DP Population Research Articles

Fractal Dimensions of Road Networks in Amman Metropolitan Districts

This paper uses Benoit software to calculate and compare five types of fractal dimensions (Box Db, Perimeter Area Dp, Information Di, Mass Dm, and Ruler Dr) in 22 districts in the Greater Amman metropolitan area. Also, each calculated fractal dimension is regressed to the district’s area, built-up areas, population, total number of roads, total length of roads, buildings area and number of buildings with a log-linear function. The results show that the five types of fractal dimensions (FD) are between1 and 2. The average values of Db, Dp, Di, Dm and Dr are 1.66, 1.25, 1.69, 1.72 and 1.15. In the same district, the values ​​of Dp and Dr are lower than the values ​​of Db, Di and Dm, while the values ​​of Db and Di are higher. The values of Dm show unevenness compared with the value of Db and Di. Statistical analysis shows strong correlation between the following pairs: Db and each of Log population and Log Buildings area. Dp and Log population. Di and each of Log population, Log number of roads, Log Buildings area and Log number of buildings. Dm and Dr show insignificant correlation with all variables.

Novel CD4+CD8+ Umbilical Cord Blood Regulatory T Cells

Background: Regulatory T cells (Treg), a well defined suppressor cell population with the phenotype of CD4+CD25+CD127- FoxP3+ are emerging as a promising adoptive therapy for a diverse subset of autoimmune diseases and inflammatory disorders. Specificlaly, cord blood (CB) Tregs have been shown to be superior to peripheral blood (PB) Tregs in their function and stability. Here, we define a novel subset of CB Tregs CD4+CD8+CD25+CD127- FoxP3+ that is not very well defined in the PB population and explore its' differentiation characteristics. Method: Tregs were isolated from CB or PB as described previously and cultured in the presence of CD3/28 beads and IL-2. On the day of harvest, cells were further sorted for CD8+ cell population and labelled as CD8+CD4+ (double positive CB Tregs: DP) and the leftover fraction as CD8-CD4+ (single positive CB Tregs: SP). Cells fractions were analyzed using flow cytometry and for cytokine assay, cells were activated (5 hours) with 1X Cell Stimulation Cocktail with Golgi inhibitors and stained for IFN-γ, IL-4, IL-17, IL-10 or TGFβ1 as per manufacturer's instructions. Additional cytokine assay was performed on the culture supernatants. DNA methylation assay was performed for the TSDR region as per manufacturer's instructions. Single cell RNAseq was performed at Albert Einstein Hospital and analyzed at Methodist Hospital. A threshold for original counts were applied to all four conditions, i.e. only transcripts with original counts>10 in all four. This step reduced the number of genes from 34626 to 16628; subsequently stringent cutoffs pairs showed two sets of genes- 1) Fold change>1.5, p-value<0.1, and 2) Fold change<2/3, p-value<0.1, this led to 73 rows, all with defined transcript names. Enrichment analysis using ConsensusPathDB for the above two gene sets were run to identify all pathways with at least one member-gene overlapping with either list, and the enrichment p-value for the pathway<0.1. Result: We cultured the Tregs isolated from CB or PB for 14 days and no evidence of a DP Tregs (0) was seen in PB as compared to a clearly defined DP Treg (8-26%) population in CB cultures. No differences were seen between the SP and DP CB Tregs in terms of the markers for mechanisms of suppression (FoxP3,Helios,CD39 ), homing (PSGL1, CLA, CD62L, CD49d), TGF-beta signaling (LAP, TGF-beta, GARP) and T cell signaling (LAG23, PD1, Icos, Tim3, CTLA-4, GITR). DP CBTregs consisted of a significantly higher expression of CCR4hiCXCR3hiCD95hipopulation. DP CB Tregs demonstrated a significantly higher IL-10 secretion compared to SP CB Tregs (Fig 1A) which correlated with the level of IL-10 secreted in the cell cuture supernatant (data not shown). While the degree of suppression of the proliferating conventional T cells was similar by the DP and SP CB Tregs (data not shown), a significantly higher demethylation of FoxP3 promoter and first intron region was observed in the DP vs SP CB Treg population (n=3) (Fig 1B). A total of 337 pathways were identified from Up-406 gene set, 80 of these pathways had enrichment p-value<0.05 and q-value<0.25 (Fig 1C). The Top 10 pathways enriched with members of Up-406 genes include (Fig 1D): TGF-β Receptor Signaling; Interleukin-10 signaling; Inflammatory bowel disease; IL12 signaling mediated by STAT4. A total of 158 pathways were identified from Down-314 gene set, 19 of these pathways had enrichment p-value<0.05 and q-value<0.25. The Top 10 pathways enriched with Down-314 genes included Immuno-regulatory interactions between a Lymphoid and a non-Lymphoid cell; ESC Pluripotency Pathways; Downstream signaling in naive CD8+ T cells; PDGF receptor signaling network; Nef Mediated CD8 Down-regulation (Fig E, F). Conclusion: We have identified a novel double positive CD4+CD8+ CB Treg population. Additional experiments for evaluating suppressor mechanism in addition to IL-10 secretion are ongoing. Figure 1 Disclosures Verma: Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria; Celgene: Honoraria; BMS: Research Funding; Janssen: Research Funding. Iyer:Genentech/Roche: Research Funding; Incyte: Research Funding; Seattle Genetics, Inc.: Research Funding; Novartis: Research Funding; Arog: Research Funding; Bristol-Myers Squibb: Research Funding. Parmar:Cellenkos Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Correlation analyses of CpG island methylation of cluster of differentiation 4 protein with gene expression and T lymphocyte subpopulation traits.

