A novel Cd8 enhancer identified as directing expression in CD44hiCD62L+ CD8T cells, including innate-like CD8+ T cells, and CD8 alpha alpha + DCs. CD8 coreceptor expression is dynamically regulated ...during thymocyte development and is tightly controlled by the activity of at least 5 different cis-regulatory elements. Despite the detailed characterization of the Cd8 loci, the regulation of the complex expression pattern of CD8 cannot be fully explained by the activity of the known Cd8 enhancers. In this study, we revisited the Cd8ab gene complex with bioinformatics and transgenic reporter gene expression approaches to search for additional Cd8 cis-regulatory elements. This led to the identification of an ECR (ECR-4), which in transgenic reporter gene expression assays, directed expression preferentially in CD44hiCD62L+ CD8+ T cells, including innate-like CD8+ T cells. ECR-4, designated as Cd8 enhancer E8VI, was bound by Runx/CBF beta complexes and Bcl11b, indicating that E8VI is part of the cis-regulatory network that recruits transcription factors to the Cd8ab gene complex in CD8+ T cells. Transgenic reporter expression was maintained in LCMV-specific CD8+ T cells upon infection, although short-term, in vitro activation led to a down-regulation of E8VI activity. Finally, E8VI directed transgene expression also in CD8 alpha alpha + DCs but not in CD8 alpha alpha -expressing IELs. Taken together, we have identified a novel Cd8 enhancer that directs expression in CD44hiCD62L+ CD8+ T cells, including innate-like and antigen-specific effector/memory CD8+ T cells and in CD8 alpha alpha + DCs, and thus, our data provide further insight into the cis-regulatory networks that control CD8 expression.
A novel Cd8 enhancer identified as directing expression in CD44hiCD62L+ CD8T cells, including innate‐like CD8+ T cells, and CD8αα+ DCs.
CD8 coreceptor expression is dynamically regulated during ...thymocyte development and is tightly controlled by the activity of at least 5 different cis‐regulatory elements. Despite the detailed characterization of the Cd8 loci, the regulation of the complex expression pattern of CD8 cannot be fully explained by the activity of the known Cd8 enhancers. In this study, we revisited the Cd8ab gene complex with bioinformatics and transgenic reporter gene expression approaches to search for additional Cd8 cis‐regulatory elements. This led to the identification of an ECR (ECR‐4), which in transgenic reporter gene expression assays, directed expression preferentially in CD44hiCD62L+ CD8+ T cells, including innate‐like CD8+ T cells. ECR‐4, designated as Cd8 enhancer E8VI, was bound by Runx/CBFβ complexes and Bcl11b, indicating that E8VI is part of the cis‐regulatory network that recruits transcription factors to the Cd8ab gene complex in CD8+ T cells. Transgenic reporter expression was maintained in LCMV‐specific CD8+ T cells upon infection, although short‐term, in vitro activation led to a down‐regulation of E8VI activity. Finally, E8VI directed transgene expression also in CD8αα+ DCs but not in CD8αα‐expressing IELs. Taken together, we have identified a novel Cd8 enhancer that directs expression in CD44hiCD62L+ CD8+ T cells, including innate‐like and antigen‐specific effector/memory CD8+ T cells and in CD8αα+ DCs, and thus, our data provide further insight into the cis‐regulatory networks that control CD8 expression.
Interindividualne razlike u metabolizmu mogu biti važan čimbenik nastanka nuspojava te varijabilnosti u učinkovitosti lijeka. Polimorfizmi gena koji kodiraju metaboličke enzime citokrome P450 (CYP) ...mogu imati značajan učinak na metabolizam lijeka i toksičnost. Ovaj pregled donosi spoznaje o tome kako polimorfizam enzima CYP2C8 i CYP2C9 utječe na bioraspoloživost i kliničke ishode liječenja ibuprofenom i diklofenakom, koji se svrstavaju među najčešće propisivane nesteroidne protuupalne lijekove. Hepatotoksičnost i gastrointestinalno krvarenje najčešće su nuspojave povezane s utjecajem varijanti CYP2C8*3 i CYP2C9*2*3 na farmakokinetiku ibuprofena i diklofenaka. Na osnovi rezultata genotipizacije CYP-a mogu biti prepoznati pacijenti koji imaju povećani rizik od razvoja nuspojava te im je nužno prilagoditi dozu lijeka ili odabrati drugi lijek koji ne dijeli isti metabolički put. Osim enzima CYP, značajan utjecaj imaju i polimorfizmi gena koji kodiraju fazu II metabolizma, osobito enzimi UGT, te transporteri, poput ABCC2, koji mogu modulirati ne samo transport na barijeri jetre i žuči nego i izlučivanje bubrezima. Stoga je u budućim istraživanjima nužan poligenski pristup. Prije uvođenja genotipizacije u redovitu kliničku praksu potrebno je provesti daljnja istraživanja koja će uključivati veće fenotipski dobro definirane skupine ispitanika za procjenu učinkovitosti ove strategije u poboljšanju liječenja ibuprofenom i diklofenakom. Zbog značajne međuetničke razlike u učestalosti polimorfizama gena CYP istraživanja treba provesti među različitim rasama i populacijama.
