double negative, and CD4 or CD8 single positive cells, but diminishes from the surface of selection-undergoing CD4 and CD8 double positive cells. This expression pattern is similar to that of anti-apoptotic molecules such as IL-7Rα and BCL-2. We hypothesised an involvement of Trojan in the regulation of apoptosis, possibly as an anti-apoptotic receptor. Our in vitro studies with a T cell line showed that upon apoptosis induction, Trojan expression rises dramatically on the surface of surviving cells and gradually decreases towards its normal levels as cells recover. When sorted based on their Trojan levels, cells with high expression appear less susceptible to apoptotic induction than those bearing no Trojan on their surface. Cells that overexpress Trojan from a cDNA plasmid show elevated steady state intracellular calcium, suggesting the molecule is able to transmit cytoplasmic signals. In addition, computational analyses pointed towards an involvement of MAPK and a possible regulatory mechanism by PKA. Trojan belongs to a novel gene family that includes two other members in the chicken. One is a receptor-type tyrosine phosphatase named Mystran, and the other – a transmembrane protein with an ITAM, named Thracian. We discovered the family in other avian species and found related genes in reptiles and coalecanth fish. We observed dynamic adaptation of their extracellular regions possibly in concert with ligand-binding, association with other surface molecules or as a response to pathogen challenges. This was coupled to largely unchanged cytoplasmic tails, suggesting a conserved signalling mechanism. The presented study shows that a novel avian leukocyte protein called Trojan possibly has an anti-apoptotic role. It belongs to a gene family that was subjected to evolutionary selection, likely linked to the molecular function of the proteins. / Tiivistelmä Trojan on uusi kanan (Gallus gallus) alkioiden kateenkorvan kypsyvistä T soluista tunnistettu molekyyli. Se on tyypin I solukalvoproteiini, jolla on solun ulkopuolinen CCP-domeeni ja kaksi FN3-domeenia. Trojanin solun sisäisessä osassa on rakenteen perusteella MAPK:n sitoutumisalue sekä PKA-fosforylaatiopaikka. Trojania ilmennetään T-solujen kehityksen aikana runsaasti CD4 ja CD8 kaksoisnegatiivisissa ja CD4 tai CD8 yksöispositiivisissa soluissa, mutta ilmentyminen on vähäinen valintaa läpikäyvissä CD4 ja CD8 kaksoispositiivisissa soluissa. Tunnetut apoptoosia eli ohjelmoitua solukuolemaa estävät molekyylit, kuten IL-7Rα ja BCL-2, noudattavat samankaltaista ilmentymistä kypsyvien T solujen pinnalla. Hypoteesimme on, että Trojanilla on rooli apoptoosin säätelyssä, mahdollisesti solukuolemaa estävänä reseptorina. In vitro apoptoosikokeet osoittivat, että aluksi Trojanin ilmentyminen lisääntyy huomattavasti soluissa, jotka välttävät apoptoottisen kuoleman, ja normalisoituu sitten muutaman solujakautumisen jälkeen. Trojania vähän ilmentävät solut ovat alttiimpia ohjelmoidulle solukuolemalle, kuin sitä paljon ilmentävät solut. Solujen sisäinen kalsiumtaso on kohonnut soluilla, jotka yliekspressoivat Trojania cDNA plasmidista. Tämä viittaa siihen, että Trojan voi toimia sytoplasman signaalinvälityksessä. Lisäksi tietokoneperusteiset ennusteet viittaavat siihen, että MAPK ja PKA voivat liittyä Trojan-signalointiin. Tutkimuksessa tunnistettiin Trojan-geeniperhe. Perheeseen kuuluu Trojanin lisäksi kaksi muuta geeniä: reseptorityyppinen tyrosiinifosfataasi Mystran ja ITAM-domeenin sisältävä solukalvon proteiini Trachian. Geeniperhe löydettiin muistakin lintulajeista, sekä niitä läheisesti muistuttavat geenit matelijoilta ja varsieväkalalta. Havaitsimme Trojan-perheen proteiineissa dynaamista sopeutumista, joka voi olla seurausta ligandien sitoutumisesta, vuorovaikutuksesta muiden pintaproteiinien kanssa tai vasteesta patogeenihaasteeseen. Proteiinien solunsisäiset alueet olivat sen sijaan suurilta osin muuttumattomia, joten ne voivat toimia solusignaloinnissa. Väitöstutkimuksessa kuvataan uusi valkosolujen proteiini Trojan, joka toiminnallisesti saattaa estää ohjelmoitua solukuolemaa. Trojan kuuluu geeniperheeseen, johon on kohdistunut sen toimintaan liittyvää valintaa.

