Primary T-cell acute lymphoblastic leukemia (T-ALL) cells require stromal-derived signals to survive. Although many studies have identified cell-intrinsic alterations in signaling pathways that ...promote T-ALL growth, the identity of endogenous stromal cells and their associated signals in the tumor microenvironment that support T-ALL remains unknown. By examining the thymic tumor microenvironments in multiple murine T-ALL models and primary patient samples, we discovered the emergence of prominent epithelial-free regions, enriched for proliferating tumor cells and dendritic cells (DCs). Systematic evaluation of the functional capacity of tumor-associated stromal cells revealed that myeloid cells, primarily DCs, are necessary and sufficient to support T-ALL survival ex vivo. DCs support T-ALL growth both in primary thymic tumors and at secondary tumor sites. To identify a molecular mechanism by which DCs support T-ALL growth, we first performed gene expression profiling, which revealed up-regulation of platelet-derived growth factor receptor beta (Pdgfrb) and insulin-like growth factor I receptor (Igf1r) on T-ALL cells, with concomitant expression of their ligands by tumor-associated DCs. Both Pdgfrb and Igf1r were activated in ex vivo T-ALL cells, and coculture with tumor-associated, but not normal thymic DCs, sustained IGF1R activation. Furthermore, IGF1R signaling was necessary for DC-mediated T-ALL survival. Collectively, these studies provide the first evidence that endogenous tumor-associated DCs supply signals driving T-ALL growth, and implicate tumor-associated DCs and their mitogenic signals as auspicious therapeutic targets.
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Phospho flow cytometry is a powerful technique to analyze signaling in rare cell populations. This technique, however, requires harsh conditions for cell fixation and permeabilization, which can ...denature surface antigens or antibody-conjugated fluorochromes. These are among several technical limitations which have been a barrier to quantify signaling in unique B cell subsets. One such immature subset, transitional B cells (TrBs), may play a role in suppressing solid organ transplant rejection, graft-versus-host disease, autoimmunity, and even the immune response to malignancy. Here we sought to optimize a protocol for quantification of signaling in human TrBs compared with mature B cell subsets.
TrBs were defined by surface marker expression as CD19+CD24hiCD38hi. Key parameters optimized included antibody clone selection, sequence of surface epitope labeling in relation to paraformaldehyde-based fixation and methanol-based permeabilization, photomultiplier tube (PMT) voltages, and compensation. Special attention was paid to labeling of CD38 with regard to these parameters, and an optimized protocol enabled reliable identification of TrBs, naïve (CD24+CD38+), early memory (CD24hiCD38−), and late memory (CD24−CD38−) B cells. Phospho flow cytometry enabled simultaneous quantification of phosphorylation among at least three different signaling molecules within the same sample. Among normal donors, transitional B cells exhibited diminished mitogen activated protein kinase/extracellular signal-regulated kinase and Akt phospho signaling upon nonspecific stimulation with phorbol 12-myristate 13-acetateand ionomycin stimulation.
We optimized an effective protocol to quantify B cell subset signaling upon stimulation. Such a protocol may ultimately serve as the basis for assessing dysfunctional B cell signaling in disease, predict clinical outcomes, and monitor response to B cell-directed therapies.
