Low-dose, sublingual AZT-monophosphate therapy for HIV+ patients?

Leukemia Associated Mutants of SHP2 Protein Tyrosine Phosphatase Inhibit Nf1-Expression by Decreasing ICSBP and IRF2 Phosphorylation.

Our research explores the potential of a panel of monoclonal antibodies targeting the tumor proliferation biomarker Thymidine Kinase 1 (TK1) for both clinical and therapeutic applications. Cancer biomarkers have become a critical component of precision medicine. TK1 is a well-known tumor proliferation biomarker that is released into the serum, is up-regulated in malignant tissues and can be found on the cell membrane of several cancer types. Notwithstanding the versatility of TK1 as a tumor biomarker, there are a limited number of available antibodies for the detection and quantification of TK1. Moreover, to the date TK1 antibody-based therapies are not being tested in the clinical setting. Thus, the generation of more sensitive TK1 antibodies could increase the availability, accuracy and options of current TK1-based diagnostics and antibody-based immuno-cell therapies. Six peptide sequences across the TK1 molecule were selected. The antibodies were generated using hybridoma technology. Seventeen clones were evaluated in ELISA with a calibration dose-response curve. The calibration curves showed R squares ranging from 0.9738-0.9980 with a 10-15 %CV. The limit of detection and quantification (LOQ, LOD) were obtained. The clones 3B2E11, 9C10, 8G2, 5F7G11, 3B4 and 3G7 showed the lowest values, being 3B2E11 the most sensitive with a LOD of 18.6 ng/ml and a LOQ of 64 ng/ml. Western blot data showed specific binding to recombinant TK1 and to multiple forms of TK1 in cell lysates, and serum samples for 14 of the 17 clones. Although, differences in the binding patterns were found in cell lysates. Flow cytometry was performed to analyze TK1 surface expression. Antibodies 8G2, 3B4 and 57G11 showed consistent binding across 4 cancer cell types in a similar percentage to the commercial TK1 antibody (Abcam91651) with a maximum percentage of binding in lung of (95.6%) followed by prostate (72.2%), colon (62.4%) and breast (49.1%). No significant binding for either the commercial or the custom TK1 antibodies was found on normal mono nuclear cells (MNC). The clones 8G2 and 3B4 were selected for testing their potential as therapeutic agents in antibody-dependent cell-mediated cytotoxicity (ADCC) experiments. Around 50% and 42% increased killing of A549 cells was observed with antibodies 8G2B and 3B4 respectively 48 hrs. after adding the antibodies when compared with isotype controls (p <0.05). Based on dose response curves 2.5 ug/ml for the clone 8G2B and 5-7 ug/ml for the clone 3B4 were the minimum concentrations required to show significant specific cell death in A549 cells. The antibodies developed have shown capacity for detection and quantification of TK1 in serum and on the membrane of cancer cells. Moreover, our in vitro ADCC experiments provide more evidence that membrane associated TK1 has potential as an immunotherapeutic target. Citation Format: Edwin J. Velazquez, Taylor D. Brindley, Gajendra Shrestha, Rachel A. Skabelund, Corbin M. Lee, Zachary D. Ewel, Eliza E. Bitter, Michelle H. Townsend, Kelsey B. Bennion, Kai Li Ong, Kiara V. Whitley, Richard A. Robison, Scott K. Weber, Kim L. O'Neill. Generation and characterization of a panel of monoclonal antibodies against the tumor biomarker Thymidine Kinase 1 for research, clinical and therapeutic applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2987.

ICSBP Directs Bipotential Myeloid Progenitor Cells to Differentiate into Mature Macrophages

A technique for the separation and cryopreservation of myeloid stem cells from human bone marrow

