Percentage Of Cells In Peripheral Blood Research Articles

A case report of Pallister-Killian syndrome with an unusual mosaic supernumerary marker chromosome 12 with interstitial 12p13.1-p12.1 duplication.

Pallister-Killian syndrome (PKS) is a rare inherited disease with multiple congenital anomalies, profound intellectual disability, and the presence in the karyotype of sSMC - i(12)(p10). The frequency of PKS may be underestimated due to problems with cytogenetic diagnosis caused by tissue-specific mosaicism and usually a low percentage of peripheral blood cells containing sSMC. Such tissue-specific mosaicism also complicates a detailed analysis of the sSMC, which, along with the assessment of mosaicism in different tissues, is an important part of cytogenetic diagnosis in PKS. Unfortunately, a full-fledged diagnosis in PKS is either practically impossible or complicated. On the one hand, this is due to problems with the biopsy of various tissues (skin biopsy with fibroblast culture is most often used in practice); on the other - a low percentage of dividing peripheral blood cells containing sSMC, which often significantly complicates the analysis of its composition and organization. In the present study, a detailed analysis of sSMC was carried out in a patient with a characteristic clinical picture of PKS. A relatively high percentage of peripheral blood cells with sSMC (50%) made it possible to perform a detailed molecular cytogenetic analysis of de novo sSMC using chromosomal in situ suppression hybridization (CISS-hybridization), multicolor FISH (mFISH), multicolor chromosome banding (MCB), array CGH (aCGH), and quantitative real-time PCR (qPCR), and short tandem repeat (STR) - analysis. As a result, it was found that the sSMC is not a typical PKS derivative of chromosome 12. In contrast to the classical i(12)(p10) for PKS, the patient's cells contained an acrocentric chromosome consisting of 12p material. Clusters of telomeric repeats were found at the both ends of the sSMC. Furthemore, the results of aCGH and qPCR indicate the presence of interstitial 8.9Mb duplication at 12p13.1-p12.1 within the sSMC, which leads to different representations of DNA from different segments of 12p within cells containing sSMC. The obtained data raise the question of the instability of the sSMC and, as a consequence, the possible presence of additional rearrangements, which, in traditional cytogenetic analysis of patients with PKS, are usually described as i(12)(p10).

T Cell Engaging Bispecific Antibodies Produce Durable Response in Mesothelin-Positive Patient-Derived Xenograft Models of Pediatric AML

BackgroundImmunotherapy development in pediatric AML is lagging because of dearth of validated AML-specific targets. We showed recently that mesothelin (MSLN) is highly expressed on the leukemia cell surface in a subset of pediatric AML patients, and validated MSLN as a therapeutic target using antibody-drug conjugates directed against MSLN (Kaeding et al., Blood Adv, 5:2350-2361, 2021). Antibody single-chain variable region (scFv) sequences derived from amatuximab recognizing MSLN and from either blinatumomab or AMG330 targeting CD3 were used to engineer and express two MSLN/CD3-targeting BsAbs: MSLN AMA-CD3 L2K and MSLN AMA-CD3 AMG respectively. Both these antibodies demonstrated anti-leukemic activity in mice engrafted with MV4;11 cells engineered to overexpress MSLN, while they failed to show any effect in mice bearing MV4;11 cells without MSLN, confirming that these antibodies specifically targeted MSLN (Gopalakrishnapillai et al., Blood, 134:3925, 2019).MethodsMSLN cell surface expression was quantitated using BD Quantibrite PE Phycoreythrin Fluorescence Quantitation kit. 3x10 6 NTPL-146 cells were injected in NSG-B2m mice and 2x10 6 DF-5 cells were injected in NSG-SGM3 mice via the tail vein. Mice were randomly assigned to treatment groups when human cells were detectable in blood. The percentage of human chimerism in mouse peripheral blood was evaluated weekly by flow cytometry. Bipsecific antibodies were administered ip at 3 mg/Kg daily for six days. Human peripheral blood pan T cells from StemCell Technologies were injected iv (3x10 6 cells per mouse) to act as effector cells. Chemotherapy (DA) consisted of 3 doses of 1.5 mg/kg daunorubicin iv and 5 doses of 50 mg/kg cytarabine ip. Mice were monitored daily and euthanized when any of the experimental endpoints were met.ResultsIn this study, we evaluated the efficacy of two bispecific antibodies in two distinct patient-derived xenograft models of pediatric AML with endogenous MSLN expression quantitated at 6617 and 7414 MSLN antibodies bound per cell in NTPL-146 and DF-5 respectively. A Kaplan-Meier survival plot based on the time when each mouse reached experimental endpoint showed that 6/8 NTPL-146 engrafted mice receiving MSLN AMA-CD3 AMG and T cells survived disease-free until the end of the experiment at day 520 whereas all the mice in control groups had died by day 138 (Fig. 1a). The AML bone marrow load of MSLN AMA-CD3 AMG-treated mice was < 0.01% at 520 days, whereas the bone marrow load of mice from the other treatment groups was greater than 90% at the time of death, consistent with marrow failure as the proximal cause of death (Fig. 1b). These data show that treatment with MSLN AMA-CD3 AMG is curative in the vast majority of mice.Treatment with MSLN AMA-CD3 L2K and T cells increased the median survival by 109.5 days compared to untreated mice while treatment with MSLN AMA-CD3 AMG showed a complete remission in 6/8 mice (**P<0.001). Thus, the survival benefit of both MSLN AMA-CD3 L2K and MSLN AMA-CD3 AMG treated mice greatly exceeded the allogeneic effect T cells alone, which only showed a 22-day improvement in median survival. Either MSLN AMA-CD3 L2K or MSLN AMA-CD3 AMG treatment alone showed no improvement in survival compared to untreated mice.The efficacy of the potent BsAb MSLN AMA-CD3 AMG in comparison with chemotherapy (DA) was evaluated in DF-5. DA treatment, like T cell infusion, did not significantly change median survival compared to untreated mice, while BsAb MSLN AMA-CD3 AMG in the presence of human T cells was curative (Fig. 1c, **P<0.005). Mice treated with only T cells showed a rapid rise in AML cell percentage in peripheral blood until day 34 when they succumbed to disease, whereas the mice receiving T cells with BsAb had AML cell burden of less than 1% (Fig. 1d, *P<0.05). Mice treated with BsAbs showed greater expansion of allogeneic human T cells compared to mice receiving T cells alone (Fig. 1e, *P<0.05). Eight weeks post treatment initiation, the surviving mice were euthanized and AML burden in the bone marrow was evaluated. In contrast with mice receiving T cells alone, mice treated with T cells and BsAbs had no detectable AML cells (Fig. 1f, **P<0.005).ConclusionThese data validate the efficacy of MSLN-targeting BsAbs in PDX models with endogenous MSLN expression. Because prior MSLN-directed therapies appeared safe in humans, MSLN-targeting BsAbs could be ideal immunotherapies for MSLN-positive pediatric AML patients. [Display omitted] DisclosuresGopalakrishnapillai: Geron: Research Funding. Correnti: Link Immunotherapeutics: Current Employment. Kaeding: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Barwe: Prelude Therapeutics: Research Funding.