ObjectiveCluster of differentiation 4 protein (CD4) gene is an important immune related gene which plays a significant role in T cell development and host resistance during viral infection.MethodsIn order to unravel the relationship of CpG island methylation level of CD4 gene with its gene expression and T lymphocyte subpopulation traits, we used one typical Chinese indigenous breed (Dapulian, DP) and one commercial breed (Landrace), then predicted the CpG island of CD4 gene, determined the methylation status of CpG sites by bisulfite sequencing polymerase chain reaction (BSP), and carried out the correlation analyses of methylation frequencies of CpG sites with mRNA expression and T lymphocyte subpopulation traits.ResultsThere was one CpG island predicted in the upstream −2 kb region and exon one of porcine CD4 gene, which located 333 bp upstream from the start site of gene and contained nine CpG sites. The correlation analysis results indicated that the methylation frequency of CpG_2 significantly correlated with CD4 mRNA expression in the DP and Landrace combined population, though it did not reach significance level in DP and Landrace separately. Additionally, 15 potential binding transcription factors (TFs) were predicted within the CpG island, and one of them (Jumonji) contained CpG_2 site, suggesting that it may influence the CD4 gene expression through the potential binding TFs. We also found methylation frequency of CpG_2 negatively correlated with T lymphocyte subpopulation traits CD4+CD8−CD3−, CD4−CD8+CD3− and CD4+/CD8+, and positively correlated with CD4−CD8+CD3+ and CD4+CD8+CD3+ (for all correlation, p<0.01) in DP and Landrace combined population. Thus, the CpG_2 was a critical methylation site for porcine CD4 gene expression and T lymphocyte subpopulation traits.ConclusionWe speculated that increased methylation frequency of CpG_2 may lead to the decreased expression of CD4, which may have some kind of influence on T lymphocyte subpopulation traits and the immunity of DP population.

Genetic Ablation of Cereblon (CRBN) Increases Long-Lived Memory T Cells

Background: Immunomodulatory drugs (IMiDs) consist of thalidomide and derivatives, pomalidomide and lenalidomide. IMiDs target the E3 ubiquitin ligase substrate receptor, cereblon (CRBN), which forms a complex with DDB1-Cul4A, to induce anti-proliferative effects in tumor cells and increase the activation of T cells. Using crbn deficient T-cells, we have shown that CRBN is a negative regulator of T cell activation in mice. Dynamic changes in the T-cell repertoire occur after thymic involution. With the reduction of thymic output with age, there is a loss of naïve T-cells in the peripheral blood, and the accumulation of a memory-like T-cell population that arises through homeostatic proliferation. In this study, we demonstrate the role of CRBN in peripheral homeostatic regulation during aging. Methods: In this study, C57BL6 crbn -/- mice were housed in pathogen-free conditions to limit foreign antigen exposure. Splenocytes were stained with T cell markers to define various memory populations from 3, 8, 10 and 13 month old crbn -/- and age-matched wild-type mice. Expression of CD8, CD44, CD127 (IL-7 receptor) and KLRG1 were determined by flow cytometry. CD44-CD127+ KLRG1- CD8+ T cells were considered naive in this analysis. Consistent with previous reports, CD44+ CD127+ KLRG1- T cells represent long-term memory T-cells while CD44+CD127-KLRG1+ T cells are effector memory cells. To better understand age-related changes in the thymus, the thymus was dissected and the absolute number of thymocytes was determined by trypan blue staining. Thymic subpopulations were defined using CD4 and CD8 as single positive, double positive, and double negative cells in 13 month old crbn -/- and WT mice. We further characterized double negative (DN) populations using CD44 and CD25 to define DN1, DN2, DN3, and DN4. Results: Splenocytes in 3 and 8 month old crbn -/- and WT mice have no significant differences in CD44+ memory cells, naïve cells (p=0.7850 and p=0.5061) or recently activated cells [CD69+ or CD25+]. Changes related to thymic involution in WT mice become evident at 10 months of age. Crbn -/- mice exhibit a similar percentage and absolute number of total memory cells (p=0.8194) at 10 months of age, as defined by CD44 expression. Evaluation of long-lived versus short-lived sub-populations of memory cells in these older mice showed that crbn deficiency is associated with significantly more CD44+ CD127+ KLRG1- CD8+ [long-lived] memory T cells compared to WT mice (p&lt;0.001). Evaluation of the thymus revealed no difference in absolute numbers of thymocytes in younger mice. Moreover, crbn deficient mice have normal distribution of SP, DP, and DN populations, with only slight changes in DN1, DN2, DN3, and DN4 subpopulations. Absolute numbers of thymocytes, however, were significantly higher in the crbn -/- mice at 10 months of age compared to age-matched controls (p=0.0182). Conclusions: The fate of developing T cells is regulated by positive and negative selection in the thymus where the coordination and selection of self-tolerant repertoire is maintained. Age-related changes caused by thymic involution impacts the protective responses of these cells against foreign pathogens and tumor cells. Our data show that the genetic germline depletion of crbn leads to an increase in long-lived memory T cells in naturally aged animals. Universally in cancer patients, and especially in MDS, the lymphocyte compartment is characterized by premature age-related changes due possibly to reduction in lymphopoiesis at the stem cell level or due to chronic antigen stimulation. Given that the CD44+ CD127+ KLRG1- population is significantly higher in aged crbn -/- mice, these mice may be more resistant to viral infection and or development of malignancy. The mechanism responsible for this phenotypic difference could be due to cell intrinsic signaling or metabolic properties. However, a role for CRBN in thymic epithelial cells must also be considered given the use of germline deficient animals in this study. Taken together, we have demonstrated that ablation of crbn results in an increase in long-lived CD8+ T cells during aging, which suggests that targeting CRBN and/or treating with lenalidomide may functionally improve memory T cell capacity in the elderly. Disclosures No relevant conflicts of interest to declare.