La leucémie lymphoïde représente 30% de tous les cancers chez l’enfant. SCL (« Stem cell leukemia ») et LMO1/2 (« LIM only protein ») sont les oncogènes les plus fréquemment activés dans les leucémies aiguës des cellules T chez l'enfant (T-ALL). L’expression ectopique de ces deux oncoprotéines dans le thymus de souris transgéniques induit un blocage de la différenciation des cellules T suivie d’une leucémie agressive qui reproduit la maladie humaine. Afin de définir les voies génétiques qui collaborent avec ces oncogènes pour induire des leucémies T-ALL, nous avons utilisé plusieurs approches. Par une approche de gène candidat, nous avons premièrement identifié le pTalpha, un gène crucial pour la différenciation des cellules T, comme cible directe des hétérodimères E2AHEB dans les thymocytes immatures. De plus, nous avons montré que pendant la différenciation normale des thymocytes, SCL inhibe la fonction E2A et HEB et qu’un dosage entre les protéines E2A, HEB et SCL détermine l’expression du pTalpha. Deuxièmement, par l’utilisation d’une approche globale et fonctionnelle, nous avons identifié de nouveaux gènes cibles des facteurs de transcription E2A et HEB et montré que SCL et LMO1 affectent la différenciation thymocytaire au stade préleucémique en inhibant globalement l’activité transcriptionnelle des protéines E par un mécanisme dépendant de la liaison à l’ADN. De plus, nous avons découvert que les oncogènes SCL et LMO1 sont soit incapables d’inhiber totalement l’activité suppresseur de tumeur des protéines E ou agissent par une voie d’induction de la leucémie différente de la perte de fonction des protéines E. Troisièmement, nous avons trouvé que Notch1, un gène retrouvé activé dans la majorité des leucémies T-ALL chez l’enfant, opère dans la même voie génétique que le pré-TCR pour collaborer avec les oncogènes SCL et LMO1 lors du processus de leucémogénèse. De plus, cette collaboration entre des facteurs de transcription oncogéniques et des voies de signalisation normales et importantes pour la détermination de la destinée cellulaire pourraient expliquer la transformation spécifique à un type cellulaire. Quatrièmement, nous avons trouvé que les oncogènes SCL et LMO1 sont des inducteurs de sénescence au stade préleucémique. De plus, la délétion du locus INK4A/ARF, un évènement retrouvé dans la majorité des leucémies pédiatriques T-ALL associées avec une activation de SCL, collabore aves les oncogènes SCL et LMO1 dans l’induction de la leucémie. Cette collaboration entre la perte de régulateurs de la sénescence suggère qu’un contournement de la réponse de sénescence pourrait être nécessaire à la transformation. Finalement, nous avons aussi montré que l’interaction directe entre les protéines SCL et LMO1 est critique pour l’induction de la leucémie. Ces études ont donc permis d’identifier des évènements collaborateurs, ainsi que des propriétés cellulaires affectées par les oncogènes associés avec la leucémie et de façon plus générale dans le développement du cancer. / Lymphoid leukemia represents 30% of all cancers in children. SCL (Stem cell leukemia) and LMO1/2 (LIM only protein) are the most frequently activated oncogenes in children T cell acute lymphoblastic leukemia (T-ALL). Ectopic expression of the SCL and LMO1 oncogenes in the thymus of transgenic mice causes T cell differentiation arrest during the preleukemic stage followed by development of aggressive leukemia that reproduce human disease. We therefore took several approaches to decipher the genetic pathway collaborating with these oncogenes in T-ALL induction. Using a candidate approach, we first identified the pTalpha, a gene crucial for T cell differentiation, as a direct target of E2A and HEB heterodimers in immature thymocytes. Moreover, we showed that during normal thymocyte differentiation, SCL inhibits E2A and HEB function and that a dosage between E2A, HEB and SCL normally determines pTalpha gene expression. Second, using both global and functional approaches, we identified novel target genes of E2A and HEB transcription factors and showed that SCL and LMO1 impairs thymocyte differentiation at the preleukemic stage by globally inhibiting E proteins transcriptional activity through a DNA binding mechanism. Moreover, we found that SCL and LMO1 oncogenes are either not totally able to inhibit E protein tumor suppressor activity or act in a different leukemic inducing pathway than E protein loss of function. Third, we found that Notch1, a gene found activated in almost all cases of pediatric T-ALL, operate in the same genetic pathway as the pre-TCR to collaborate with the SCL and LMO1 oncogenes in leukemogenesis. Moreover, this collaboration between oncogenic transcription factors and normal signalling pathways important for cell fate determination might explain cell-type specific transformation. Fourth, we found that the SCL and LMO1 oncogenes are inducers of senescence at the preleukemic stage. Moreover, deletion of INK4A/ARF, an event found in almost all cases of SCL associated pediatric T-ALL, collaborate with SCL and LMO1 oncogenes in leukemogenesis. This collaboration with loss of senescence regulators suggests that a bypass of senescence response would be necessary for transformation. Finally, we also showed that SCL and LMO1 direct interaction is critical for leukemia induction. These studies permitted the identification of collaborating events and cellular properties affected by oncogenes associated with leukemia and more generally in cancer development.