The unique characteristic in peripheral immune cells in patients with advanced hepatocellular carcinoma

Recent progress in cancer immunology provides more insight in immune evasion of cancer cells. Cancer cells may achieve immune evasion through several ways including ineffective antigen presentation, T cell checkpoint utilization, immunosuppressive cytokines secretion and immunosuppressive cells recruitment. However, few literatures mentioned about the change of peripheral blood immune cells in advanced hepatocellular carcinoma (HCC) patients. To answer this question, we initiated a pilot study through detailed flow cytometry. We enrolled patients with advanced HCC patients who had informed consent to the collection of their peripheral blood. We also recruited healthy individuals for the control group. Using flow cytometry, we analyzed lymphocyte subclasses and the PD-1 or PD-L1 positivity of immune cells in peripheral blood from HCC patients and healthy individuals. Twenty-four HCC patients were enrolled and twenty healthy individuals were enrolled. Most of the HCC patients were HBV carrier (58.3%), and the mean age was 61 years old. Among 55 immune cell parameters we examined in peripheral blood, 16 were significantly different between advanced HCC patients and healthy individuals by univariate analysis. Multivariate analysis was then conducted by fitting logistic regression model and showed that CD69-CD25- Naïve CD4αβT cell percentage and dendritic cell percentage can reasonably predict the advanced HCC status from peripheral blood. By our regression model, the adjusted generalized R2=0.918 and the estimated area under the Receiver Operating Characteristic (ROC) curve was 0.99. CD69-CD25- Naïve CD4αβT cell percentage and dendritic cell percentage in peripheral blood are highly correlated with the advanced HCC status. The change may result from immune evasion initiated by hepatocellular carcinoma cells and further investigation is warranted. Validation study is ongoing and this mechanism may be utilized to treat advanced HCC patient in the future.

516P - Proportion of peripheral lymphocyte subsets correlates with the progression-free survival and metastatic status of pancreatic neuroendocrine tumour patients

BackgroundNeuroendocrine system plays a critical role in regulating the immune system, and therefore the progression and prognosis of pancreatic neuroendocrine tumors (pNETs) may be largely influenced and reflected by immune cell levels. Lymphocytes are crucial immune cells that correlate with tumorigenesis and tumor progression, and lymphocyte subsets in peripheral blood, which include CD4+ and CD8+ T cells, B cells and NK cells, are reported to reflect tumor prognosis and progression in various cancer types. We thus speculated that peripheral lymphocyte subset proportion may reflect the state of progression and predict tumor prognosis of pNETs. MethodsA retrospective, follow-up cohort study consisting 73 patients diagnosed as pNETs was conducted. Flow cytometry was performed to analyze peripheral lymphocyte subsets. Ultivariate analyses were performed using the stepwise Cox hazards model, and Kaplane-Meier methods and log-rank tests were used for the comparison of survival rates, Mann-Whitney U test were used for analyses of the correlation between lymphocyte subset percentage and clinicopathologic parameters. ResultspNET patients with distant metastasis were associated with lower CD3+ T cell (p=0.003), lower CD4+ T cell (p=0.006) and higher NK cell (p=0.003) percentage in peripheral blood. Lower percentage of CD3+, CD4+ T cells, B cells and higher percentage of NK cells in peripheral blood were associated with shorter progression-free survival (PFS). Distant metastasis (HR=29.194, 95%CI: 6.865 to 124.144 p<0.001) and lower percentage peripheral B cells turned out to be independent risk factors for PFS both in all patient group (HR=12.232 95%CI: 2.883 to 51.907, p=0.001) and in group of patients without distant metastasis (HR=6.744, 95%CI: 1.257 to 36.172, p=0.026). ConclusionsCD3+, CD4+ T cell and NK cell percentage in peripheral blood may reflect the the status of distant metastasis in pNET patients. The percentage of peripheral B cells cells may independently predict the progression of pNET patients with or without distant metastasis, making them promising clinical indicators to instruct the frequency for reexaminations after primary medical treatment. Legal entity responsible for the studyYitao Gong. FundingHas not received any funding. DisclosureThe author has declared no conflicts of interest.

