Organochlorine pesticides, such as DDT, methoxychlor, and their metabolites, have been characterized as endocrine disrupting chemicals (EDCs); suggesting that their modes of action involve ...interaction with or abrogation of endogenous endocrine function. This study examined whether embryonic thymocyte death and alteration of differentiation induced by the primary metabolite of methoxychlor, HPTE, rely upon estrogen receptor binding and concurrent T cell receptor signaling. Estrogen receptor inhibition of ERα or GPER did not rescue embryonic thymocyte death induced by HPTE or the model estrogen diethylstilbestrol (DES). Moreover, adverse effects induced by HPTE or DES were worsened by concurrent TCR and CD2 differentiation signaling, compared with EDC exposure post-signaling. Together, these data suggest that HPTE- and DES-induced adverse effects on embryonic thymocytes do not rely solely on ER alpha or GPER but may require both. These results also provide evidence of a potential collaborative signaling mechanism between TCR and estrogen receptors to mediate adverse effects on embryonic thymocytes, as well as highlight a window of sensitivity that modulates EDC exposure severity.
Endocrine-disrupting chemicals (EDC) are widespread in the built and natural environments. Heightened public awareness of their potential danger has led to concern about whether EDC and their ...metabolites have significant negative biological effects. Studies have shown that EDC like DDT and other organochlorine pesticides, such as methoxychlor (MXC), have adverse effects on immune cells, but no studies have addressed the impact of HPTE, the primary metabolite of MXC. To elucidate the presence and significance of HPTE adverse effects, this study explored the impact of HPTE on a critical window and component of immune system development, embryonic T-cell development. Lesions at this phase of development can lead to lifelong immune dysfunction and increased incidence of immune disease, such as autoimmunity. Embry-onic thymocytes (GD 16-18) from C57BL/6 mice were subjected to an in vitro differentiation culture that mimicked early steps in thymocyte development in the presence of 0.005, 0.05, 0.5, 5, or 50 μM HPTE, or a model endocrine disruptor, DES. The results indicated that compared to the vehicle control, HPTE- and DES-induced death of thymocytes. Annexin-V staining and Caspase 8, markers of programed cell death, revealed that the loss of cells was due at least in part to induction of apoptosis. Moreover, HPTE-induced cell death not only resulted in selective loss of double positive thymocytes, but also loss of developing CD4 intermediate cells (post-double positive partially differentiated thymocyte population). Phenotypic analysis of thymocyte maturation (T-cell receptor, TCR) and TCR ligation (CD5) surface markers revealed that surviving embryonic thymocytes expressed low levels of both. Taken together these data demonstrate that immature embryonic thymocytes are sensitive to HPTE exposure and that HPTE exposure targets thymocyte populations undergoing critical differentiation steps. These findings suggest HPTE may play a pivotal role in MXC exposure-induced immune dysfunction.
Bcl-x is a member of the Bcl2 family and has been suggested to be important for the survival and maturation of various cell types including the erythroid lineage. To define the consequences of Bcl-x ...loss in erythroid cells and other adult tissues, we have generated mice conditionally deficient in the Bcl-x gene using the Cre-loxP recombination system. The temporal and spatial excision of the floxed Bcl-x locus was achieved by expressing the Cre recombinase gene under control of the MMTV-LTR. By the age of five weeks, Bcl-x conditional mutant mice exhibited hyperproliferation of megakaryocytes and a decline in the number of circulating platelets. Three-month-old animals suffered from severe hemolytic anemia, hyperplasia of immature erythroid cells and profound enlargement of the spleen. We demonstrate that Bcl-x is only required for the survival of erythroid cells at the end of maturation, which includes enucleated reticulocytes in circulation. The extensive proliferation of immature erythroid cells in the spleen and bone marrow might be the result of a fast turnover of late red blood cell precursors and accelerated erythropoiesis in response to tissue hypoxia. The increase in cell death of late erythroid cells is independent from the proapoptotic factor Bax, as demonstrated in conditional double mutant mice for Bcl-x and Bax. Mice conditionally deficient in Bcl-x permitted us for the first time to study the effects of Bcl-x deficiency on cell proliferation, maturation and survival under physiological conditions in an adult animal.
Tec family kinases are implicated in T cell receptor (TCR) signaling, and combined mutation of inducible T cell kinase (Itk) and resting lymphocyte kinase (Rlk)/Txk in mice dramatically impairs ...mature T cell function. Nonetheless, mutation of these kinases still permits T cell development. While itk(-)(/)- mice exhibit mild reductions in T cells with decreased CD4/CD8 cell ratios, rlk(-)(/)-itk(-)(/)- mice have improved total T cell numbers yet maintain decreased CD4/CD8 ratios. Using TCR transgenics and an in vitro thymocyte deletion model, we demonstrate that mutation of Tec kinases causes graded defects in thymocyte selection, leading to a switch from negative to positive selection in rlk(-)(/)-itk(-)(/)- animals. The reduction in both positive and negative selection and decreased CD4/CD8 ratios correlates with decreased biochemical parameters of TCR signaling, specifically defects in capacitive Ca(2+) influx and activation of the mitogen-activated kinases extracellular signal-regulated kinase 1 and 2. Thus, Tec kinases influence cell fate determination by modulating TCR signaling, leading to altered thresholds for thymocyte selection. These results provide support for a quantitative model for thymic development and provide evidence that defects in negative selection can substantially alter thymic cellularity.