The use of epidermal growth factor receptor inhibitors (EGFRI) for the treatment of solid tumors is increasing due to elevated expression of epidermal growth factor receptors (EGFR) in the ...stimulation of tumor development. EGFR inhibitors have shown to be effective in the treatment of neoplasms of the head, neck, colon, and lung. Inhibition of EGFR may cause cutaneous reactions in more than 50% of patients. The most common skin manifestations are papulopustular lesions in the seborrhoeic areas (upper torso, face, neck, and scalp). Other cutaneous side effects include xerosis and hair and nail changes. The onset of eruption is usually within one to three weeks after starting therapy, although in some cases it may occur much later. All dermatologic side effects are reversible and generally resolve after adequate therapy. However, for a minority of patients side effects are severe and intolerable, demanding dose reduction or even interruption of therapy. A positive correlation has been demonstrated between the degree of cutaneous toxicity and the antitumor response. For dermatologists the goal is to provide treatment of symptoms, so that the patient may continue to benefit from the EGFRI therapy. However, frequent cutaneous manifestations, even though related to a better antitumor response, may limit use of the therapy considering the interference with patient quality of life. Early management of cutaneous side effects of EGFRI may prevent severe, extensive symptoms, the need for dose reduction, or antitumor therapy interruption. This indicates a dermatologist should play a role in early stages of treatment.
CD4/CD8 lineage specification of thymocytes is linked with coreceptor expression. Previously, the transcription factor MAZR was identified as an important regulator of
Cd8
gene expression. Here we ...show that variegated CD8 expression by loss of
Cd8
enhancers is reverted in MAZR-deficient mice, confirming that MAZR negatively regulates the
Cd8
loci during the transition to the double-positive (DP) stage. Moreover, loss of MAZR led to a partial redirection of MHC class I-restricted thymocytes into CD4
+
helper-like T cells correlating with derepression of ThPOK, a central transcription factor for helper-lineage development. MAZR bound the ThPOK silencer, indicating direct regulation of the
ThPOK
locus by MAZR. Thus, MAZR is part of the transcription factor network regulating CD8 lineage differentiation of DP thymocytes.
Abstract 1019
Reactivation of fetal γ-globin is of outstanding demand in patients with β-hemoglobinopathies. B-Cell/Lymphoma 11A (BCL11A) is a well-known repressor of γ-globin, and its expression is ...directly activated by Kruppel-Like Factor 1 (KLF1). KLF1 is a major regulator of human fetal to adult hemoglobin switching and reduced expression of KLF1 due to mutations is associated with hereditary persistence of fetal hemoglobin (HPFH). Analysis of the HPFH phenotype has led to the proposal that KLF1 has a dual role in γ-globin suppression, through its preferential activation of the β-globin gene and as a key activator of expression of the BCL11A repressor protein. To study regulation of erythropoiesis and globin expression by KLF1 and BCL11a in an in vivo model, we used mice carrying a human β-globin locus transgene with combinations of Klf1 haploinsufficiency, and Bcl11a floxed and EpoRCre knockin alleles. We measured hematological parameters of the mutant mice. With the exception of a small reduction in MCV (mean corpuscular volume), parameters of Klf1wt/ko animals were similar to those observed in the control animals. Bcl11acko/cko animals displayed a small but significant reduction of HCT (Hematocrit), RBC (red blood cell count) and HGB (hemoglobin) values. The reductions in these values were more pronounced in the Klf1wt/ko::Bcl11acko/cko animals. In addition, Klf1wt/ko::Bcl11acko/cko mice displayed small but significantly increased values for MCV, MCH (mean corpuscular hemoglobin) and MCHC (mean corpuscular hemoglobin concentration). We observed a higher concentration of erythropoietin in Bcl11acko/cko and compound Klf1wt/ko::Bcl11acko/cko animals suggesting a mild compensated anemia. To extend these observations, we analyzed embryonic blood and fetal livers at day E18.5, just prior to birth. Flow cytometry analysis of E18.5 blood revealed no difference in the CD71+Ter119+ population in peripheral blood of Bcl11acko/cko embryo’s. This percentage was increased in Klf1wt/ko blood samples and was highest in blood from the compound Klf1wt/ko::Bcl11acko/cko embryos. Similar results were obtained following flow cytometry of E18.5 fetal liver cells. Consequently, the percentage of mature CD71−/Ter119+ cells in fetal liver and peripheral blood of E18.5 Klf1wt/ko::Bcl11acko/cko embryos was significantly lower than that observed in Klf1wt/ko, Bcl11acko/cko and control embryos. Analysis of Klf1wt/ko, Bcl11acko/cko and Klf1wt/ko::Bcl11acko/cko mutant embryos demonstrated increased expression of mouse embryonic α - and β-like globins during fetal development. Expression of human γ-globin remained high in Bcl11acko/cko embryos during fetal development, and this was further augmented in Klf1wt/ko::Bcl11acko/cko embryos. After birth, expression of human γ-globin and mouse embryonic globins decreased in Bcl11acko/cko