Acute Myeloid Leukemia Alters Natural Killer Cell Maturation and Functional Activation

Acute myeloid leukemia (AML) is a devastating disease primarily affecting adults. Immune evasion is a major mechanism of AML persistence, and represents a barrier for long-term clinical success. Natural killer (NK) cells are a key component of the innate immune system and hold promise as a tool for effective anti-leukemia therapy. However, to date, the clinical success of NK therapy has been disappointing, indicating additional immune evasion strategies may affect the ability of NK cells to function in patients. We hypothesized that AML may evade the innate immune system by inhibiting NK cell maturation.To evaluate NK function and maturation during AML progression, our lab utilized a novel knock-in model of AML that expresses both Flt3-ITD and Mll-PTD mutations (PTD/ITD), and develops AML with 100% penetrance and recapitulates human disease. For these studies, both primary PTD/ITD mice and transplanted AML blasts were used. For transplant studies, AML or wild-type (WT) control cells (CD45.2+) were transplanted into irradiated C57BL/6 (CD45.1+) naïve recipients. Normal (CD45.1+) NK cells were defined as NK1.1+/CD3- by flow cytometry, and expression of receptors was determined within this population. NK cells in leukemic mice exhibited a reduction in the activating receptors Ly49D and Ly49H in the spleen, blood, LN, and bone marrow. There was also an increase in the inhibitory receptor Ly49C/I and NKG2C/A/E (both p<0.05). However, these alterations were not consistent with all activating and inhibitory receptors, as CD69 was elevated, while NKp46 expression was decreased (both p<0.05). NK function was determined by ELISA measurement of soluble interferon-gamma (IFN-g) production. While there was no change in IFN-g production at baseline, there was surprisingly a significant elevation in IFN-g detected upon stimulation with interleukin-12 and interleukin-18 in the NK cells isolated from leukemic mice as compared to WT control mice (p=0.0004).NK maturation was then evaluated by examining CD11b and CD27 protein expression on NK1.1+/CD3- cells in the spleen, bone marrow, blood, and LN. CD11b+/CD27+ (DP) NK cells were reduced in the spleen as well as bone marrow, blood and LN (p<0.02 for all organs). AML splenocytes had a corresponding increase in NK cells negative for both proteins (DN) when compared to WT control splenocytes (p=0.0004). Previous studies indicate the DP population of NK cells have the highest cytotoxic and cytokine producing functions, while DN NK cells are functionally immature. These alterations were more distinct as AML burden increased in the mice (p=0.0004). Interestingly, there was no significant difference in the most mature CD11b+/CD27- NK population in the spleen, but it there was a significant increase in the CD11b+/CD27- population in the blood (p=0.002). Additional analysis of two transcription factors known to regulate NK development, T-bet and Eomes, was also performed. A reduction in expression of both transcription factors was seen in total NK cells of leukemic mice, as well as NK subsets in the spleen (p<0.02). These data suggest that NK maturation is altered in the presence of AML, and that this alteration becomes more pronounced as leukemic burden increases. Studies evaluating the mechanistic link between AML and NK maturation are ongoing. Future studies will evaluate the efficacy of combinatorial therapies addressing both this imbalance in maturation as well as enhancing recognition to improve NK killing of AML cells. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2005: Aberrant hedgehog signaling is responsible for the highly invasive behavior of a subpopulation of hepatoma cells