The nature of the T-cell response to antigen is governed by the activation state of the antigen-presenting dendritic cell (DC). Immature or resting DCs have been shown to induce T-cell responses that may protect against the development of autoimmune disease. Effectively harnessing this "tolerogenic" effect of resting DCs requires that it be disease-specific and that activation of DCs by manipulation ex vivo is avoided. We reasoned that this could be achieved by transferring in vivo partially differentiated myeloid progenitor cells encoding a disease-specific autoantigen. With the aim of preventing autoimmune diabetes, we transferred myeloid progenitor cells encoding proinsulin into NOD mice. Bone marrow (BM) was cultured in granulocyte macrophage colony-stimulating factor (GM-CSF) and transforming growth factor-beta1, a cytokine combination that expands myeloid cells but inhibits terminal DC differentiation, to yield Gr-1(+)/CD11b(+)/CD11c(-) myeloid progenitor cells and a minor population of CD11c(+)/CD11b(+)/CD86(lo) immature DCs. After transfer, Gr-1(+) myeloid cells acquired the characteristics of resting DCs (CD11c(+)/MHC classII(int)/CD86(lo)/CD40(lo)). Gr-1(+) myeloid cells generated from transgenic NOD mice that expressed proinsulin controlled by a major histocompatibility complex (MHC) class II promoter, but not from wild-type NOD mice, transferred into 4-week-old female NOD mice significantly suppressed diabetes development. The transfer of DC progenitors encoding a disease-specific autoantigen is, therefore, an effective immunotherapeutic strategy that could be applied to humans.

To investigate the clinical relevance of common myeloid progenitor (CMP) cells in breast tumor microenvironment (TME). The role of rare cells in TME is less studied. In Silico transcriptomic analyses of real-world data enable us to detect and quantify rare cells, including CMP cells. A total of 5176 breast cancer (BC) patients from SCAN-B, METABRIC, and 5 single-cell sequence cohorts were analyzed using the xCell algorithm. The high group was defined as more than two-thirds of the CMP scores in each cohort. CMP cells consist of 0.07% to 0.25% of bulk breast tumor cells, more in estrogen receptor-positive (ER+) compared with triple-negative (TN) subtype (0.1% to 0.75%, 0.18% to 0.33% of immune cells, respectively). CMP cells did not correlate with any of the myeloid lineages or stem cells in TME. CMP infiltration was higher in smaller tumors, with lower Nottingham grade, and in ER+/HER2- than in TNBC consistently in both SCAN-B and METABRIC cohorts. High CMP was significantly associated with a lower risk of brain metastasis and with better survival, particularly in ER+/HER2-. High CMP enriched epithelial-to-mesenchymal transition and angiogenesis pathways, and less cell proliferation and DNA repair gene sets. High CMP ER+/HER2- was associated with less immune cell infiltration and cytolytic activity ( P <0.001). CMP infiltration correlated with neoadjuvant chemoimmunotherapy response for both ER+/HER2- and TNBC in the ISPY-2 cohort (AUC=0.69 and 0.74, respectively). CMP in BC is inversely associated with cell proliferation and brain metastasis, better response to immunotherapy, and survival. This is the first to report the clinical relevance of CMP infiltration in BC.

Inhibitory effect of 2-chlorodeoxyadenosine on granulocytic, erythroid, and T-lymphocytic colony growth

Downstream of hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs), common myeloid progenitors (CMPs) are thought to represent a progenitor population that contributes to the development of myeloid and erythroid cells. CMPs do not contribute to T cell development in mice, as upon intrathymic transfer they fail to generate T cells in the thymus. However, when co-cultured on OP9 stromal cells expressing Delta-like Notch ligands, CMPs generated T cells, though less efficiently than HSCs or MPPs. Such inefficiency was demonstrated by the increased sensitivity of CMPs to GSI blockade. This defect correlated with reduced expression of Notch1 and also Lunatic fringe. Consistent with this finding, overexpression of ICN1 to mimic constitutively active Notch1 signaling was sufficient to drive CMPs into the T lineage developmental pathway in vivo, and resulted in development of T cell leukemia. This study shows CMPs are still receptive to Notch1 signaling and Notch-mediated T cell leukemogenesis. We are currently exploring mechanisms that might signal downregulation of Notch1 and Lunatic fringe in CMPs, and so initially constrain T potential. We are also reconstituting expression of Notch1 and Lunatic fringe in CMPs using retroviruses, and will determine whether such manipulations are sufficient to confer T potential to CMPs in vivo, or whether additional mechanisms that constrain T potential exist.

