bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Search for Genes Specifying HSCs through the Development of a New Retroviral-Based System That Creates Nested Deletions in ES cells.
Authors: Bilodeau Melanie , Sauvageau Guy 
Published: October 2019

Abstract
With the intention of identifying novel DNA segments essential for hematopoietic stem cell (HSC) specification, we have taken advantage of retroviral-based delivery of loxP sequences to ES cells in order to create large genomic deletions. Cre-induced recombination between the integrated loxP-containing proviruses is selected based on the reconstitution of a functional neomycin resistance cassette. A unique set of complementary retroviruses (virus A and D) was finally identified after the generation and testing of more than 10 different cassettes. The nature of the rearrangements produced (deletions, inversions, etc.) was determined according to the sensitivity to selection marker genes incorporated inside the viruses and confirmed by Southern blot analysis of genomic DNA extracted from selected clones. The new technology was first tested on 11 different clones infected at a low MOI of virus D (to obtain one integrant per cell). This virus, selected under puromycin, contains also a neomycin resistance gene (neor), devoid of an initiation codon (ATG) and of a functional promoter. The proviral integration sites are now easily determined by inverse PCR using primers anchored at the neor gene. The second retrovirus (virus A), selected with hygromycin, integrates randomly another loxP site and a pgk promoter which precedes the ATG-loxP cassette. For the 11 primary clones tested to date, the number of secondary clones derived ranged between 7000 and 37 000. Recombination between the loxP sites, upon Cre addition, is selected by the functional reconstitution of the neomycin gene (i.e. pgk-ATG-loxP-neor). Out of 107cells electroporated with the cre plasmid, neomycin resistant clones were obtained for each population (ranging between 7 and 188). Rearranged tertiary clones (i.e., virus D+A+Cre) derived from a selected primary clone (no.9) have been studied extensively by Southern Blot analysis. Chromosomal rearrangements correlated with bands of expected sizes (3.0 kb compare to 3.4 kb for the unrearranged primary and secondary clones, using KpnI digests and a neomycin probe). Clonal analysis for integration of the second retrovirus (virus A) revealed at least 13 different deletions out of 34 tested. In one experiment, very preliminary results suggest that 3 out of these 13 clones bearing independent deletions failed to differentiate upon LIF removal. Among the other 10 primary clones saturated to date, 9 gave tertiary clones sensitive to puromycin (undergoing selection). Overall, these results document that our novel retroviral-based strategy is amenable to functional screening of genes that specify HSC in differentiating ES cells. Moreover, this technology is efficient (11 out of 11 clones tested), rapid and applicable to any cellular system.
HoxA10 Dramatically Alters the Immunophenotype of Early Hematopoietic Progenitors Derived In Vitro from ES Cells.
Authors: Iacovino Michelina , Osawa Mitsujiro , Kyba Michael K. 
Published: October 2019

Abstract
Hox genes are important regulators of normal hematopoiesis, where each step in differentiation from stem cell to differentiated progeny is associated with a particular Hox expression pattern. In addition, abnormal or disregulated Hox gene expression is often associated with leukaemia. In order to understand how Hox gene transcriptional networks govern normal and leukemic hematopoiesis, we have generated ES cell lines in which expression of representative Hox genes can be induced with doxycycline during in vitro differentiation. Previous work showed that the relatively 3′ Hox gene, HoxB4, promotes self-renewal of hematopoietic stem cells, as well as enabling a primitive to definitive hematopoietic transition. We report here the generation of an ES cell line with inducible expression of a relatively 5′ Hox gene, HoxA10. When expressed during the time at which the hematopoietic lineage first segregates from the endothelial lineage (day 4 of embryoid body (EB) differentiation), HoxA10 dramatically alters the immunophenotype of the EB hematopoietic compartment. Whereas 100% of hematopoietic colony forming cells (CFC) from a wild-type day 6 EB are double positive for c-kit and CD41, HoxA10 expression from day 4 to day 6 eliminates the CD41-positive population, while increasing the c-kit-positive population. CFCs were present at relatively normal numbers. In contrast, HoxB4 did not dramatically alter either the frequency or the immunophenotype of this early hematopoietic population, but significantly enhanced the frequency of mixed-lineage CFCs. We further compared the ability of these two Hox genes to promote the outgrowth of an undifferentiated hematopoietic population on OP9 from day 6 EBs. Whereas HoxB4 induced a rapid expansion of this population, concomitantly with ongoing myeloid commitment, HoxA10 induced a much slower outgrowth characterized by a prominent B-lymphoid component as well as myeloid and erythroid elements. We are currently assaying this HoxA10-induced hematopoietic population for adult repopulating activity.
Increased Non-Canonical Splicing of Autoantigen Transcripts Provides the Structural Basis for Expression of Untolerized Epitopes.
Authors: Yang Xiao-Feng , Ng Bernard , Yang Fan , Huston David P. , Peterson Leif E. , Wang Hong 
Published: October 2019