Long-term effects of omalizumab on peripheral blood cells and C-reactive protein levels in patients with chronic spontaneous urticaria.

Omalizumab's mechanism of action is not well-understood yet despite its strong therapeutic efficacy in chronic spontaneous urticaria (CSU). To determine the overall effect of omalizumab on peripheral blood cell counts and serum C-reactive protein levels (sCRP) during a 1-year follow-up in patients with CSU. Data of 74 patients (male/female: 20/54) were reviewed from medical charts. Leucocyte counts, percentages of peripheral blood cells(lymphocyte, monocyte, neutrophil [PPBN], eosinophil, basophil [PPBB]) and sCRP were recorded at baseline, 3rd, 6th, 12th months of omalizumab treatment. Although a dramatic increase in the mean PPBB (±SD) was observed at the 3rd month, PPBB (%) gradually decreased after the 3rd month (PPBB: 0.38 ± 0.21 [baseline] vs. 0.59 ± 0.3 [3rd month], p = .002). However, 12th month PPBB remained higher than baseline (PPBB:0.38 ± 0.21 [baseline] vs. 0.46 ± 0.27 [12th month], p = .03). A dramatic decrease in the mean PPBN (%) was noticed within the first 3 months (PPBN:62.85 ± 8.97 [baseline] vs. 58.37 ± 9.07 [3rd month], p = .04), and 12th month PPBN remained lower than baseline values (PPBN: 62.85 ± 8.97 [baseline] vs. 60.31 ± 8.02 [12th month], p = .045).Mean sCRP (mg/dL) decreased rapidly within the first 3 months (sCRP: 1.09 ± 1.53 [baseline] vs. 0.56 ± 0.45 [3rd month], p = .17) and 12th month sCRP still remained lower than baseline levels (sCRP: 1.09 ± 1.53 [baseline] vs. 0.83 ± 1.06 [12th month], p = .01). Omalizumab substantially increases PPBB,and reduces PPBN accompanied by a reduction in sCRP especially in the first 3 months; however, these effects may continue in the long-term. The alterations in peripheral blood cell ratios and sCRP may contribute to the therapeutic effect of omalizumab in CSU.

Association of absolute lymphocyte count and peripheral blood lymphocyte subsets percentage with minimal residual disease at the end of induction in pediatric B cell acute lymphoblastic leukemia

Absolute lymphocyte count (ALC) has been associated with overall survival (OS) and event-free survival, but we do not know if ALC is associated with minimal residual disease (MRD) at the end of induction (EOI) and whether it can be used as surrogate marker in resource limited settings. Immunological differences between MRD-positive and MRD-negative B ALL patients at the EOI are not known at present. This prospective study evaluated the association of ALC and peripheral blood lymphocyte subset percentage at the EOI with MRD. ALC was done at baseline, day 8, and day 15 and at EOI. Assessment for MRD and peripheral blood lymphocyte subset was done at EOI. In 2-year study duration, 197 B cell acute lymphoblastic leukemia (ALL) patients were recruited out of which 150 were analyzed. Peripheral lymphocyte subset percentage was available for 58 patients. We found that ALC at baseline, day 8, day 15, and EOI was not associated with MRD. Day 8 ALC was significantly higher in poor steroid responders (day 8 blasts > 1 × 109 cells/l) (p < 0.0001). At the EOI, CD4−CD8+ cell percentage in peripheral blood were significantly higher in MRD-positive patients than MRD-negative patients (p = 0.01). Our study suggests that ALC at any point is not a surrogate marker for MRD. Immunologically MRD-positive and MRD-negative patients differ in CD4−CD8+ cells. The role of CD8+T and TCRαβCD3+T cells in eliminating residual leukemic cells need to be studied further by functional assays.

Inhibition of CXCR4 by MicroRNA-1192 Reduces the Activation of Th17 Cells and Expression of Inflammation Factors in a Mouse Model of Vulvovaginal Candidiasis