Abstract Background: Hepatoma consists of heterogeneous subpopulations in terms of their cell surface markers, tumorigenicity, invasion and metastatic capability. In our previous study, we indicated that the CD133-/EpCAM- hepatoma subpopulation was more metastatic than its counterpart; however the controlling mechanisms are unexplored (J Hepatol 2011;55:838-845). The present study aims to delineate the significance of aberrant hedgehog signaling in the development of metastases. Methods: Huh-7 cells were FACS-enriched into CD133+/EpCAM+ (double positive, DP) and CD133-/EpCAM- (double negative, DN) subpopulations. The double negative cells further underwent Transwell-selection for metastatic cells (Transwell-selected, TS). The metastatic rate, matrix metalloproteinase (MMP) gene expression, epithelial mesenchymal transition (EMT) markers, and hedgehog signaling activities, as well as truncated Gli1 were determined in these subpopulations. Results: TS cells displayed much greater metastatic activity as evidenced by an increased Transwell invasion rate (105 vs. 68.7 and 58.0%, p&amp;lt;0.01), extremely elevated expression of MMP1, 2 and 9 genes (36-3000-fold, p&amp;lt;0.05-0.001) compared to DN and DP populations. There was a nearly 2-fold increase in TGF-β1 gene expression in TS cells compared to DN and DP subpopulations. TS cells lost E-cadherin and were all vimentin-positive as shown by immunocytochemistry in contrast to DP cells. There was a transitional increase in Gli1 gene expression levels from DP, DN to TS subpopulations, which is consistent with elevated Gli-2 and Twist1 levels in the nuclear fraction as detected by Western blot analysis and quantitative RT-PCR. Furthermore, truncated Gli1, which has been indicative of mediating expression of the molecules involved in metastasis, was detected in highly invasive hepatoma cell subpopulations and was the highest in the TS subpopulation; whereas, it did not appear in primary human hepatocytes. Flow cytometric analysis verified that these TS cells maintained a negative cell surface marker profile in their sub-passages. Conclusions: The highly metastatic capability of unique Transwell-selected cells is associated with negative expression of CD133 and EpCAM surface molecules, significant EMT, enhanced hedgehog activity and aberrant expression of truncated Gli1. The appearance of truncated Gli1 is probably responsible for highly invasive behavior of the transwell-selected double-negative subpopulation. Further exploration of molecular mechanisms of abnormal hedgehog signaling in modulating metastases will establish molecular targets for the therapeutic intervention of hepatoma metastases. Citation Format: Yahan Fan, Jia Ding, Jian Wu. Aberrant hedgehog signaling is responsible for the highly invasive behavior of a subpopulation of hepatoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2005. doi:10.1158/1538-7445.AM2014-2005

Elevated T Cell Receptor Signaling Identifies a Thymic Precursor to the TCRαβ+CD4−CD8β− Intraepithelial Lymphocyte Lineage

The origin and developmental pathway of intestinal T cell receptor αβ(+) CD4(-)CD8β(-) intraepithelial lymphocytes (unconventional iIELs), a major population of innate-like resident cytolytic T cells, have remained elusive. By cloning and expressing several TCRs isolated from unconventional iIELs, we identified immature CD4(lo)CD8(lo)(DP(lo))CD69(hi)PD-1(hi) thymocytes as the earliest postsignaling precursors for these cells. Although these precursors displayed multiple signs of elevated TCR signaling, a sizeable fraction of them escaped deletion to selectively engage in unconventional iIEL differentiation. Conversely, TCRs cloned from DP(lo)CD69(hi)PD-1(hi) thymocytes, a population enriched in autoreactive thymocytes, selectively gave rise to unconventional iIELs upon transgenic expression. Thus, the unconventional iIEL precursor overlaps with the DP(lo) population undergoing negative selection, indicating that, concomitant with the downregulation of both CD4 and CD8 coreceptors, a balance between apoptosis and survival signals results in outcomes as divergent as clonal deletion and differentiation to the unconventional iIEL lineage.

CD5-CK2 binding domain regulates TCR signals and apoptosis during thymic development. (HEM4P.232)

Abstract CD5 has been previously shown to play an important role in thymic development by downregulating TCR signaling. The receptor contains two major signaling domains in its cytoplasmic tail. The first domain is a functional ITIM domain that recruits proteins such as Ras-Gap, PI3K, Zap 70 and others. The second domain is a CKII Binding Domain (CD5-CKII BD), which in peripheral T cells promotes survival, proliferation and Th differentiation. However, the role of these CD5 signaling domains in early T cell development is poorly understood. In this work we determined the function of CD5-CKII BD in differentiation of thymocyte subpopulations, activation of TCR downstream molecules and regulation of apoptosis using a knock-in mouse in which the CK2 BD is selectively ablated (CD5ΔCKII-BD). Our data show that in non TCR-Tg and in OTII TCR-Tg the loss of CD5-CKII signaling results in a decrease in DP populations in proportion and total cell number and an increase in percentage of CD4+ thymocytes. This reduction in DP thymocytes of OTII.CD5ΔCKII-BD mice was associated with increase in antigen-induced acute activation of ERK phosphorylation relative to OTII.wild-type mice. Additionally, DP and CD4+ thymocytes in CD5ΔCKII-BD mice had increased apoptosis along with elevated levels Caspase 3. Collectively, these results demonstrate that CD5-CKII BD is an important regulator of TCR signals during thymic development and also is a major regulator of the apoptotic events in the thymus.

Abstract 5007: Delineating human fetal CNS stem cell hierarchy reveals a progenitor cell of origin for human Gliobastoma Multiforme.