Abstract
Alternative splicing is important for increasing the complexity of the human proteome from a limited genome. Previous studies have shown that for some autoantigens, there is differential immunogenicity among alternatively spliced isoforms. In this report, we tested the hypothesis that alternative splicing is a common feature for transcripts of autologous proteins that are autoantigens. The corollary hypothesis tested was that non-autoantigen transcripts have a lower frequency of alternative splicing. We compared the extent of alternative splicing within 45 randomly selected self-proteins associated with autoimmune diseases to 9554 randomly selected proteins encoded in human genome using bioinformatics analysis. Isoform specific regions that result from alternative splicing were studied for their potential to be epitopes for antibodies or T cell epitopes. Our results demonstrate for the first time that alternative splicing occurred in 100% of the autoantigen transcripts. This is significantly higher that the approximately 42% rate of alternative splicing observed in 9554 randomly selected human gene transcripts. In addition, the majority of isoform-specific regions of these autoantigens encoded MHC restricted T cell antigen epitopes and autoantibody binding epitopes. Furthermore, 80% of the autoantigen transcripts underwent non-canonical alternative splicing, which is also significantly higher than the less than 1% rate in randomly selected gene transcripts. These studies suggest that non-canonical alternative splicing may be an important mechanism for the generation of untolerized epitopes that may lead to autoimmunity. Furthermore, the product of a transcript that does not undergo alternative splicing is unlikely to be a target antigen in autoimmunity.
Analysis of CD161+ T Cells in Human Peripheral Blood.
Authors: Takahashi Tsuyoshi , Dejbakhsh-Jones Sussan , Strober Samuel 
Published: October 2019

Abstract
CD161 (NKR-P1) is a natural killer receptor of C-type lectin superfamily found on natural killer (NK) cells. A significant number of both human CD4+ and CD8+ T cells express CD161. However, little is known about the extended phenotype or function of CD161+ T cells. Here, we analyzed this population in normal human peripheral blood by immunofluorescent staining, and multi-color flow cytometric analysis (N=15). Amongst CD4+ T cells, a mean of 22% showed intermediate staining for CD161 (CD161int) and the remainder were CD161−. Almost all the CD4+CD161int TCRαβ+ T cells had the CD45RO+ memory phenotype, and did not express the CD16 or CD56 NK markers (<5%). CD4+CD161− T cells were a mixture of CD45RO+ and CD45RA+ cells. Invariant natural killer T (NKT) cells with the Vα24+/Vβ11+ TCR are known to express CD161, but CD4+CD161int T cells contained less than 1 % of these invariant NKT cells. After in vitro stimulation with αCD3 and αCD28 mAb, CD4+CD161int T cells produced larger amounts of both Th1 and Th2 type cytokines compared with CD4+CD161− T cells, especially IFN-γ, IL-4 and IL-10. However, there was no difference in the proliferative response between CD161− and CD161int CD4+ T cells, and CD4+CD161int T cells had no suppressor functions against autologous cell proliferation. In CD8+ T cells, there were two populations that were CD161 positive. One was CD161hi (mean; 11%) and another was CD161int (mean; 9%). Almost all CD161int and CD161hi CD8+ T cells had the CD45RO+ memory phenotype, and few expressed the Vα24+/Vβ11+ TCR NKT cell marker (<1%) or CD16 (<5%). CD8+CD161hi cells contained around 15% of CD56+ and CD8+CD161int cells contained around 5% of CD56+. CD8+CD161hi T cells had decreased expression of CD8β and 40% of CD8+CD161hi T cells expressed only CD8α. Conversely, most of the CD8α+CD8β− T cells in human peripheral blood expressed CD161. After in vitro stimulation with αCD3 and αCD28 mAb, CD8+CD161hi T cells secreted no IFN-γ, TNF-α or IL-2. Both CD8+CD161− and CD8+CD161int T cells produced all three cytokines. CD8+CD161hi T cells failed to proliferate after αCD3+αCD28 stimulation, though CD8+CD161− and CD8+CD161int T cells made vigorous proliferative responses. Thus, it appeared that CD8+CD161hi T cells were anergic. This anergic state could not be reversed by addition of IL-2. CD8+CD161+ T cells had no suppressor function against autologous cell proliferation nor cytotoxicity against the NK or NKT sensitive tumor cell lines, K562 and Jurkat. In conclusion, the CD161 marker can be used to identify subsets of CD4+ and CD8+ T cells that differ in their extended phenotypes and functions. In addition, we identified a unique subset of anergic CD8+CD161hi T cells that express a memory phenotype with a low level of CD8β.
The Spectrum of Human T Cell Receptor (TCR)-Vβ Frequencies Are Established Prior to Thymic Selection.
Authors: Melenhorst Jan J. , Follmann Dean , Fujiwara Hiroshi , Barrett Austin J. 
Published: October 2019