Vulvovaginal candidiasis (VVC) is a disease commonly occurring in sexually active women. The involvement of microRNAs in several kinds of infectious diseases has been highlighted in a number of researches. Therefore, we conducted the present study in order to investigate whether microRNA-1192 (miR-1192) would significantly target CXCR4 in Th17 cells as well as inflammatory factors in mouse models suffering from VVC. Seventy-five mice were selected as test subjects for this study, of which twenty-five were used as the normal control, while the rest were treated with estradiol or oil-treated in order to establish VVC mouse models (each n = 25). Protein expressions of CXCR4, IL-6, IL-17, and IL-23 were all measured using both an immunohistochemistry and ELISA. The Th17 cell percentage in peripheral blood and the expression of RORγt in Th17 cells were detected using a flow cytometry. Mouse vaginal epithelial cells were isolated from normal mice, after which the mice were treated with estradiol to regulate their estrogen, followed by treatments involving the miR-1192 mimic, miR-1192 inhibitor, siRNA-CXCR4, and miR-1192 inhibitor + si-CXCR4. The cell cycle, apoptosis, and proliferation were all examined by using an additional flow cytometry as well as the employment of the MTT assay. The miR-1192, CXCR4, IL-6, IL-17, and IL-23 expressions in tissues and cells were both measured using both RT-qPCR and western blot assay techniques. The mice treated with either estradiol or oil had presented to us lowered levels in miR-1192 expression as well as higher levels in both Th17 cell percentage and expression of RORγt in Th17 cells, along with mRNA and protein expressions of CXCR4, IL-6, IL-17, and IL-23. In cell experiments, the mouse vaginal epithelial cells that had been treated with miR-1192 inhibitor had shown us a decreased cell proliferation rate and contrarily increased expressions of CXCR4, IL-6, IL-17, and IL-23 mRNA, protein, and cell apoptosis rate; these results were opposite to the ones found in the mice treated with miR-1192 mimic. Our results provided significant evidence that miR-1192 could directly development and progression of VVC by restraining the CXCR4 gene in the VVC mice.

Distinctive effects of pilose antler on mouse peripheral blood immune cell populations

ABSTRACTPilose antler is a rare animal-sourced traditional Chinese medicine that has been demonstrated to cause a change in total immunity. Little is known about its specific effect on immune cells. In this study, the immunological modulatory effects of pilose antler extract on mice were investigated. Mice were gavaged daily for 7 days with pilose antler extracts, and peripheral blood was analysed by flow cytometry. Our results show that pilose antler extract exerted distinctive effects on the different immune cell types analysed. Pilose antler gavage transiently increased T cell percentage and decreased B cell percentage in peripheral blood. After treatment, the proliferation of killer and helper T cells was stimulated in the short term, but in the longer term, NK and memory T cell populations were increased. These results suggest that pilose antler treatment has both transient and long-term effects on the immune system in mice.

Abstract TP270: Ischemic Preconditioning-afforded Neuroprotection Against Ischemic Stroke is Associated With Altered Peripheral Immune Responses

The limb remote ischemic preconditioning (RIPC) is considered a clinical feasible protective strategy against ischemia-reperfusion injury after stroke. However, its mechanisms of protection remain elusive. Here, we used a rat model of non-invasive RIPC by tourniquet to study the impact of RIPC on the immune cell and cytokine profiles after transient middle cerebral artery occlusion. Our data indicates that RIPC protects against focal ischemia and preserves neurological functions 3 days after stroke. Meanwhile, RIPC results in dramatic changes in several immune cell populations after stroke. Specifically, the RIPC ameliorated the post-MCAO reduction of blood CD3+CD8+ T cells and abolished the reduction of CD3+/CD161a+ NKT cells. Another striking change is that RIPC significantly elevated the B cell percentage in peripheral blood, which reversed the otherwise reduced B cell population after stroke. RIPC also markedly elevated the percentage of CD43+/CD172a+ non-inflammatory residential monocytes, while showing no effect on the percentage CD43-/CD172a+ inflammatory monocytes. In addition, we found that RIPC induced IL-6 expression and enhanced the elevation of TNFα after stroke. Collectively, our results reveal potential immune mechanisms of RIPC-afforded neuroprotection against cerebral ischemia. Further exploration about these immune elements in RIPC-afforded ischemic tolerance may lead to the development of systemically administered immune therapies for stroke patients.

Arhgap21 Expression in Bone Marrow Niche Is Crucial for Hematopoietic Progenitor Homing and Short Term Reconstitution after Transplantation

The microenvironment of the bone marrow (BM) is essential for retention and migration of hematopoietic progenitor cells. ARHGAP21 is a negative regulator of RhoGTPAses, involved in cellular migration and adhesion, however the role of ARHGAP21 in hematopoiesis is unknown.In order to investigate whether downregulation of Arhgap21 in microenvironment modulates bone marrow homing and reconstitution, we generated Arhgap21+/- mice using Embryonic Stem cell containing a vector insertion in Arhgap21 gene obtained from GeneTrap consortium and we then performed homing and bone marrow reconstitution assays.Subletally irradiated (9.5Gy) Arhgap21+/- and wild type (WT) mice received 1 x 106 BM GFP+cells by IV injection. For homing assay, 19 hours after the transplant, Lin-GFP+ cells were analyzed by flow cytometry. In reconstitution and self-renew assays, the GFP+ cell percentage in peripheral blood were analyzed 4, 8, 12 and 16 weeks after transplantation. Hematopoietic stem cells [GFP+Lin-Sca+c-Kit+ (LSK)] were counted after 8 and 16 weeks in bone marrow after primary transplant and 16 weeks after secondary transplant.The percentage of Lin-GFP+ hematopoietic progenitor cells that homed to Arhgap21+/- recipient (mean± SD) (2.07 ± 0.85) bone marrow was lower than those that homed to the WT recipient (4.76 ± 2.60); p=0.03. In addition, we observed a reduction (WT: 4.22 ±1.39; Arhgap21+/-: 2.17 ± 0.69; p=0.001) of Lin- GFP+ cells in Arhgap21+/-receptor spleen together with an increase of Lin- GFP+ population in Arhgap21+/-receptor peripheral blood (WT: 8.07 ± 3.85; Arhgap21+/-: 14.07 ±5.20; p=0.01), suggesting that hematopoietic progenitor cells which inefficiently homed to Arhgap21+/- bone marrow and spleen were retained in the blood stream.In bone marrow reconstitution assay, Arhgap21+/-receptor presented reduced LSK GFP+ cells after 8 weeks (WT: 0.19 ±0.03; Arhgap21+/-0.12±0.05; p=0.02) though not after 16 weeks from primary and secondary transplantation. The reduced LSK percentage after short term reconstitution was reflected in the lower GFP+ cells in peripheral blood 12 weeks after transplantation (WT: 96.2 ±1.1; Arhgap21+/-94.3±1.6; p=0.008). No difference was observed in secondary transplantation, indicating that Arhgap21reduction in microenvironment does not affect normal hematopoietic stem cell self-renewal.The knowledge of the niche process in regulation of hematopoiesis and their components helps to better understand the disordered niche function and gives rise to the prospect of improving regeneration after injury or hematopoietic stem and progenitor cell transplantation.In previous studies, the majority of vascular niche cells were affected after sublethal irradiation, however osteoblasts and mesenchymal stem cells were maintained (Massimo Dominici et al.; Blood; 2009.). RhoGTPase RhoA, which is inactivated by ARHGAP21 (Lazarini et al.; Biochim Biophys acta; 2013), has been described to be crucial for osteoblasts and mesenchymal stem cell support of hematopoiesis (Raman et al.; Leukemia; 2013). Taken together, these results suggest that Arhgap21 expression in bone marrow niche is essential for homing and short term reconstitution support. Moreover, this is the first study to investigate the role of Arhgap21 in bone marrow niche. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Regulatory T cells in chronic lymphocytic leukemia