One of the greatest challenges instructors face is getting students to connect with the subject in a manner that encourages them to learn. In this essay, we describe the redesign of our Developmental ...Biology course to foster a deeper connection between students and the field of developmental biology. In our approach, we created a community of scientific practice focused on the investigation of environmental impacts on embryonic development and informed by popular and scientific media, the students' own questions, and the instructor. Our goals were to engage students in meaningful ways with the material, to develop students' science process skills, and to enhance students' understanding of broad principles of developmental biology. Though significant challenges arose during implementation, assessments indicate using this approach to teach undergraduate developmental biology was successful. (Contains 5 tables and 2 boxes.)
Course-based undergraduate research experiences (CUREs) are an alternative pedagogical approach to the apprenticeship model for high-impact research immersion experiences. A W.M. Keck-funded Research ...Immersion Program at a Hispanic-serving institution in Southern California proposed to expand the benefits of the CURE model by developing lower- and upper- division CUREs across a variety of STEM (biology, computer science, mathematics, and physics) and associated disciplines (anthropology, kinesiology, sociology, rhetoric, and communications). A subset of these courses moved completely online mid-semester of spring 2020 due to the COVID-19 pandemic. The goal of the study presented here is to understand the overall experiences of students and faculty members in the CURE courses in light of the transition to online learning. We present data here that show gains in skills development and understanding research design and stability in science opinions and self-perception in the spring 2020 semester, despite the transition to remote learning. We also report faculty perceptions regarding the challenges, supports, and successes of transitioning and implementing their CUREs in a remote learning environment.
Mutations affecting the Tec kinases Itk and Rlk decrease T cell receptor-induced Ca
2+ mobilization and Erk kinase activation and impair both positive and negative thymic selection.
Itk
−/−
and
Rlk
...−/−Itk
−/−
mice also have decreased CD4:8 T cell ratios, suggestive of altered CD4:8 lineage commitment. Nonetheless, we find that CD8 single-positive (SP) thymocytes and peripheral CD8
+ T cells in these mice do not resemble conventional CD8
+ T cells. Instead, these cells express memory markers, rapidly produce interferon-γ, and can be selected on hematopoietically derived cells, similar to MHC class Ib-restricted “innate-type” lymphocytes. Itk deficiency also greatly increases the number of cells selected by MHC class Ib. Expression of a hypersensitive Erk2 mutant partially corrects the CD8
+ T cell phenotypes in
Itk
−/−
mice, arguing that altered signaling permits development of this innate-type CD8
+ cell population. Our results suggest that Tec kinases differentially regulate development of conventional versus nonconventional lymphocytes.
Abstract
The incidence of allergy and asthma among children has risen 150% in the last two decades in children under five years old, and environmental pollutants are suspected to be contributors to ...this rise in incidence. Environmental pollutants, such as pesticides, are known to have a negative impact on the environment as well as many living organisms. However, there are a limited number of studies that have investigated the impact pesticides have on the immune system of mammals. Futhermore, even fewer studies exist that examine the impact of environmental toxicants on the immune system of the developing embryo and young animal, though the immune systems of the young are more vulnerable to perturbation than that of adults. Therefore risk assessment studies addressing fetal immune development are important tools for better understanding whether pesticides and other environmental toxicants contribute to immune system diseases that are widely common today. We set up an in vitro model system to assess the risk of exposure to the pesticide Malathion. Malathion was chosen for the study because of its widespread use in agriculture and the known role it plays as an acetylcholinesterase inhibitor. We analyzed the response of day 16–18 embryonic thymocytes to varying concentrations of Malathion. We hypothesized that greater concentrations of the pesticide would have a greater toxicity in T cells. Flow cytometric analysis was used to determine the level of toxicity and maturation of the embryonic T cells following culture.
J.V. is a ULV undergraduate researcher.
Abstract
Endocrine disrupting chemicals (EDCs) with estrogenic activity are ubiquitous in the environment. Studies of select EDCs have suggested that human exposure alters the immune system leading ...to increases in infection and autoimmune disease frequency. How these alterations occur is unclear. Exogenous and pregnancy-related increases in estrogen have been shown to induce atrophy and alterations in thymocyte development, suggesting a potential mechanism for EDC alteration of the immune system. Diethylstilbestrol (DES), a synthetic estrogen once prescribed to pregnant women, and HPTE, the metabolite of the once widely applied pesticide Methoxychlor (a replacement for DDT) are EDCs of interest. Using an in vitro assay, our research probed whether HPTE and DES alter thymocyte development in embryonic C57Bl/6 mice and at what concentrations. We also examined the mechanism by which DES and HPTE affect thymocyte differentiation. Our preliminary data show a dose-dependent decrease in thymocyte viability and alterations in thymocyte population distribution. Over the range of doses tested, increasing DES and HPTE concentration led to significant decreases in CD4+ intermediate and DP T-cells. Furthermore, we designed a time course experiment to probe the cell death mechanism induced by DES and HPTE using Annexin V and PI staining. Our work suggests that one mechanism of action of DES and HPTE on embryonic thymocytes is induction of cell death through apoptosis.
...while the department was a collective of passionate faculty, we lacked a shared vision, and several members were hesitant to change. ...the Keck/PKAL model itself was a critical piece for us.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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