Abstract
Human T cell development is characterized by three phases of clonal selection and expansion: (1) β-selection which occurs independent of ligand recognition; (2) Positive thymic selection resulting in the outgrowth of T cell clones with an intermediate affinity for self-MHC/ self-peptide complexes; (3) Post-thymic antigen-driven expansion. The T cell receptor (TCR) Vβ frequencies in the peripheral blood could therefore be determined by T cell selection and expansion at one or all phases. To determine whether Vβselection patterns originate at the first phase of thymic proliferation we used flow cytometric analysis to quantitate 21 TCRVβ proteins in peripheral blood T cells of 23 HLA-disparate healthy adults. A common rank order of TCRVβ allele frequencies was found in both CD4 and CD8 T cell subsets (figure A). We found the same spectrum of HLA-independent TCR Vβ frequencies in three published studies of healthy adults. Furthermore, the percentages of CD4 cells expressing a particular Vβ were almost identical in a total of 151 data points, and was significantly correlated in CD8 cells (R2 = 0.79; slope 0.61; p < 0.00001). A significant correlation in TCRVβ allele frequencies in CD4 cells and CD8 cells was identified in all data sets (R2 = 0.73, slope 0.54, p < 0.00001)(figure B). Individuals over 50 years of age showed a 5-fold increase in variability of Vβ frequency in CD4 cells than younger subjects. The difference in the median values was statistically significant (p = 0.03) using a permutation test, indicating that post-thymic antigen recognition skewed the mature αβ T cell repertoire predominantly in elderly individuals. Our data therefore indicate that the patterns of TCRVβ protein frequencies are shaped during β-selection, conserved throughout life and are only weakly modified by antigen-driven thymic and post-thymic selection and expansion. A genetic basis for this underlying TCR Vβ frequency pattern remains undefined since there was no association of TCRVβ expression frequencies with chromosomal (5′-3′) organization. However, the reproducible pattern of TCR Vβ frequencies between individuals of diverse HLA types and distinct origins suggests that they are determined by a common genetic mechanism.
Figure Figure
The Human Plasma Cell Gene Expression Program.
Authors: De Vos John , Hose Dirk , Reme Thierry , Goldschmidt Hartmut , Rossi Jean-Francois , Cremer Friedrich , Klein Bernard 
Published: October 2019