Abstract Glioblastoma Multiforme (GBM) is the most common and highly aggressive human brain tumor. Even with great advancement of surgical procedures and treatment regimens with radiation and chemotherapy, malignant gliomas remain incurable. This is due to their resistance to all conventional therapies and the diffuse infiltrative nature of the tumor cells, which makes complete tumor resection impossible. One of the major difficulties for the identification of the cell of origin for gliomas is the complex cellular composition of this disease. It is now hypothesized that the different subtypes of high-grade glioma may have different cell of origin and in mouse models it has been shown that the cell of origin resides within the progenitor populations. In order to identify the cell of origin we decided to first dissect out the various stages of CNS stem cell differentiation. After testing a panel of 34 antibodies to prospectively isolate neurospheres forming cells from human fetal brain tissue we identified a panel of four antibodies (i.e CD15, Notch1, EGFR, and CD90). Subpopulations of cells derived after FACS were analyzed for single cell lineage potency and the ability to generate neurospheres. Using an in-vitro limiting dilution assay, we ascertained that double-positive (DP; CD15+Notch1+EGFR-CD90-)) populations have the highest frequency of self-renewing cells, followed by quadruple-positive (QP) populations (CD15+Notch1+EGFR-CD90-). In-vitro differentiation assays reveal both DP and QP to be multipotent in nature however serial passaging showed that only the DP cells have a consistent self-renewing capacity, whereas QP derived cells were unable to survive after 5-6 passages. In glioma samples however we observed by limiting dilution and serial passaging assays that the tumorsphere forming frequency was predominant in the QP population and not in the DP populations. Furthermore the GBM derived DP cells serially passaged if grown under normal neural stem cell growth condition suggesting that this population is still at a pre-malignant state. In-vivo tumorigenic assay via orthotopic implation of as few as a 1000 cells into mouse brain showed significantly higher tumor formation from the QP cells and not in the DP cells. Taken together our data suggests the presence of “normal” or pre-malignant stem cells in glioblastoma, which suggests a possible mechanism of relapse. These pre-malignant cells could comprise a cellular reservoir that may need to be targeted for future therapies. Citation Format: Siddhartha S. Mitra, Sharareh Gholamin, Abdullah Feroze, Samuel H. Cheshier, Irving L. Weissman. Delineating human fetal CNS stem cell hierarchy reveals a progenitor cell of origin for human Gliobastoma Multiforme. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5007. doi:10.1158/1538-7445.AM2013-5007

Disease resistance and genetic diversity analysis in selected populations ofMacrobrachium rosenbergii

16 selected populations were injected with bacteria(Vibrio alginolyticus) suspension to know deeply about disease resistance of Macrobrachium rosenbergii. Bacterial challenge experiments demonstrated that there were big differences in disease resistance among the 16 selected populations. Some families exhibited strong disease resistance to bacterial infection,while some families were not resistant to bacterial challenge. Among the 16 selected populations,3 populations( population SCR11-6,SCR11-11 and SCR1116) with strong disease resistance with a survival rate of 80% were identified,7 populations with secondly strong disease resistance,4 populations with normal disease resistance with a survival rate of 65%-70%, and 2 populations with low disease resistance with a survival rate of 49.53% and 35.0% were identified. The genetic structure of 16 selected populations was analyzed assessed by 8 microsatellite markers. The results showed that the number of total alleles was 53. The numbers of allele at each locus ranged from 5 to 9 with an average of 6.625,and polymorphic information content(PIC) was 0.6294- 0.8294,and the average was 0.7323. The mean polymorphism information content in 16 selected populations was 0.4932- 0.6956. The average observed and expected heterozygosity of the 16 selected populations varied from 0.5062 to 0.6513 and from 0.5519 to 0.7332,respectively,and the average genetic similarity coefficient and genetic distance between them was 0.6552 and 0.4344,respectively. Besides,after PCR amplification on the individuals of DP and SP populations,differential bands were calculated and the correlation betw een growth trait and microsatellite loci was established. The results showed that 5 loci were correlated with disease resistant trait,of which MRMB11 had extreme significance of 0.282. These results indicated that 4 of 16 selected populations had strong disease resistance and high genetic diversity. The development of these 8 populations lays foundation for selective breeding of M. rosenbergii with disease resistance.

Synergism of resistance to phosalone and comparison of kinetic properties of acetylcholinesterase from four field populations and a susceptible strain of Colorado potato beetle

The susceptibility to phosalone and biochemical characteristics of acetylcholinesterase (AChE) were compared between susceptible (SS) strain and four field populations of Colorado potato beetle (CPB) collected from commercial potato fields of Hamedan Province in west of Iran. Bioassays involving topical application of phosalone to fourth instars revealed up to 252 fold resistance in field populations compared with the SS strain. Synergism studies showed that although esterase and/or glutathione S-transferase metabolic pathways were present and active against phosalone, they were not selected for and did not have a major role in resistance. It is likely that piperonyl butoxide (PBO) reduced phosalone toxicity by inhibiting bio-activation of phosalone. The affinity ( K m) and hydrolyzing efficiency ( V max) of AChE to selected substrates, namely, acetylthiocholine iodide (ATC), propionylthiocholine iodide (PTC), and butyrylthiocholine iodide (BTC) were examined. AChE inhibition by higher substrate concentration was evident only in the SS strain. In resistant field populations, Aliabad (Aa), Bahar (B) and Dehpiaz (Dp), substrate inhibition at higher concentrations was not seen. There was no definite optimal concentration found for any of the substrates examined. When ATC, PTC, and BTC were used as substrate, the reaction rates of AChE from Yengijeh (Yg) population increased as the concentration of all three substrates were increased, but were almost constant at concentration of ATC ⩾ 3.98, PTC ⩾ 2.8, and BTC ⩾ 5 mM. The susceptible form of AChE had the most efficient ATC hydrolysis but very low BTC hydrolysis activity. In contrast, AChEs from field populations elicited relatively reduced ATC hydrolysis, but relatively increased BTC hydrolysis. The in vitro inhibition potency of some organophosphates (OPs), on AChEs of the field populations and SS strain was determined. The rank order from the most potent inhibitor to the least as determined by their bimolecular reaction constants ( K i) was ethyl paraoxon > diazoxon > methyl paraoxon for AChE from Aa, B, Dp, and Yg populations, respectively, whereas the rank order for the susceptible strain was methyl paraoxon > ethyl paraoxon > diazoxon.