Controversial results concerning the prognostic value of the regulatory T (Treg) cell percentage in peripheral blood of chronic lymphocytic leukemia (CLL) patients have been reported in the previous studies. This study aimed to estimate the prognostic relevance of Treg cells in untreated CLL patients at diagnosis. CLL patients showed significantly higher Treg cell percentage and Treg cell counts as compared to that identified in healthy normal controls (P < 0.01 for both). Furthermore, CLL patients with high LDH, β2 microglobulin levels, and those positive for CD38% express significantly higher Treg cell percentage as compared to those patients with normal LDH, β2 microglobulin levels, and negative CD38%. Also, the percentage of Treg cells was significantly higher among CLL patients having autoimmune cytopenias. In conclusion, Treg cell percentage is higher in CLL patients as compared to normal healthy controls and related to advanced stages as well as poor prognostic markers. Treg cell manipulation may represent a future strategy for management of CLL patients.

[Differential analysis of BM cell morphology, immunophenotypic, cytogenetic characters and prognosis between myeloblastic and lymphoblastic crisis of CML].

This study was purposed to investigate the difference of morphology, immunophenotype, cytogenetic features and prognosis between myeloid blast crisis and lymphoid blast crisis of chronic myelogenous leukemia (CML). A total of 31 patients with CML in blastic crisis in Department of Hematology, the First Affiliated Hospital of Xi'an Jiaotong University school of Medicine from 2009 January to 2014 January were enrolled in this study. Out of 31 CML patients, 24 cases were patients with myeloid blast crisis and other 7 cases were patients with lymphoblastic crisis. The clinical data, blast cell percentage in peripheral blood and bone marrow, eosinophil and basophil percentage, immunophenotype, cytogenetic characteristics and prognosis were analyzed. The results indicated that there was no significant difference of blastic cell percentage in peripheral blood and bone marrow of CML with myeloid blast crisis, and the eosinophil and basophil cells could be easily detected. The ratio of blastic cells in BM was higher than that in PB in lymphoid blastic crisis of CML, eosinophil and basophil cells were rare. 7 cases of CML with lymphoid blastic crisis were B ALL with CD10, CD19, CD34, HLA-DR expression, and 2 cases with CD13 and CD33 expression. The lymphoid score was in all CML patients with lymphoid blastic crisis was greater than or equal to 1.5;and 2 patients with CD13 and CD33 expression, and with 1 myeloid score.24 cases of myeloid blastic crisis of CML patients mainly expressed CD33, CD13, CD38, CD34, CD11b and HLA-DR, and their myeloid score greater than or equal to 2, among them the lymphoid scores of 2 patients were 0.5 and 1 score, respectively. All the 31 patients showed 100% Ph(+) chromosome, among them 3 cases also showed other new chromosome aberrations. There was no significant difference of overall survival rate between lymphoid and myeloid blastic crisis of CML, but the overall survival rate of patients treated with tyrosine kinase inhibitor (TKI ) was higher than that in the patients without TKI treatment. It is concluded that eosinophil and basophil cells in peripheral blood of lymphoid blastic crisis were less than that of CML patients with myeloid blastic crisis. Lymphoid blastic crisis of CML patients occurred mostly in B ALL cases with expression of CD10 and CD19. Patients with myeloid blastic crisis of CML mainly expressed CD33, CD13, CD38, CD34, CD11b and HLA-DR, and could be accompanied by other lineage antigen expression, but the score was less than 2. New chromosome aberration is easily observed in myeloid blastic crisis of CML. There is no significant difference of overall survival rate of between CML patients with lymphoid and myeloid blastic crisis, but the overall survival rate of patients treated with TKI is higher than the patients without TKI treatment.