La leucémie lymphoïde représente 30% de tous les cancers chez l’enfant. SCL (« Stem cell leukemia ») et LMO1/2 (« LIM only protein ») sont les oncogènes les plus fréquemment activés dans les leucémies aiguës des cellules T chez l'enfant (T-ALL). L’expression ectopique de ces deux oncoprotéines dans le thymus de souris transgéniques induit un blocage de la différenciation des cellules T suivie d’une leucémie agressive qui reproduit la maladie humaine. Afin de définir les voies génétiques qui collaborent avec ces oncogènes pour induire des leucémies T-ALL, nous avons utilisé plusieurs approches. Par une approche de gène candidat, nous avons premièrement identifié le pTalpha, un gène crucial pour la différenciation des cellules T, comme cible directe des hétérodimères E2AHEB dans les thymocytes immatures. De plus, nous avons montré que pendant la différenciation normale des thymocytes, SCL inhibe la fonction E2A et HEB et qu’un dosage entre les protéines E2A, HEB et SCL détermine l’expression du pTalpha. Deuxièmement, par l’utilisation d’une approche globale et fonctionnelle, nous avons identifié de nouveaux gènes cibles des facteurs de transcription E2A et HEB et montré que SCL et LMO1 affectent la différenciation thymocytaire au stade préleucémique en inhibant globalement l’activité transcriptionnelle des protéines E par un mécanisme dépendant de la liaison à l’ADN. De plus, nous avons découvert que les oncogènes SCL et LMO1 sont soit incapables d’inhiber totalement l’activité suppresseur de tumeur des protéines E ou agissent par une voie d’induction de la leucémie différente de la perte de fonction des protéines E. Troisièmement, nous avons trouvé que Notch1, un gène retrouvé activé dans la majorité des leucémies T-ALL chez l’enfant, opère dans la même voie génétique que le pré-TCR pour collaborer avec les oncogènes SCL et LMO1 lors du processus de leucémogénèse. De plus, cette collaboration entre des facteurs de transcription oncogéniques et des voies de signalisation normales et importantes pour la détermination de la destinée cellulaire pourraient expliquer la transformation spécifique à un type cellulaire. Quatrièmement, nous avons trouvé que les oncogènes SCL et LMO1 sont des inducteurs de sénescence au stade préleucémique. De plus, la délétion du locus INK4A/ARF, un évènement retrouvé dans la majorité des leucémies pédiatriques T-ALL associées avec une activation de SCL, collabore aves les oncogènes SCL et LMO1 dans l’induction de la leucémie. Cette collaboration entre la perte de régulateurs de la sénescence suggère qu’un contournement de la réponse de sénescence pourrait être nécessaire à la transformation. Finalement, nous avons aussi montré que l’interaction directe entre les protéines SCL et LMO1 est critique pour l’induction de la leucémie. Ces études ont donc permis d’identifier des évènements collaborateurs, ainsi que des propriétés cellulaires affectées par les oncogènes associés avec la leucémie et de façon plus générale dans le développement du cancer. / Lymphoid leukemia represents 30% of all cancers in children. SCL (Stem cell leukemia) and LMO1/2 (LIM only protein) are the most frequently activated oncogenes in children T cell acute lymphoblastic leukemia (T-ALL). Ectopic expression of the SCL and LMO1 oncogenes in the thymus of transgenic mice causes T cell differentiation arrest during the preleukemic stage followed by development of aggressive leukemia that reproduce human disease. We therefore took several approaches to decipher the genetic pathway collaborating with these oncogenes in T-ALL induction. Using a candidate approach, we first identified the pTalpha, a gene crucial for T cell differentiation, as a direct target of E2A and HEB heterodimers in immature thymocytes. Moreover, we showed that during normal thymocyte differentiation, SCL inhibits E2A and HEB function and that a dosage between E2A, HEB and SCL normally determines pTalpha gene expression. Second, using both global and functional approaches, we identified novel target genes of E2A and HEB transcription factors and showed that SCL and LMO1 impairs thymocyte differentiation at the preleukemic stage by globally inhibiting E proteins transcriptional activity through a DNA binding mechanism. Moreover, we found that SCL and LMO1 oncogenes are either not totally able to inhibit E protein tumor suppressor activity or act in a different leukemic inducing pathway than E protein loss of function. Third, we found that Notch1, a gene found activated in almost all cases of pediatric T-ALL, operate in the same genetic pathway as the pre-TCR to collaborate with the SCL and LMO1 oncogenes in leukemogenesis. Moreover, this collaboration between oncogenic transcription factors and normal signalling pathways important for cell fate determination might explain cell-type specific transformation. Fourth, we found that the SCL and LMO1 oncogenes are inducers of senescence at the preleukemic stage. Moreover, deletion of INK4A/ARF, an event found in almost all cases of SCL associated pediatric T-ALL, collaborate with SCL and LMO1 oncogenes in leukemogenesis. This collaboration with loss of senescence regulators suggests that a bypass of senescence response would be necessary for transformation. Finally, we also showed that SCL and LMO1 direct interaction is critical for leukemia induction. These studies permitted the identification of collaborating events and cellular properties affected by oncogenes associated with leukemia and more generally in cancer development.