Abstract
Seven purified peripheral blood memory B-cells (BM), seven in-vitro-generated polyclonal plasmablastic cells (PPC) and seven purified bone marrow mature plasma cells (BMPC) were studied by oligonucleotide microarrays. All samples were obtained from healthy volunteers. The gene expression profiling of these samples was determined with Affymetrix pan genomic U133A + B arrays (44 928 oligonucleotide probesets). We determined that 2313 genes were differentially expressed between these three B cell categories (P 〈 0.01 by a Kruskal-Wallis test and a ratio between two categories 〉 3).
These 2313 genes were classified into six categories, according to the expression profile: early plasma cell genes (EPC), late plasma cell genes (LPC), genes lost early during plasma cell differentiation (LEPC), genes lost late during plasma cell differentiation (LLPC), genes upregulated only in plasmablasts (PBO) and genes lost only in plasmablasts (LPBO). As expected, Ig transcripts where essentially classified as EPC. As a corollary, genes involved in protein synthesis or degradation, transmembrane transporters and metabolism genes were overrepresented in EPC genes. Interestingly, genes involved in intercellular communication and extracellular matrix were enriched in LPC, highlighting the fact that mature plasma cells develop tight interactions with the bone marrow environment. Of note, genes involved in cell cycle are upregulated mainly in plasmablasts, whereas antiapoptotic genes are lost in plasmablasts only.
Mains genes known to be involved in plasma cell differentiation display an expression profile in agreement with published data, as illustrated for transcription factors in Figure 1, validating this DNA microarray dataset. However most of these 2313 genes have either never been described yet or have no yet been linked to plasma cell differentiation. The description of those genes among our genome whose expression vary most during plasma cell differentiation will be an essential step in understanding the biology of a cell type essential to immune defenses and involved in deadly diseases.
Figure 1: Transcription factors involved in plasma cell differentiation. Color indicates the expression profile category. For each gene is given the ratio of the mean expression value in plasma cell samples (PPC and BMPC) to the mean expression value in BM. UPR: Unfolded Protein Response. Figure 1:. Transcription factors involved in plasma cell differentiation. Color indicates the expression profile category. For each gene is given the ratio of the mean expression value in plasma cell samples (PPC and BMPC) to the mean expression value in BM. UPR: Unfolded Protein Response.
VCAM-1 Expression in B-Lymphopoiesis and Leukemia.
Authors: Young Faith M. , Felgar Raymond E. , Eyssallenne Antonia P. , Bottaro Andrea , Bushnell Timothy P. 
Published: October 2019

Abstract
Vascular Cell Adhesion Molecule-1 (VCAM; CD106), a member of the Ig Superfamily of molecules, binds to the β-1 integrin, Very Late Antigen-4 (VLA-4; CD49d); this interaction plays an integral role in leukocyte trafficking as well as lymphocyte-stromal cell interactions. VCAM can be shed from the surface of cells, and, in humans, serum levels of soluble VCAM (sVCAM) parallel activity and remission states in acute lymphocytic leukemia (ALL) and inflammation. Although widely investigated as a stromal-cell associated molecule, our lab and others have recently identified VCAM expression on normal bone-marrow derived B-lymphoid cells.
Using FACS technology, we found that surface expression of VCAM is closely modulated at specific stages of B cell development, with relatively high levels on the pro-B cell population, down-modulation in pre-B cells at the onset of immunoglobulin (Ig) gene rearrangement, and subsequent re-expression at variable levels in immature and mature peripheral B cell subsets. We have verified VCAM transcripts by cDNA PCR in highly purified populations of murine precursor B cells. Normal human bone marrow precursor B-lymphoid populations (hematogones) also demonstrate VCAM surface protein expression. Finally, in an animal model of BCR/ABL+ ALL, we found that VCAM expression is dramatically increased on lymphoblasts when compared to normal reference populations in bone marrow and spleen. VCAM expression in human lymphoid malignancies is currently under investigation.
Antibody-mediated VCAM cross-linking on primary B-cell precursors ex-vivo generates intracellular reactive oxygen species, demonstrating that signaling through this molecule has functional consequences. Intriguingly, in-vivo, VCAM expression is limited to B-lymphoid cells harvested from tissues such as bone marrow, spleen and lymph node; since, in the same animal, peripheral blood lymphocytes and peritoneal cells do not express readily detectable levels of the surface antigen. VCAM-expressing B-lymphoid cells cultured ex-vivo gradually lose surface expression over 24 hours. The tissue-associated modulation of VCAM expression is preserved in the murine Ph+ lymphoblasts; leukemia cells isolated from the peripheral blood express very low levels of surface VCAM compared to those harvested from bone marrow or spleen. Our data suggests that VCAM expression is dependent on tissue-specific microenvironmental signals in-vivo.
B-lymphoid expression of both VCAM and its ligand VLA-4 is a surprising finding that has broad implications regarding leukemic cell interaction with endothelial cells, the bone marrow retention and trafficking of precursor- and leukemic-B cell populations, and the interpretation of an extensive experimental database predicated on the stromal-cell specificity of VCAM expression and function.
Direct Real Time Visualization of Platelet Calclium Signaling In Vivo: Role of Platelet Activation and Thrombus Formation in a Living Mouse.
Authors: Dubois Christophe , Panicot-Dubois Laurence , Furie Barbara C. , Furie Bruce 
Published: October 2019