•Phospho flow cytometry is a powerful technique to analyze signaling.•Technical limitations are a barrier to quantify B cell subset signaling.•B cell surface marker labeling was best after fixation but before permeabilization.•CD38 labeling required careful PMT voltage and compensation optimization.•Diminished signaling in human CD24hiCD38hi TrBs after stimulation
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
The thymus and parathyroid glands arise from a shared endodermal primordium in the third pharyngeal pouch (3rd pp). Thymus fate is specified in the ventral 3rd pp between E9.5 and E11, whereas ...parathyroid fate is specified in the dorsal domain. The molecular mechanisms that specify fate and regulate thymus and parathyroid development are not fully delineated. Previous reports suggested that Tbx1 is required for thymus organogenesis because loss of Tbx1 in individuals with DiGeorge syndrome and in experimental Tbx1 deletion mutants is associated with thymus aplasia or hypoplasia. However, the thymus phenotype is likely to be secondary to defects in pharyngeal pouch formation. Furthermore, the absence of Tbx1 expression in the thymus-fated domain of the wild-type 3rd pp suggested that Tbx1 is instead a negative regulator of thymus organogenesis. To test this hypothesis, we generated a novel mouse strain in which expression of a conditional Tbx1 allele was ectopically activated in the thymus-fated domain of the 3rd pp. Ectopic Tbx1 expression severely repressed expression of Foxn1, a transcription factor that marks the thymus-fated domain and is required for differentiation and proliferation of thymic epithelial cell (TEC) progenitors. By contrast, ectopic Tbx1 did not alter the expression pattern of Gcm2, a transcription factor restricted to the parathyroid-fated domain and required for parathyroid development. Ectopic Tbx1 expression impaired TEC proliferation and arrested TEC differentiation at an early progenitor stage. The results support the hypothesis that Tbx1 negatively regulates TEC growth and differentiation, and that extinction of Tbx1 expression in 3rd pp endoderm is a prerequisite for thymus organogenesis.
English language proficiency of students in the Philippines is a point of concern by many institutions particularly as it was highlighted when the results of the first participation of the country in ...the Program for International Student Assessment (PISA) was released. The use of technology-aided educational applications dedicated to language learning, particularly those that can be accessed through mobile devices, is heavily studied internationally. However, in the Philippines, there is a limited number of developed mobile-supported applications that assist acquisition and learning of language, specifically English. Thus, the study aims were to develop a mobile learning application intended to assist students in learning a least mastered topic in English. The study utilized mixed-method descriptive research design. For the quantitative part, it employed a content-validated questionnaire to survey 132 fifth grade students, while for the qualitative part; it interviewed five English language teachers in the Philippines. A mobile learning application intended for smartphones in an Android OS was developed with focus on subject-verb agreement (SVA) as identified topic which was divided into 10 sub-topics. The application comprises two main parts: The Learn and Play Mode. Additionally, the application was also able to integrate an animated character named Alvin to simulate interaction. Alvin interacts with the user by narrating lessons, giving remarks, providing basic corrective feedback in the form of text, and expressing feelings based on the user’s inputs when doing exercises. The survey from students aged 10 to 12 years old showed that they have very often access to technological devices (i.e., desktop, laptops, tablet, smartphone) (M = 4.33) and that their technology aptitude in using them is high (M = 3.45). Furthermore, results also showed that students mostly prefer smartphones for their learning as it is their most available device at home. It is recommended to subject the developed mobile application to a user acceptance and usability testing in different educational contexts. Additionally, it is suggested to explore newer technologies like artificial intelligence to make the application intelligent and more responsive to students’ needs and inputs. Future studies may also include addition of new topics as well as developing similar applications that are localized and culturally-aware.
Thymus organogenesis requires coordinated interactions of multiple cell types, including neural crest (NC) cells, to orchestrate the formation, separation, and subsequent migration of the developing ...thymus from the third pharyngeal pouch to the thoracic cavity. The molecular mechanisms driving these processes are unclear; however, NC-derived mesenchyme has been shown to play an important role. Here, we show that, in the absence of ephrin-B2 expression on thymic NC-derived mesenchyme, the thymus remains in the cervical area instead of migrating into the thoracic cavity. Analysis of individual NC-derived thymic mesenchymal cells shows that, in the absence of ephrin-B2, their motility is impaired as a result of defective EphB receptor signaling. This implies a NC-derived cell-specific role of EphB–ephrin-B2 interactions in the collective migration of the thymic rudiment during organogenesis.