Clinical Value of Serum Soluble CD30 Levels in Adult T-Cell Leukemia/ Lymphoma

Adult T-cell leukemia/lymphoma (ATLL) is an incurable neoplasm of mature T-cells with a median survival time of approximately one year. The aim of this study was to compare the clinical value of soluble CD30 (sCD30) levels with soluble IL-2 receptor α chain (sIL-2R) levels in two different clinical settings of ATLL patients; before an initial therapy of chemotherapy or gastric resection (n=32), and before allogeneic hematopoietic stem cell transplantation (HSCT; n=24). All patients completed the 2-year follow-up. Both sIL-2R (p=0.016) and sCD30 (p=0.030) levels were significant predictors of overall survival before the initial therapy. The number of ATLL cells in peripheral blood (PB) was correlated with sCD30 levels (Spearman’s correlation coefficient, ρ=0.46; p=0.009) but not with sIL-2R levels (ρ=0.16; P=0.38). Patients who survived for longer than two years, and for whom the percentage of ATLL cells in PB were <5%, displayed relatively low sCD30 levels, but this tendency for low sCD30 levels was not observed in longterm survivors whose ATLL cells constituted ≥5% of PB cells. sIL-2R (p=0.041) and sCD30 (p=0.0003) values were both significant predictors of overall survival in HSCT, but sCD30 levels predicted more patients with early death than were predicted by sIL-2R levels. Combination of sCD30 and CRP levels showed high sensitivity and specificity for detection of early death (within 100 days) following HSCT (81.8% and 84.6%, respectively). These results suggest that analysis of sCD30 levels may be useful for prediction of overall survival in ATLL patients, especially before HSCT. These findings would alter the treatment strategy and improve the prognosis of ATLL.

Immunophenotypic characteristics of peripheral blood cells in normal elderly men

This study was aimed to distinguish abnormal cells and to diagnose hematologic diseases through recognizing antigen expression pattern and percentage of peripheral blood cells in normal elderly men. Antigen expression of blast cells, granulocytes, monocytes, lymphocytes, nucleated red blood cells and plasma cells was detected by seven-color flow cytometry in a total of 88 peripheral blood samples from normal elderly men, aged median 82 years old, from 70 to 98 years. Groups were divided according to age, region and underlying diseases, and the percentages of different subgroup cells were examined to confirm whether the differences were significant or not. The results showed that the median proportion of CD34(+) blast cells in peripheral blood from normal elderly men were 0.017% (0.015%-0.020%), with high expression of HLA-DR, CD33, CD13 and CD117, low expression of myeloid antigens, such as CD15, CD11b and CD16, while lymphoid antigens were seldom positive, including CD7, CD19 and CD56. Dim-expression of CD38 was found in peripheral blood blast cells, CD38(dim)+/- cell percentage in blast cells was 61.36% ± 18.26%. In the differentiation and development of granulocytes, CD16(-), CD13(+) CD16(+) (intermediate) and CD16(+) (strong) CD13(+) cells appeared in sequence from immature to mature granulocytes, whose median proportions in nuclear cells were 0.04%, 0.30% and 61.30%, respectively. The percentages of immature monocytes, such as CD64(+) CD14(-) and HLA-DR(+) CD11b(-) cells, were from 0.00% to 0.10% and from 0.07% to 0.68%, separately. No significant differences were found between different subgroups (P > 0.05). It is concluded that the immunophenotypic characteristics and referential percentages of CD34(+) blast cells, granulocytes and monocytes with different development stages in peripheral blood from normal elderly men are recognized, which can help to discriminate abnormal cells.

Non-Canonical Wnt Signaling Regulates Hematopooetic Repopulation Through the Ryk Receptor