Abstract
Intracellular calcium mobilization plays a critical role in platelet signaling. Upon platelet activation, an intracellular calcium mobilization leads to the activation of various intracellular and membrane proteins, including integrins involved in both platelet shape change and aggregation. The goal of the present study was to monitor platelet calcium mobilization in vivo in an intact animal and to determine which intracellular pathways are dominant in platelet accumulation into the developing thrombus. Platelets were isolated from mice, washed, loaded with a calcium-sensitive fluorochrome, Fura2-AM and then infused into a recipient mouse. We studied Fura2-AM loaded platelet incorporation during arterial thrombus development following laser injury of the vessel wall in the cremaster microcirculation of living mice using high speed intravital widefield digital microscopy. Fura-2 loaded platelets were monitored by excitation at 380 nm; this fluorescence reports the basal calcium levels in platelets. Calcium mobilization was monitored by excitation at 340 nm where the fluorescence intensity reflects Fura2-calcium complex formation. We observed that platelets bind to the growing thrombus independent of calcium mobilization. However, the stable incorporation of platelets into the thrombus correlated with a significant intracellular calcium increase. Once the thrombus reached maximal size at about 100 seconds, the calcium mobilization also reached maximal intensity. Subsequently, platelets that did not mobilize calcium dissociated from the thrombus. We confirmed these observations by using platelets treated with the calcium chelators, BAPTA-AM or EGTA-AM. We observed a significant inhibition of platelet accumulation into the thrombus, indicating that the intracellular calcium increase is necessary in vivo for the stable accumulation of the platelets into the thrombus. We also evaluated the involvement in vivo of two platelet agonists, ADP and thromboxane A2 (TxA2), on calcium mobilization and platelet incorporation into thrombi. When platelets were treated with aspirin or with the P2Y1 antagonist A3P5P (adenosine 3′-phosphate-5′-phosphate), we observed a partial decrease in both calcium mobilization and platelet accumulation into the thrombus. These results indicate that TxA2 and ADP via the P2Y1 receptor are involved in vivo in platelet activation upon vessel wall injury in this thrombosis model. When platelets were treated with both compounds, we completely inhibited the calcium increase and the incorporation of platelets into the thrombus. Altogether, our results directly show, for the first time in vivo, the importance of the calcium mobilization on platelet accumulation into the developing thrombus. The platelet agonists TxA2 and ADP both play an important and complementary role on platelet activation by acting on the mobilization of the intracellular calcium.
Immunoglobulin G Fc Receptor Polymorphisms and Clinical Course in Follicular Lymphoma Patients.
Authors: Weng Wen-Kai , Rosenberg Abby , Levy Ronald 
Published: October 2019

Abstract
Patients with low-grade B cell lymphoma, such as follicular lymphoma usually have an indolent clinical course. In most of the cases, patients do not require therapy at the time of diagnosis. Instead, patients can be monitored from months to years before the need for therapy. However, the time interval from diagnosis to the first therapy in these patients can vary significantly. In addition, the overall clinical course is heterogeneous in these patients with variable length of survival after diagnosis. The pathobiological basis of this hetergenisity is not known. We have recently found that IgG Fc receptor (FcγR) polymorphisms correlated with clinical outcome after immunotherapies in patients with follicular lymphoma. In one case, FcγRIIIa 158 V/F and FcγRIIa 131 H/R polymorphisms predicted the response to anti-CD20 antibody, rituximab therapy probably due to their role in the antibody-dependent cellular cytotoxicity. One question is whether the FcγR polymorphisms are correlated with the general clinical course of follicular lymphoma due toq their role in innate immunity. We therefore tested if FcγRIIIa 158 V/F, FcγRIIa 131 H/R and FcγRIIb 232 I/T polymorphisms predict clinical course of a group of 307 patients with follicular lymphoma. None of the three FcγR polymorphisms tested correlated with the time interval from diagnosis to first therapy, or with overall survival in this group of patients. We then tested whether FcγR polymorphisms have predictive value on clinical response to chemotherapy. Of 307 patients, only 158 patients received chemotherapy alone as first therapy, while the rest of the patients have received additional idiotype vaccination or rituximab. In the analysis of these 158 patients, there was no correlation between the FcγR polymorphisms and response rate to chemotherapy or time to progression after chemotherapy. Our results did not find any association between the three FcγR polymorphisms tested and clinical course of disease or response to chemotherapy in patients with follicular lymphoma, suggesting that the predictive value of FcγR polymorphisms on the clinical responses to rituximab is specific to the monoclonal antibody therapy.