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Embryos that are homozygous for Splotch, a null allele of Pax3, have a severe neural crest cell (NCC) deficiency that generates a complex phenotype including spina bifida, exencephaly and cardiac ...outflow tract abnormalities. Contrary to the widely held perception that thymus aplasia or hypoplasia is a characteristic feature of Pax3Sp/Sp embryos, we find that thymic rudiments are larger and parathyroid rudiments are smaller in E11.5–12.5 Pax3Sp/Sp compared to Pax3+/+ embryos. The thymus originates from bilateral third pharyngeal pouch primordia containing endodermal progenitors of both thymus and parathyroid glands. Analyses of Foxn1 and Gcm2 expression revealed a dorsal shift in the border between parathyroid- and thymus-fated domains at E11.5, with no change in the overall cellularity or volume of each shared primordium. The border shift increases the allocation of third pouch progenitors to the thymus domain and correspondingly decreases allocation to the parathyroid domain. Initial patterning in the E10.5 pouch was normal suggesting that the observed change in the location of the organ domain interface arises during border refinement between E10.5 and E11.5. Given the well-characterized NCC defects in Splotch mutants, these findings implicate NCCs in regulating patterning of third pouch endoderm into thymus- versus parathyroid-specified domains, and suggest that organ size is determined in part by the number of progenitor cells specified to a given fate.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Gut microbial imbalance (dysbiosis) can contribute to intestinal inflammation. Inflammation-driven colon cancer is a leading cause of death in the aged. We hypothesize that intestinal ...dysbiosis occurs with age and contributes to colon cancer susceptibility. We found that aged mice (~20 mos) had increased basal colon inflammation. Young mice (~3 mos) treated with AOM/DSS, an established model of inflammation-driven colon cancer, got transient colitis followed by colon cancer as expected. By striking contrast, aged mice on AOM/DSS developed severe, fatal, acute colitis preventing carcinogenesis studies. We also found that the colon microbiome is age-dependent. Gut flora-ablating antibiotics (abx) in young mice on AOM/DSS did not alter acute colitis or cancer. Strikingly, old mice on abx were highly protected from AOM/DSS-induced colitis and colon cancer, consistent with age-related dysbiosis causing increased risk for colitis and inflammation driven-colon cancer. PD-1 regulates gut flora in young mice and B7-H1 is its ligand. Aged B7-H1-/- mice given AOM/DSS, but no abx, did not develop acute colitis or colon cancer, suggesting that B7-H1 mediates age-related gut dysbiosis, inflammation and colon cancer risk. Taken together, our data suggest that age-related, B7-H1-dependent gut dysbiosis increases colon inflammation and susceptibility to colon carcinogenesis. Ongoing co-housing (WT/B7-H1 and young/aged) and other studies are defining specific immune mechanisms.
Abstract
As thymocytes develop, they modulate expression of receptors, such as chemokine receptors, that promote migration into distinct thymic microenvironments. Within these thymic regions, ...developing thymocytes have the opportunity to interact with unique stromal cells that provide survival, selection, and/or differentiation signals. Thymocyte: stromal interactions are essential for proper thymocyte development, implicating the molecules involved in regulating intrathymic migration as key regulators of T cell development. Previously, we have used 2-photon microscopy to study real-time migration of thymocytes within live thymic slices. These studies revealed that immature thymocyte progenitors were restricted to the cortex while only post-positive selection thymocytes were able to enter the medulla. The ability to migrate within cortex versus medulla was regulated by multiple mechanisms, including G-protein coupled receptor mediated chemotaxis and substrate accessibility. We hypothesize that the molecular cues that mediate normal intrathymic migration may be disrupted during thymic lymphomagenesis, contributing to the initiation and/or progression of disease. Consistent with this hypothesis, we find that the thymic microenvironment is disorganized in a murine model of thymic lymphoma. Furthermore, using 2-photon microscopy, we find that migration of lymphoma cells within thymic tissue is abnormal, suggesting that aberrant thymocyte: stromal interactions may contribute to disease.
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