and Klf1wt/ko::Bcl11acko/cko mice, but the levels remained much higher than those observed in control animals. We find that haploinsufficiency for KLF1 delays γ- to β-globin switching leading to a ∼2-fold increase in the γ/(γ+β) ratio at E14.5 and E18.5. Part of this increase can be explained by diminished BCL11A expression in embryos with KLF1 insufficiency. Collectively, these data support the proposed role of the KLF1-BCL11A axis in γ-globin regulation. In conclusion, our results suggest that haploinsufficiency for KLF1 prolongs reticulocyte maturation, and that this phenotype is further exacerbated in combination with BCL11A deficiency. Despite this, the impact on erythropoiesis is modest and none of the compound mutant mice suffer from overt anemia even at prenatal stages when the demand for erythroid expansion is high. Collectively, our data support an important role for the KLF1-BCL11A axis in erythroid maturation and developmental regulation of globin expression and importantly in the absence of BCL11A, KLF1 still preferentially activates the β-globin gene.
No relevant conflicts of interest to declare.
Emersion of Websites that enable users to easily participate in creation of their content moved individuals on a scale rarely seen before. Web 2.0 transformed the passive reader into an active user ...and millions of users were drawn into a community previously reserved for professionals only. Users became able to experiment with data, collaborate with other users, and add value to a community of users. A similar revolution is needed in the electrical engineering education. In this field, courses offer a significant amount of theory and generally an unstimulating content to the students. Remote laboratories (RLs) could, however, make a difference. Instead of being passive collectors of the theory, students could become active builders of their own knowledge. At this point, the design of such a laboratory becomes important. Without a detailed user-oriented design, RL could have a counterproductive effect, generating frustration instead of motivation. A team of researchers used the QFD method to translate multidimensional and interdependent user requirements into the RL design model-MIRACLE. The MIRACLE model is based on survey results, instructional design, and good e-learning practice, and as such this model brings satisfaction, raises effectiveness and motivation, and makes electrical engineering courses appealing to students.
Abstract
Cell fate specifications during thymocyte differentiation are linked with the tightly controlled expression of CD4 and CD8. We have previously identified that the transcriptional regulator ...MAZR is an important regulator of the activation of the Cd8ab gene complex during the DN to DP transition. To investigate the role of MAZR in more detail, MAZR-/- mice were generated. MAZR-/- mice were smaller in size and were born at reduced mendelian frequency. MAZR-/- DN thymocytes did not show premature expression of CD8, indicating that the deletion of MAZR is not sufficient to activate CD8 expression in DN cells. However, variegated CD8 expression in DP thymocytes of Cd8 enhancer E8I,E8II doubly deficient mice was dramatically reduced in the absence of MAZR. In addition, we observed elevated CD4/CD8 ratios in thymocytes and in peripheral T cells of MAZR-/- mice. Using MHC class I-restricted TCR transgenic mice, we demonstrated that the altered CD4/8 ratio in MAZR-/- mice was, in part, due to redirected differentiation of a fraction of MHC-class I restricted thymocytes into the CD4 lineage. Thus, our data provide genetic evidence that MAZR controls CD8 expression during thymocyte development. In addition, our results may indicate that MAZR is part of the transcription factor network that controls CD4/CD8 cell fate decisions of DP thymocytes.
Supported by the Austrian Science Fund (P19930, SFB-F2305 and START Program Y-163).
Emersion of Websites that enable users to easily participate in creation of their content moved individuals on a scale rarely seen before. Web 2.0 transformed the passive reader into an active user ...and millions of users were drawn into a community previously reserved for professionals only. Users became able to experiment with data, collaborate with other users, and add value to a community of users. A similar revolution is needed in the electrical engineering education. In this field, courses offer a significant amount of theory and generally an unstimulating content to the students. Remote laboratories (RLs) could, however, make a difference. Instead of being passive collectors of the theory, students could become active builders of their own knowledge. At this point, the design of such a laboratory becomes important. Without a detailed user-oriented design, RL could have a counterproductive effect, generating frustration instead of motivation. A team of researchers used the QFD method to translate multidimensional and interdependent user requirements into the RL design model-MIRACLE. The MIRACLE model is based on survey results, instructional design, and good e-learning practice, and as such this model brings satisfaction, raises effectiveness and motivation, and makes electrical engineering courses appealing to students.
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