Abstract 852Proper regulation of the balance between quiescence and proliferation is implicated in long-term maintenance of hematopoietic stem cell (HSC) function. Our lab has demonstrated that Wnt5a, a member of the Wnt family of ligands, induces HSC quiescence in vitro and enhances short and long-term hematopoietic repopulation (Nemeth, et al. 2007). To determine the mechanisms of Wnt5a signaling in HSCs we sought to identify potential Wnt5a receptors. The Ryk gene encodes a non-canonical Wnt receptor in lineage-negative, Sca-1+, c-kit+ (LSK) cells. Previous research using neural crest cells identified Ryk as a receptor for Wnt5a. We hypothesized that the ability of Wnt5a to enhance HSC quiescence and hematopoietic repopulation required the Ryk receptor. To test this hypothesis, we cultured C57BL/6 LSK cells in serum-free media with 25ng/ml SCF and Flt3, 250ng/ml recombinant Wnt5a (rWnt5a), and 1.0 μg/ml anti-Ryk neutralizing antibody (α-Ryk) for four days. We observed that compared to control treatment, rWnt5a significantly inhibited cellular expansion (control: 5.9 ± 0.1-fold; rWnt5a: 3.1 ± 0.3-fold; n = 4, p <0.01). The effect of Wnt5a on cellular expansion was blocked by the inhibition of Ryk with α-Ryk (α-Ryk + rWnt5a: 3.1 ± 0.3-fold; n = 4, p = 0.01 compared to rWnt5a alone). We confirmed these data using a genetic approach in which we transduced LSK cells with a lentiviral vector expressing a dominant-negative Ryk mutant; LSK cells transduced with this vector did not respond to rWnt5a treatment as measured by cell expansion. We measured the effects of rWnt5a and α-Ryk treatment on the cell cycle. rWnt5a treatment resulted in a modest decrease in the percentage of cells in the S/G2/M phases (control: 35.0 ± 1.9%; rWnt5a: 29.1 ± 0.5%, n=3, p < 0.01). Treatment with α-Ryk partially restored this response (α-Ryk + rWnt5a; 32.2 ± 2.0%, n=3, p = 0.06 compared to rWnt5a). We then tested the effects of rWnt5a and α-Ryk treatment on cell quiescence. As expected, rWnt5a treatment increased the percentage of cells in G0 compared to control (control: 11.3 ± 6.7%; rWnt5a: 26.1 ± 0.9%, n=3, p < 0.01). The effect of rWnt5a on quiescence was blocked by the addition of α-Ryk (α-Ryk + rWnt5a; 21.1 ± 1.4%, n=3, p < 0.05 compared to rWnt5a). To test the effects of inhibiting Ryk on Wnt5a-mediated enhancement of hematopoietic repopulation, we performed competitive bone marrow transplant assays. We cultured C57BL/6 LSK cells for 4 days and transplanted 1 × 104 cells along with 5 × 105 whole bone marrow cells from Ubc-GFP transgenic mice (C57BL/6 background) into lethally irradiated B6.SJL mice. At 4 and 16 weeks, we measured the contribution of cultured LSK cells to hematopoietic repopulation by determining the percentage of peripheral blood cells that were CD45.2+, GFP-. rWnt5a increased the contribution of cultured LSK cells to hematopoietic repopulation after 4 weeks (control: 5.3 ± 1.4%, n = 8, rWnt5a: 19.5 ± 7.1%, n = 6, p < 0.05) and 16 weeks (control: 4.5 ± 1.6% n = 8, rWnt5a: 32.0 ± 13.1%, n = 6, p = 0.01). Inhibiting Ryk blocked the effects of rWnt5a on reconstitution after 4 and 16 weeks (4 weeks: α-Ryk + rWnt5a; 5.8 ± 2.1% n = 6, p < 0.05 compared to rWnt5a; 16 weeks: α-Ryk + rWnt5a; 11.9 ± 7.1%, n = 6, p < 0.05 compared to rWnt5a). These data indicate that the effects of rWnt5a on hematopoietic cell proliferation and HSC engraftment are regulated by the Ryk receptor. To determine a mechanism by which Wnt5a induces quiescence and hematopoietic repopulation, we tested whether Wnt5a regulates production of reactive oxygen species (ROS). Excessive ROS production is linked to decreased HSC quiescence and reduced long-term function. We measured the level of ROS in our sorted LSK cells using the ROS probe dihydrodichlorofluorescein. We observed that treatment of LSK cells with rWnt5a resulted in a 1.6 ± 0.3-fold reduction in ROS levels compared to control (n = 3); this reduction was comparable to that observed using the proto-typical anti-oxidant N-acetyl cysteine. This reduction in ROS production was inhibited by the addition of α-Ryk (1.04 ± 0.1-fold compared to control, n = 3). Together, our data indicate that the Ryk receptor functionally interacts with Wnt5a to regulate hematopoietic stem and progenitor quiescence and function and that regulation of ROS production is a potential mechanism by which Wnt5a regulates HSC function. Disclosures:No relevant conflicts of interest to declare.

Selective activation of T cells in newly diagnosed insulin-dependent diabetic patients: evidence for heterogeneity of T cell receptor usage

Cell surface phenotyping of 58 newly diagnosed diabetic children and 25 controls confirmed the presence of activated T cells, expressing HLA class II antigens or receptors for interleukin-2 (IL-2R, CD25) in the majority of the patients. Some of these cells putatively include those involved in islet cell destruction, as reported previously. Monoclonal antibodies recognizing three families of the variable regions of the beta chain (V beta) of the T cell receptor were used to determine the percentage of peripheral blood cells expressing those specific gene segment products. The number of the activated T cells from each V beta family was compared with that of the resting T cells of the same family in the patients and the controls. In 18 out of 58 (31%) of these patients there was evidence of oligoclonal proliferation of activated T cells as judged by marked increases in cells expressing a V beta family in the IL-2R+ T cell pool, compared with the total T cell pool. However, different V beta families were augmented in individual patients, indicating considerable heterogeneity of T cell activation in different patients. These results are in contrast to murine models of autoimmunity, where virtually monoclonal T cell activation, restricted to a single V beta family has been reported.

Expression of a HoxC4 Retroviral Vector Mediates Expansion of Murine Hematopoietic Stem Cells with Normal Hematopoiesis in Transplanted Mice.