N- and O-Glycans Modulate Galectin-1 Binding, CD45 Signaling, and T Cell Death

Galectin-1, a beta-galactoside-binding protein highly expressed in the thymus, induces apoptosis of specific thymocyte subsets and activated T cells. Galectin-1 binds to N- and O-glycans on several glycoprotein receptors, including CD7, CD43, and CD45. Here we show that galectin-1 signaling through CD45, which carries both N- and O-glycans, is regulated by CD45 isoform expression, core 2 O-glycan formation and the balance of N-glycan sialylation. Regulation of galectin-1 T cell death by O-glycans is mediated through CD45 phosphatase activity. While galectin-1 signaling in cells expressing low molecular weight isoforms of CD45 requires expression of core 2 O-glycans (high affinity ligands for galectin-1), galectin-1 signaling in cells expressing a high molecular weight isoform of CD45 does not require core 2 O-glycans, suggesting that a larger amount of core 1 O-glycans (low affinity ligands for galectin-1) is sufficient to overcome lack of core 2 O-glycans. Furthermore, regulation of galectin-1 signaling by alpha2,6-sialylation of N-glycans is not solely dependent on CD45 phosphatase activity and can be modulated by the relative expression of enzymes that attach sialic acid in an alpha2,6- or alpha2,3-linkage. Thus, N- and O-glycans modulate galectin-1 T cell death by distinct mechanisms, and different glycosylation events can render thymocytes susceptible or resistant to galectin-1.

T lineage precursors from diabetes-prone NOD mice exhibit defective alpha-beta and enhanced gamma-delta T cell development (99.20)

Abstract Because defects in various T lineages have been suggested to contribute to autoimmune diabetes in NOD mice, we sought to determine if early T cell precursors from NOD mice exhibit developmental abnormalities, which might affect later T cell lineage choices and responses. We previously reported that early T cells from NOD.Rag-/- mice undergo a spontaneous breakthrough at the first TCR-dependent checkpoint, a trait that appears to map near to two diabetes susceptibility genetic regions. We have also found that purified early T cell subsets from wild-type NOD mice undergoing differentiation in vitro exhibit profound defects in the generation and maintenance of DP populations, while gamma-delta T cell development is enhanced. NOD thymocytes also exhibit defects in development to the DP stage in vivo. These results show that NOD T cells have a lineage-specific abnormality at the first TCR-dependent checkpoint, which may select for DP cells with skewed signaling and/or survival characteristics, potentially altering positive and negative selection thresholds, T lineage choices, peripheral responses, and autoimmunity. (Supported by Juvenile Diabetes Foundation International and NIH R01AI064590 (to MAY).)

KLF13 influences multiple stages of both B and T cell development

The Kruppel-like factor, KLF13, is a member of a family of transcription factors shown to be involved in haematopoietic development. Here we show that KLF13 is involved in the development of B and T cells at multiple stages. Expression of KLF13 in the thymus was maximal in the DP population and in KLF13-/- deficient mice there was an accumulation of DP thymocytes and reduction of CD4+SP cells. Cell-surface expression of CD3high, CD8, CD5 and HSA were altered on KLF13-/- DP cells, consistent with a defect in TCR signalling and at the DP to SP transition in KLF13-/- mice. KLF13 is also expressed in peripheral T-cells and peripheral T cell activation was impaired in KLF13-/- mice. Analysis of early B cell development in the bone marrow (BM) revealed a partial arrest of B cells at the transition from CD43+ to CD43- pre-B cell, a transition that requires signalling through the pre-BCR. The proportion of IgM+/IgD+ mature B cells was also increased in the BM of the KLF13-/- mice. This finding is consistent with a reduction in the strength of BCR signal or an accumulation of recirculating B cells from the periphery. Analysis of splenocytes isolated from KLF13-/- mice revealed an increase in the expression of CD21 and CD23 on B220+ B cells, demonstrating a negative regulatory role for KLF13 in co-regulation of expression of CD21 and CD23. Thus KLF13 is involved at multiple different checkpoints in development that require signalling through the TCR, pre-BCR or mature BCR.

A NUP98-HOXD13 Fusion Gene Impairs Differentiation of T and B Lymphocytes.