The proliferation of human myeloid progenitor cells is negatively regulated in the presence of certain members of the chemokine family of molecules. This includes interleukin 8 (IL-8) and platelet factor 4 (PF4), which in combination are able to synergize, resulting in cell suppression at very low concentrations of these molecules. A series of PF4 and IL-8 mutant proteins were analyzed in an in vitro colony formation assay for myeloid progenitor cells to assess domains of these proteins that are required for activity. Mutation of either of the two DLQ motifs within PF4 resulted in an inactive protein. Perturbations within the IL-8 dimer interface region also resulted in mutants that were incapable of suppressing colony formation. A class of chimeric mutants consisting of domains of either PF4 and IL-8, Gro-alpha and PF4, or Gro-beta and PF4 were observed to inhibit myeloid cell proliferation at concentrations which were between 500- and 5000-fold lower than either the IL-8 or PF4 wild-type proteins alone. These chimeric mutants possessed activities that were comparable to or better than the activity observed when IL-8 and PF4 were added together in vitro. One of these highly active chimeric proteins was observed to be 1000-fold more active than either IL-8 or PF4 alone in suppressing not only the proliferation but also the cell cycling of myeloid progenitor cells following intravenous injection of the mutant into mice. Examination of additional IL-8-based mutants in the colony formation assay, which centered on the perturbation of the amino-terminal "ELR" motif, resulted in the observation that the highly active IL-8 mutant required both aspartic acid at amino acid residue 4 and either glutamine or asparagine at residue 6. Single mutations at either of these positions resulted in mutants with myelosuppressive activity equivalent to wild-type IL-8. Mutants such as IL-8M1 and IL-8M10 were observed to be significantly reduced in their ability to activate isolated human neutrophils, suggesting that separate mechanisms may exist by which myeloid progenitor cells and neutrophils are affected by chemokines.

The RAR-RXR as well as the RXR-RXR pathway is involved in signaling growth inhibition of human CD34+ erythroid progenitor cells

Objective To investigate the expression and clinical significance of P-selectin (CD62P) in bone marrow hematopoietic stem cells of patients with acute leukemia (AL). Methods The CD62P expression in bone marrow mononuclear cells of 15 healthy donors and 56 untreated patients with AL, were examined by flow cytometry. Results The average rate of CD62P expression was (6.72±7.64) % in hematopoietic stem cells (CD+45 CD+34 CD-38) of the 38 patients with acute myeloid leukemia (AML), was (3.46±2.51) % in hematopoietic stem cells (CD+45 CD+34 CD+19) of the 12 patients with B-acute lymphoblastic leukemia (B-ALL), and was (6.23±4.95) % in hematopoietic stem cells (CD+45 CD+34 CD+7) of 6 patients with T-acute lymphoblastic leukemia (T-ALL). The expression rates in those AL patients were higher than that in the healthy controls (1.04 ±1.23) % (t = 2.847, 3.284, 3.091, respectively, P ＜0.01), while there was no difference between the control group and the group who reach CR after routine treatment (t =1.932, P ＞0.05). Furthermore, the leukocyte,hemoglobin and platelet count in CD+62P patients with AML and T-ALL were significantly higher than CD-62P ones (t =4.153, 8.095, 8.289, 7.235, 8.692, 9.832, respectively, P ＜0.05), but there was no significant difference between CD+62P and CD-62P patients with B-ALL (t =0.340, 1.142, 0.019, respectively, P ＞0.05).Conclusion The CD62P is one of the markers of platelet activation, and its expression varies in different types of AL. The CD62P in hematopoietic stem cells of AL could be regarded as a new sign for the leukemic stem cells, as well as a helpful prognostic indicator in treatment response assessment. Key words: Pselectin; Myeloid progenitor cells; Flow cytometry

Expression of Ia Antigens on Myeloid Progenitor Cells in Chronic Myeloid Leukemia: Direct Analysis Using Partially Purified Colony-Forming Cells

Administration of Granulocyte Colony-Stimulating Factor Does Not Restore Defective Expression of bcl-2 and bcl-2-xL in Myeloid Progenitor Cells of Patients with Severe Congenital Neutropenia (Kostmann Syndrome).

EARLY T-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA IN CHILDHOOD

Rapid Discrimination of Early CD34+ Myeloid Progenitors Using CD45-RA Analysis