INTRODUÇÃO: A molécula HIG2 pode atuar como agonista da via Wnt/beta-catenina, pois se liga ao receptor Frizzled 10 e induz a expressão de genes da mesma. Dados recentes do nosso grupo mostraram expressão diferencial do gene HIG2 em células mononucleares do sangue periférico e em especial linfócitos T CD4+ naïve, mas não em células diferenciadas de memória em indivíduos sadios. Também observamos in vitro em linfócitos T CD4+ de indivíduos saudáveis que o peptídeo sintético HIG2 induziu a ativação da via Wnt/beta-catenina, produção de HIG2 e outros produtos da via, além da proliferação de células T CD4+ naïve sugerindo um papel do HIG2 na proliferação homeostática de linfócitos T CD4+. HIPÓTESE: Como as células T CD4+ naïve são diretamente exportadas pelo timo, os níveis aumentados de HIG2 neste tipo celular sejam decorrentes da ativação da via Wnt/?-catenina nos estágios tardios da diferenciação de timócitos. Portanto, as células T CD4+ naïve e timócitos simples positivos para CD4 (SP CD4) apresentariam perfil semelhante de expressão de HIG2 e genes da via Wnt/beta-catenina, incluindo receptores, fatores de transcrição, genes estruturais da via e alvos quando comparadas as demais populações celulares. OBJETIVO: Avaliar a expressão de HIG2 e outros genes da via Wnt/beta-catenina em timócitos e linfócitos T CD4+ naïve e memória de camundongos. MÉTODOS: Isolamos timócitos duplo negativos (DN), timócitos duplo positivos (DP), simples positivos para CD4 e CD8 (SP CD4 e SP CD8) de timo e também células T CD4+ naïve e memória do baço dos mesmos camundongos pelo procedimento de citometria de fluxo. Analisamos a expressão de vários genes da via Wnt/beta-catenina por PCR em tempo real. RESULTADOS: Em timócitos DN há expressão significativa dos genes que codificam para Frizzled 6, LRP5, TCF-1 e TCF-4 em relação as outras populações celulares. Nos timócitos DP há maior expressão dos genes que codificam para LRP5, LRP6, beta-catenina, GSK-3beta, TCF-1 e Bcl-XL em relação às demais populações. Em timócitos SP CD4 foi detectada expressão diferencial de genes que codificam para Frizzled 10, LRP6, beta-catenina, LEF-1 e HIG2 enquanto que na população de timócitos SP CD8 não observamos expressão significativa de nenhum gene da via Wnt/beta-catenina. Nas células T CD4+ naïve há expressão significativa de Frizzled 5 e Frizzled 10 quando comparadas a timócitos SP CD8 e células T CD4+ de memória . Já nos linfócitos T CD4+ de memória, detectamos maior expressão de Frizzled 6, TCF-4, Bcl-XL e ciclina D1 em relação as demais populações. CONCLUSÃO: Cada população apresenta um perfil distinto de expressão gênica. As maiores semelhanças ocorrem entre os timócitos DN e DP onde as principais diferenças são a expressão de Frizzled 6 e Ciclina D1.Os timócitos SP CD4 e as células T CD4+ naïve não apresentaram níveis semelhantes de expressão gênica de elementos da via Wnt canônica, o que não corrobora a hipótese de que o perfil transcripcional de timócitos SP CD4 e linfócitos T CD4+ naïve é semelhante. Ainda, não observamos expressão aumentada de HIG2 em linfócitos T CD4+ naïve comparados aos de memória, o que contrasta com os resultados obtidos anteriormente por nosso grupo com amostras humanas sugerindo que camundongos não regulam a expressão de HIG2 em linfócitos T CD4+ como os seres humanos / INTRODUCTION: HIG2 molecule can act as an agonist of Wnt/?-catenin pathway, because it able to bind to Frizzled 10 receptor and induce the expression of the genes related to this pathway. Recent data from our group have shown differential expression of the HIG2 gene in peripheral blood mononuclear cells, and particularly in naive CD4 + T cells, but not in memory T cells in healthy individuals. We have also observed that inducing the CD4 + T lymphocytes from healthy individuals with HIG2 synthetic peptide in vitro, led to the activation of Wnt/beta-catenin pathway, HIG2 production and expression of other target genes of this pathway and the proliferation of naïve CD4 + T cells, suggesting that HIG2 may play a role in homeostatic proliferation of CD4+ T cells. HYPOTHESIS: As naïve CD4 + T cells are directly exported from the thymus, we have hypothesized that increased levels of HIG2 in this cell type is due to the activation of Wnt/beta-catenin pathway in the later stages of thymocyte differentiation. Therefore, naïve CD4 + T cells and CD4 single-positive thymocytes (CD4 SP) may share a similar pattern of gene expression of HIG2 and Wnt/beta-catenin