Chromosomal translocations leading to NUP98 gene fusions are associated with a wide range of hematologic malignancies, including acute myelogenous leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), and myelodysplastic syndrome (MDS). The NUP98-HOXD13 (NHD13) gene fusion was first identified in a patient with a MDS that progressed to AML. Recently, we used an NHD13 fusion gene to develop a mouse model of MDS that recapitulates all of the key findings of the human disease, including ineffective hematopoiesis leading to peripheral blood cytopenias, dysplasia, and progression to AML. In addition to the features noted above, we observed that the NHD13 mice were lymphopenic, and 10–30% of these mice, depending on the strain background, progressed to T-ALL. These findings prompted us to define the lymphocyte development of NHD13 mice. CBCs obtained from clinically healthy NHD13 mice showed lymphopenia (2.21 vs. 8.72 K/μL, p<0.01) compared with wild type (WT) littermate controls. This lymphopenia was due to a decrease in both T and B cells, as FACS analysis of peripheral blood (PB) from NHD13 mice revealed a marked decrease in CD4 single positive (SP) and B220+/IgM+ cells (p<0.05) compared to WT controls; similar findings were observed in the spleen (p<0.01). The percentage of CD8 SP cells was not different between the NHD13 and WT mice in either the PB or spleen. To investigate the cause for the B-cell lymphopenia, we determined the Hardy fractions of bone marrow B cells. Although the pro-B cell (B220+/CD43+, Hardy fractions A–C) populations showed no difference between NHD13 and WT BM, the NHD13 BM showed decreased pre-B and B cell (B220+/CD43−, Hardy fractions D–F) populations, suggesting impaired differentiation at the pro-B to pre-B stage. Thymi from NHD13 mice (n=7, median age=7 months) showed grossly decreased size and decreased total number of thymocytes (1.75×107 vs. 9.21×107, p<0.01). The CD4/CD8 DN population was markedly increased (p<0.001) and the CD4/CD8 DP population markedly decreased (p<0.001) in NHD13 compared to WT mice. In addition, there was a variable increase in the DN1 and DN2 population, as well as a decrease in the DN3 and DN4 population in thymi from the NHD13 thymus, suggesting a partial block at the DN2 to DN3 transition. To determine clonality of the NHD13 thymocyte population, we used degenerate RT-PCR to identify clonal TCRβ gene rearrangements. As expected, the WT thymi showed polyclonal TCRβ gene rearrangements. However, 5 of 6 NHD13 thymus samples showed clonal DJ rearrangements, with over half of the TCRβ rearrangements in the thymus showing an identical D-J junction, but distinct V-D junctions. This finding suggested that there was a massive clonal expansion of DN2 cells that had undergone a DJ rearrangement, but not completed a VDJ rearrangement, further supporting the contention of a partial block at the DN2 to DN3 transition. Interestingly, analysis of TCRβ gene rearrangements in NHD13 spleens showed no evidence of cells with clonal DJ rearrangements, suggesting that the thymocytes with clonal DJ rearrangements did not mature and migrate from the thymus. Finally, we noted that although there is a marked increase in DN thymocytes from the NHD13 mice, the T-ALL that developed in these mice were typically DP or CD4 SP, suggesting that a rare cell that “escapes” the T-cell differentiation block is susceptible to leukemic transformation. Taken together, these findings demonstrate that the NHD13 transgene inhibits lymphoid as well as myeloid and erythroid differentiation, and is oncogenic in lymphoid as well as myeloid cells.

Microsatellite marker analysis of genetic diversity and phylogenetic relationships in three populations of Cyprinus carpio L. y Shandong Province

利用微卫星标记技术对3个鲤鱼群体(DP、WS和SY)的遗传多样性及亲缘关系进行研究.结果表明.3个群体的遗传多样性总体水平较高,其中SY群体最高,WS群体次之,DP群体最低.根据基因型频率(P)检验了各位点的Hardy- Weinberg平衡情况.Fis值表明3个群体中共有32个微卫星位点的杂合度观测值过剩.3个群体间,WS和SY群体间的遗传相似系数最大(0.8320),遗传距离最小(0.1680),表明这两个群体亲缘关系较近;WS和DP群体间的遗传相似系数最小(0.8288),遗传距离最大(0.1712),表明这两个群体亲缘关系较远.;The genetic diversity and the phylogenetic relationships of three Cyprinus carpio L. populations were studied by using microsatellite marker technology. It showed that the genetic diversity level of three Cyprinus carpio L. populations was high, population was the highest, WS population was lower,and DP population was the lowest. In order to check the depature of microsatellite loci from HWE, the genotypic frequencies were calculated. Furthermore,32 cases of observed heterozygosity excess(Fis <0) were observed in three populations. The genetic similarity index between WS population and SY population was the highest(0.8320) among the three populations, and their genetic distance was the lowest (0.1680),which indicated that the phylogenetic relationship between these two populations was quite close. On the other hand, the genetic similarity index between WS population and DP population was the Iowest(0.8288),and their genetic distance was the highest(0.1712),which indicated that the phylogenetic relationship between ihese two populations was quite far.