genes (genes that encodes receptors and co-receptors, transcription factors, structural and target genes) when compared to other cell populations. AIM: our major aim is to evaluate the expression of HIG2 and other genes of the Wnt/beta-catenin in thymocytes, naïve CD4 + T lymphocytes and memory CD4+ T cells from mice. METHODS: We have isolated thymocytes double negative (DN) T cells, positive double positive (DP) T cells, CD4 and CD8 single-positive thymocytes (CD4 SP and CD8 SP) of thymus from BALB/c mice and we have also isolated naïve CD4 + T cells and memory CD4+ T cells of the spleen from the same mice we have used the thymus. We have analysed the expression of several genes of Wnt/beta-catenin by real time PCR RESULTS: In DN cells there was expression of the Frizzled 6, LRP5, TCF-1 and TCF-4 genes compared to other cell populations. In DP thymocytes it could be observed a greater expression of LRP5, LRP6, beta-catenin, GSK-3beta, TCF-1 and Bcl-XL genes compared to other populations. In CD4 SP thymocytes, it was detected differential expression of the Frizzled 10, LRP6, beta-catenin, LEF-1 HIG2 genes and in CD8 SP cells we could not observe significant expression of any gene of Wnt/?-catenin pathway. In naïve CD4 + T cells there was a significant expression of Frizzled5 and Frizzled 10 genes when compared to all the samples. In memory CD4 + T cells, we have detected higher expression of Frizzled 6, TCF-4, Bcl-XL and cyclin D1 genes than in any other populations. CONCLUSION: Each population has a distinct gene expression pattern. The biggest similarities occur between DN and DP thymocytes where the main differences are the expression of Frizzled 6 and cyclin D1.However, the pattern of gene expression in SP thymocytes is not similar to those presented by naïve CD4+ T cells. Moreover, we have not observed increased expression of HIG2 in naïve CD4 + lymphocytes compared to memory CD4+ T cells, which contrasts the results obtained previously by our group with human samples suggesting that mice might not regulate the HIG2 expression in CD4 + T lymphocytes as human beings do

Transendothelial migration is a key process in lymphocyte trafficking. It is known that growth hormone (GH) modulates intrathymic T cell migration and that directly or indirectly influences the biology of endothelial cells and thymocytes. In this context, we evaluated the effect of GH on the transendothelial migration of thymocytes, in the presence or absence the chemokine CXCL12. For the study, we applied the murine thymic endothelial cell line (tEnd.1) and thymocytes freshly isolated from male C57BL/6 mice aged 4-6 weeks. Through in vitro studies of transendothelial migration of thymocytes, and analyses by flow cytometry, we observed a statistically significant reduction in the numbers of transmigrated thymocytes, mainly seen for CD4+CD8+ cells, across endothelial barriers treated with GH [100 ng/ml], for 8 h. Importantly, this effect was reverted when thymocytes were treated with GH [100 ng/ml] for 1 h. Similar effects were noted in transmigration through joint action of GH and CXCL12. Despite the fact that all the CD4/CD8-defined subsets have been affected, we observed a statistically significant increase in the number of transmigrated thymocytes, mainly of CD4+CD8+ and CD4+CD8- cells, when they were treated with GH and allowed to migrate in response to CXCL12, when compared to untreated controls in response to CXCL12. We demonstrated, by real-time PCR, that GH at 100 ng/ml did not modulate the expression of VCAM-1 and CXCL12 by tEnd.1 cells treated for 8 h. Furthermore, we found by cytofluorimetric assay that GH alone did not change the membrane expression of VLA-4 and CXCR4 receptor in transmigrated thymocytes. Through joint action of GH and CXCL12, was observed increase in expression of VLA-4 on CD4-CD8+ cells from the group of GH-treated thymocytes, when compared to groups in which endothelial cells were treated. As for expression of CXCR4, we found that the GH/CXCL12 decreased the expression of this receptor on transmigrated thymocytes, mainly in CD4+CD8+ cells from the group in which the thymocytes were treated. In conclusion, our results reinforce that GH influence the thymus physiology and modulate the biology of endothelial cells and thymocytes, with differential effects in the process of endothelial transmigration of murine thymocytes. / Fundação de Amparo a Pesquisa do Estado de Alagoas / A transmigração endotelial é um processo essencial no trânsito de linfócitos. É sabido que o hormônio do crescimento (GH) está envolvido na migração intratímica de células T, e que influencia direta ou indiretamente a biologia das células endoteliais e dos timócitos. Neste contexto, avaliamos a ação do GH na migração transendotelial de timócitos, frente ou não à quimiocina CXCL12. Para o estudo, utilizamos a linhagem endotelial tEnd.1 e timócitos recém isolados de camundongos machos C57BL/6 de 4-6 semanas. Através do ensaio de transmigração endotelial in vitro de timócitos, e análise por citometria de fluxo, verificamos uma redução estatisticamente significativa no número de timócitos transmigrados, principalmente nas subpopulações CD4+CD8+, frente a barreiras endoteliais tratadas com GH [100 ng/ml], por 8 horas. Este efeito foi revertido quando os timócitos foram tratados com GH [100 ng/ml], por 1 hora. Efeitos similares na transmigração foram notados pela ação do GH frente a CXCL12. Apesar de todas as subpopulações CD4/CD8 terem sido afetadas, observamos um aumento estatisticamente significativo no número de timócitos transmigrados, principalmente das células CD4+CD8+ e CD4+CD8-, quando foram tratados com GH e colocados para migrar em presença da CXCL12, em relação ao grupo controle não tratado e em presença da quimiocina. Demonstramos, ainda, por RT-PCR, que o GH na concentração de 100 ng/ml, não modulou a expressão de VCAM-1 e CXCL12 por células tEnd.1 tratadas, por 8 horas. Além disso, verificamos por análise citofluorimétrica, que o GH sozinho não alterou a expressão dos receptores VLA-4 e CXCR4 nos timócitos transmigrados. Pela ação conjunta do GH e CXCL12, foi registrado aumento na expressão de VLA-4 sobre células CD4-CD8+ no grupo experimental em que os timócitos foram tratados com GH, quando comparamos aos grupos onde as células endoteliais foram tratadas. Quanto à expressão de CXCR4, verificamos que o GH/CXCL12 diminuiu a expressão deste receptor sobre timócitos transmigrados, principalmente em células CD4+CD8+, nos grupos experimentais em que os timócitos foram tratados. Em conclusão, nossos dados fortalecem o postulado de que o GH influencia a fisiologia do timo, e modula a biologia das células endoteliais e dos timócitos, com efeitos diferenciais no processo de transmigração endotelial de timócitos murinos.

INTRODUÇÃO: A molécula HIG2 pode atuar como agonista da via Wnt/beta-catenina, pois se liga ao receptor Frizzled 10 e induz a expressão de genes da mesma. Dados recentes do nosso grupo mostraram expressão diferencial do gene HIG2 em células mononucleares do sangue periférico e em especial linfócitos T CD4+ naïve, mas não em células diferenciadas de memória em indivíduos sadios. Também observamos in vitro em linfócitos T CD4+ de indivíduos saudáveis que o peptídeo sintético HIG2 induziu a ativação da via Wnt/beta-catenina, produção de HIG2 e outros produtos da via, além da proliferação de células T CD4+ naïve sugerindo um papel do HIG2 na proliferação homeostática de linfócitos T CD4+. HIPÓTESE: Como as células T CD4+ naïve são diretamente exportadas pelo timo, os níveis aumentados de HIG2 neste tipo celular sejam decorrentes da ativação da via Wnt/?-catenina nos estágios tardios da diferenciação de timócitos. Portanto, as células T CD4+ naïve e timócitos simples positivos para CD4 (SP CD4) apresentariam perfil semelhante de expressão de HIG2 e genes da via Wnt/beta-catenina, incluindo receptores, fatores de transcrição, genes estruturais da via e alvos quando comparadas as demais populações celulares. OBJETIVO: Avaliar a expressão de HIG2 e outros genes da via Wnt/beta-catenina em timócitos e linfócitos T CD4+ naïve e memória de camundongos. MÉTODOS: Isolamos timócitos duplo negativos (DN), timócitos duplo positivos (DP), simples positivos para CD4 e CD8 (SP CD4 e SP CD8) de timo e também células T CD4+ naïve e memória do baço dos mesmos camundongos pelo procedimento de citometria de fluxo. Analisamos a expressão de vários genes da via Wnt/beta-catenina por PCR em tempo real. RESULTADOS: Em timócitos DN há expressão significativa dos genes que codificam para Frizzled 6, LRP5, TCF-1 e TCF-4 em relação as outras populações celulares. Nos timócitos DP há maior expressão dos genes que codificam para LRP5, LRP6, beta-catenina, GSK-3beta, TCF-1 e Bcl-XL em relação às demais populações. Em timócitos SP CD4 foi detectada expressão diferencial de genes que codificam para Frizzled 10, LRP6, beta-catenina, LEF-1 e