Critical Function for SIP, a Ubiquitin E3 Ligase Component of the β-Catenin Degradation Pathway, for Thymocyte Development and G1 Checkpoint

Beta-catenin has been implicated in thymocyte development because of its function as a coactivator of Tcf/LEF-family transcription factors. Previously, we discovered a novel pathway for p53-induced beta-catenin degradation through a ubiquitin E3 ligase complex involving Siah1, SIP (CacyBP), Skp1, and Ebi. To gain insights into the physiological relevance of this new degradation pathway in vivo, we generated mutant mice lacking SIP. We demonstrate here that SIP-/- thymocytes have an impaired pre-TCR checkpoint with failure of TCRbeta gene rearrangement and increased apoptosis, resulting in reduced cellularity of the thymus. Moreover, the degradation of beta-catenin in response to DNA damage is significantly impaired in SIP-/- cells. SIP-/- embryonic fibroblasts show a growth-rate increase resulting from defects in G1 arrest. Thus, the beta-catenin degradation pathway mediated by SIP defines an essential checkpoint for thymocyte development and cell-cycle progression.

The Essential of PI3Kγ and PI3Kδ in Thymocyte Survival.

Class I phosphoinositide 3-kinases (PI3Ks), consisting of PI3Kα, β, γ and δ, are a family of intracellular signalling molecules that play an important role in both innate and adaptive immune responses. In thymocytes, however, their role is less clear as PI3Kγ, but not other class I members, has been postulated to be partially involved in pre-TcR-dependent differentiation without effecting overall T cell numbers. We now report that PI3Kγ in conjunction with its delta counterpart are essential for thymocyte survival and ultimately T cell production. Surprisingly, genetic deletion of the both p110γ and p110δ catalytic subunits resulted in a dramatic reduction in thymic size, cellularity, and lack of cortico-medullary differentiation. Total thymocyte counts in these animals were 27-fold less than observed for wild-type (WT) controls due to a diminished number of DP cells and was associated with T cell depletion in both peripheral blood and secondary lymphoid organs. This phenotype could also be recapitulated in vitro as incubation of thymi harvested from day 14 p110γ−/− embryos with the p110δ specific inhibitor IC87114, but not vehicle control, yielded identical results. Moreover, the observed reduction in the DP population appears to be intrinsic to thymocytes as reconstitution of animals deficient in both p110γ and p110δ catalytic subunits with WT fetal liver cells restored the proportions of DN, DP, SP to that observed in WT controls. The observed defects, however, appear to be due to an excessive amount of apoptosis of the DP thymocyte population, while TCRbeta expression, pre-TCR selection and DP cell production appear to proceed unperturbed. Thus, this study provides evidence that these two distinct class I PI3Ks isoforms work in concert to protect developing thymocytes from apoptotic signals rather than by participating directly in thymocyte differentiation.

Expression of the HPV16E7 oncoprotein by thymic epithelium is accompanied by disrupted T cell maturation and a failure of the thymus to involute with age.

Transgenic mice expressing the E7 protein of HPV16 from the keratin 14 promoter demonstrate increasing thymic hypertrophy with age. This hypertrophy is associated with increased absolute numbers of all thymocyte types, and with increased cortical and medullary cellularity. In the thymic medulla, increased compartmentalization of the major thymic stromal cell types and expansion of thymic epithelial cell population is observed. Neither an increased rate of immature thymocyte division nor a decreased rate of immature thymocyte death was able to account for the observed hypertrophy. Thymocytes with reduced levels of expression of CD4 and/or CD8 were more abundant in transgenic (tg) mice and became increasingly more so with age. These thymic SP and DP populations with reduced levels of CD4 and/or CD8 markers had a lower rate of apoptosis in the tg than in the non-tg mice. The rate of export of mature thymocytes to peripheral lymphoid organs was less in tg animals relative to the pool of available mature cells, particularly for the increasingly abundant CD4lo population. We therefore suggest that mature thymocytes that would normally die in the thymus gradually accumulated in E7 transgenic animals, perhaps as a consequence of exposure to a hypertrophied E7-expressing thymic epithelium or to factors secreted by this expanded thymic stromal cell population. The K14E7 transgenic mouse thus provides a unique model to study effects of the thymic epithelial cell compartment on thymus development and involution.

Facilitation of beta selection and modification of positive selection in the thymus of PD-1-deficient mice.

PD-1 is an immunoglobulin superfamily member bearing an immunoreceptor tyrosine-based inhibitory motif, and disruption of the PD-1 gene results in the development of lupus-like autoimmune diseases. In this study, we examined effects of the PD-1 deficiency on the thymocyte differentiation at the clonal level using T cell receptor (TCR)-beta (Vbeta8) and TCR-alpha/beta (H-Y and 2C) transgenic mice. In these TCR transgenic lines, PD-1 expression in the thymus was variably augmented, but as in the normal mice, confined largely to the CD4(-)CD8(-) thymocytes. The transgenic mice crossed with PD-1(-/)- mice in the neutral genetic backgrounds exhibited selective increase in the CD4(+)CD8(+) (DP) population with little effect on other thymocytes subsets. Similarly, the absence of PD-1 facilitated expansion of DP thymocytes in recombination activating gene (RAG)-2(-/)- mice by anti-CD3epsilon antibody injection. On the other hand, H-Y or 2C transgenic PD-1(-/)- mice with the positively selecting background showed significantly reduced efficiency for the generation of CD8(+) single positive cells bearing the transgenic TCR-alpha/beta in spite of the increased DP population. These results collectively indicate that PD-1 negatively regulates the beta selection and modulates the positive selection, and suggest that PD-1 deficiency may lead to the significant alteration of mature T cell repertoire.