HIG2 enquanto que na população de timócitos SP CD8 não observamos expressão significativa de nenhum gene da via Wnt/beta-catenina. Nas células T CD4+ naïve há expressão significativa de Frizzled 5 e Frizzled 10 quando comparadas a timócitos SP CD8 e células T CD4+ de memória . Já nos linfócitos T CD4+ de memória, detectamos maior expressão de Frizzled 6, TCF-4, Bcl-XL e ciclina D1 em relação as demais populações. CONCLUSÃO: Cada população apresenta um perfil distinto de expressão gênica. As maiores semelhanças ocorrem entre os timócitos DN e DP onde as principais diferenças são a expressão de Frizzled 6 e Ciclina D1.Os timócitos SP CD4 e as células T CD4+ naïve não apresentaram níveis semelhantes de expressão gênica de elementos da via Wnt canônica, o que não corrobora a hipótese de que o perfil transcripcional de timócitos SP CD4 e linfócitos T CD4+ naïve é semelhante. Ainda, não observamos expressão aumentada de HIG2 em linfócitos T CD4+ naïve comparados aos de memória, o que contrasta com os resultados obtidos anteriormente por nosso grupo com amostras humanas sugerindo que camundongos não regulam a expressão de HIG2 em linfócitos T CD4+ como os seres humanos / INTRODUCTION: HIG2 molecule can act as an agonist of Wnt/?-catenin pathway, because it able to bind to Frizzled 10 receptor and induce the expression of the genes related to this pathway. Recent data from our group have shown differential expression of the HIG2 gene in peripheral blood mononuclear cells, and particularly in naive CD4 + T cells, but not in memory T cells in healthy individuals. We have also observed that inducing the CD4 + T lymphocytes from healthy individuals with HIG2 synthetic peptide in vitro, led to the activation of Wnt/beta-catenin pathway, HIG2 production and expression of other target genes of this pathway and the proliferation of naïve CD4 + T cells, suggesting that HIG2 may play a role in homeostatic proliferation of CD4+ T cells. HYPOTHESIS: As naïve CD4 + T cells are directly exported from the thymus, we have hypothesized that increased levels of HIG2 in this cell type is due to the activation of Wnt/beta-catenin pathway in the later stages of thymocyte differentiation. Therefore, naïve CD4 + T cells and CD4 single-positive thymocytes (CD4 SP) may share a similar pattern of gene expression of HIG2 and Wnt/beta-catenin genes (genes that encodes receptors and co-receptors, transcription factors, structural and target genes) when compared to other cell populations. AIM: our major aim is to evaluate the expression of HIG2 and other genes of the Wnt/beta-catenin in thymocytes, naïve CD4 + T lymphocytes and memory CD4+ T cells from mice. METHODS: We have isolated thymocytes double negative (DN) T cells, positive double positive (DP) T cells, CD4 and CD8 single-positive thymocytes (CD4 SP and CD8 SP) of thymus from BALB/c mice and we have also isolated naïve CD4 + T cells and memory CD4+ T cells of the spleen from the same mice we have used the thymus. We have analysed the expression of several genes of Wnt/beta-catenin by real time PCR RESULTS: In DN cells there was expression of the Frizzled 6, LRP5, TCF-1 and TCF-4 genes compared to other cell populations. In DP thymocytes it could be observed a greater expression of LRP5, LRP6, beta-catenin, GSK-3beta, TCF-1 and Bcl-XL genes compared to other populations. In CD4 SP thymocytes, it was detected differential expression of the Frizzled 10, LRP6, beta-catenin, LEF-1 HIG2 genes and in CD8 SP cells we could not observe significant expression of any gene of Wnt/?-catenin pathway. In naïve CD4 + T cells there was a significant expression of Frizzled5 and Frizzled 10 genes when compared to all the samples. In memory CD4 + T cells, we have detected higher expression of Frizzled 6, TCF-4, Bcl-XL and cyclin D1 genes than in any other populations. CONCLUSION: Each population has a distinct gene expression pattern. The biggest similarities occur between DN and DP thymocytes where the main differences are the expression of Frizzled 6 and cyclin D1.However, the pattern of gene expression in SP thymocytes is not similar to those presented by naïve CD4+ T cells. Moreover, we have not observed increased expression of HIG2 in naïve CD4 + lymphocytes compared to memory CD4+ T cells, which contrasts the results obtained previously by our group with human samples suggesting that mice might not regulate the HIG2 expression in CD4 + T lymphocytes as human beings do

No description available.

Es konnte ein neuer Cluster von insgesamt sieben T-Zell-Rezeptor delta-Rekombinationselementen (deltaRec2.1-2.7), welcher sich innerhalb des TCRD-Gens, 2,6-5,2 kb strangabwärts des variablen Gensegments Vdelta2, befindet. Gezeigt wurde, dass diese isolierten recombination signal sequences sowohl mit Ddelta3- als auch mit Jdelta1-Segmenten des TCRD-Gens und pseudo joining-Gensegmenten des TCRA-Gens rearrangiert werden. Rearrangements, die deltaRec2-Elemente enthielten, wurden in allen zehn Proben peripherer Leukozyten gesunder Spender gefunden, obwohl sie hundertfach seltener sind als klassische deltaRec-Rearrangements. Die absolute Frequenz der deltaRec2-Rearrangements in PB-T-Zellen war, verglichen mit der in Thymozyten, niedriger, was nahe legt, dass sie während der T-Zell-Entwicklung deletiert werden. Am auffälligsten war das Verschwinden von deltaRec2-Ddelta3-Umlagerungen, deren Häufigkeit in PB-T-Zellen elffach niedriger als in Thymozyten war. Die überwiegende Mehrheit der deltaRec2-Jdelta1-Rearrangements enthielt das Ddelta3-Element an der Verknüpfungsstelle. Dies bedeutet, dass sie sich aus deltaRec2-Ddelta3-Rearrangements entwickelt haben könnten. Im Gegensatz dazu steht, dass die Mehrheit der deltaRec2-pseudoJalpha–Umlagerungen kein Ddelta3-Segment enthielt, was vermutlich daher rührt, dass sie direkte Rearrangements darstellen. Die Umlagerungen deltaRec2-Jdelta1 und deltaRec2-pseudoJalpha scheinen T-Zell-spezifisch zu sein, deltaRec2-Ddelta3-Rearrangements hingegen wurden auch in B-Lymphozyten und NK-Zellen in geringer Zahl nachgewiesen. Zusammenfassend zeigen diese Daten, dass deltaRec2-Rearrangements wahrscheinlich vorübergehende Schritte des Rekombinationsprozesses des TCRAD-Lokus sind und durch nachfolgende Valpha-Jalpha-Rearrangements deletiert werden können. Ähnlich wie die klassischen deltaRec-Umlagerungen, spielen vermutlich auch deltaRec2-Rearrangements eine Rolle auf dem Wege der Differenzierung von T-Lymphozyten hin zur reifen TCRalpha/beta-Zelle. Somit könnten die hier erstmals beschriebenen weiteren Rekombinationselemente in der MRD-Diagnostik maligner lymphatischer Erkrankungen die bisher genutzten Rearrangements ergänzen und ebenso als Verlaufsmarker genutzt werden. / A new cluster of 7 T-cell receptor delta recombining elements (deltaRec2.1-2.7) located within the T-cell receptor delta (TCRD) gene, 2.6-5.2 kb downstream from the variable region (Vdelta2) gene segment could be identified. The deltaRec2 elements are isolated recombination signal sequences to rearrange with the Ddelta3, Jdelta1 segments of the TCRD gene as well as with the pseudo joining segment of the TCRA gene. Rearrangements involving the deltaRec2 elements were found in all peripheral blood (PB) samples from 10 healthy individuals, although their frequency was about 100-fold lower than classical deltaRec rearrangements. Compared with thymocytes, the total frequency of deltaRec2 rearrangements was lower in PB T-cells, suggesting that they are deleted during T-cell development. The decrease in frequency of the deltaRec2-Ddelta3 rearrangements was most prominent: 11 times lower in PB T lymphocytes than in thymocytes. Since the deltaRec2-Jdelta1 rearrangements contained the Ddelta3 segment in the junctional region, it could be assumed that they are derived from the deltaRec2-Ddelta3 rearrangements. In contrast, the majority of deltaRec2-pseudoJalpha rearrangements did not contain the Ddelta3 segment, indicating that they are single step rearrangements. The deltaRec2-Jdelta1 and deltaRec2-pseudoJalpha rearrangements seem to be T-lineage specific, but the deltaRec2-Ddelta3 rearrangements were also found at very low frequencies in B-lymphocytes and NK-cells. The results suggest that deltaRec2 rearrangements are transient steps in the recombinatorial process of the TCRAD locus and are probably deleted by subsequent Valpha-Jalpha rearrangements. Similarly to the classical deltaRec rearrangements, also the deltaRec2 rearrangements most likely play a role in T-cell differentiation towards the TCRalpha/beta cell. These recombination elements could also complete the rearrangements used so far as marker in diagnosis of minimal residual disease of malignant lymphatic diseases.

No description available.