Homeobox (Hox) transcription factors are important regulators of hematopoietic cell proliferation and differentiation. Of them, HoxB4 is of particular interest because overexpression promotes rapid expansion of mouse hematopoietic stem cells (HSCs) without causing neoplastic transformation. Despite the effects of HoxB4 overexpression on HSCs, mice that are homozygous for HoxB4 gene deletion have only subtle defects in HSCs and progenitor cells. We hypothesized that other paralogs of HoxB4 may also be capable of inducing HSC expansion could thereby compensate for loss of HoxB4 function. To test this hypothesis, we have studied the effects of retroviral overexpression of a HoxC4 gene in murine progenitors and HSCs. The murine HoxC4 cDNA was cloned and inserted into an MSCV vector that co-expresses an IRES-YFP reporter gene. We transduced murine bone marrow cells with a MSCV-HoxC4-YFP vector and compared the secondary replating efficiency of myeloid colonies (CFU-Cs) to that seen using either a MSCV-HoxB4-GFP or an MSCV-GFP vector. This assay tests for progenitor cell self-renewal which is increased using HoxB4-expressing vectors. Cells transduced with the MSCV-HoxC4-YFP vector formed 20–40 times more secondary CFU-Cs than with cells transduced with the MSCV-GFP control vector. This increase in CFU-C replating efficiency was equivalent to that seen with the MSCV-HoxB4-IRES-GFP vector. To test the in vivo effects of the MSCV-HoxC4-YFP vector on self-renewal of HSCs, we transplanted lethally irradiated mice with a mixture of cells; 20% transduced with the MSCV-HoxC4-YFP vector and 80 % mock-transduced. Peripheral blood analysis of the transplanted recipients up to 28 weeks post-transplantation showed that the percentage of cells transduced with the MSCV-HoxC4-YFP vector was 70–85% in both lymphoid and myeloid cells in the peripheral blood. A similar degree of chimerism was noted in concurrent controls using the MSCV-HoxB4-GFP vector. In contrast, the percentages of peripheral blood cells transduced with the MSCV-GFP vector was only 15–25%, paralleling the input ratios of transplanted cells. Secondary transplantation experiments showed stable levels of chimerism in both HoxC4 and HoxB4 groups, indicating that the expansion seen with the MSCV-HoxC4-YFP vector occurred at the HSC level. These results indicate that retroviral-mediated expression of HoxC4, like HoxB4, can cause significant expansion of HSCs in vivo. Because several other Hox genes can cause hematopoietic abnormalities and leukemia when expressed from a retroviral vector, we transplanted lethally irradiated mice with 4x106 cells that were transduced with the MSCV-HoxC4-YFP vector and monitored the animals for survival and complete blood counts. Now, at 33 weeks post transplantation, no tumor formation was observed in mice expressing either the HoxB4 or the HoxC4 vector, and peripheral blood counts have remained normal. Our results show that retroviral overexpression of HoxC4 can induce a significant expansion of the HSCs in vivo, and suggest that expression of HoxC4 may compensate for the loss of HoxB4 in knockout mice. We are currently analyzing the effects of HoxA4 and HoxD4 to determine if they share the same functional characteristics, and are also determining whether HoxB4 and HoxC4 are modulating the same downstream genes using microarray analysis of transduced murine bone marrow cells.

Syngeneic Bone Marrow Transplantation Without Conditioning in a Patient With Paroxysmal Nocturnal Hemoglobinuria: In Vivo Evidence That the Mutant Stem Cells Have a Survival Advantage

A 10-year-old girl with paroxysmal nocturnal hemoglobinuria (PNH) received an infusion of syngeneic bone marrow without preparative marrow ablation or immunosuppression. Following transplant, the patient became asymptomatic in concordance with an increase in the percentage of peripheral blood cells with normal expression of glycosyl phosphatidylinositol-anchored proteins (GPI-AP). However, molecular analysis suggested engraftment of a relatively small number of donor stem cells and persistence of an abnormal stem cell with mutant PIG-A. During 17 months of observation, the percentage of cells with normal GPI-AP expression gradually decreased, while intravascular hemolysis progressively increased. Approximately 16.5 months post-transplant, the patient once again became symptomatic. Together, these results indicate that syngeneic marrow infusion provided a clinical benefit by increasing the proportion of erythrocytes with normal expression of GPI-anchored complement regulatory proteins without supplanting the abnormal stem cells. However, evidence of insidious disease progression following the marrow infusion implies that the abnormal stem cells have a survival advantage relative to the transplanted stem cells. Thus, these studies contribute in vivo data in support of the hypothesis that PNH arises as a consequence of a pathological process that selects for hematopoietic stem cells that are GPI-AP-deficient.

Changes in lymphocyte and macrophage subsets due to morphine and ethanol treatment during a retrovirus infection causing murine aids

Infection by LP-BM5 murine leukemia virus (MuLV) suppressed significantly the percentage of peripheral blood cells showing surface markers for macrophages, lymphocytes and activated lymphoid cells. Chronic administration of a 7% (36% calories) ethanol diet or injection of 1.9 mg/mouse/day of morphine for a 7 day period were followed by 3 week periods of abstinence and then 1 week periods of consumption of 5% ethanol diets or morphine injection to female C57BL/6 mice resulted in changes in the numbers of macrophages and lymphocyte subsets. The number of lymphocytes of various subsets were not significantly changed by the ethanol exposure except those showing activation markers which were reduced. The percentage of peripheral blood cells showing markers for macrophage functions and their activation were significantly reduced after “binge” use of ethanol. Ethanol retarded suppression of cells by retroviral infection. However by 25 weeks of infection there was a 8.6% survival in the ethanol fed mice infected with retrovirus which was much less than virally infected controls (45.0%). Morphine treatment also increased the percentage of cells with markers for macrophages and activated macrophages in virally infected mice, while suppressing them in uninfected mice. The second and third morphine injection series suppressed lymphocyte T-helper and T-suppressor cells, but not total T cells. However, suppression by morphine was significantly less during retroviral disease than suppression caused by the virus only. At 25 weeks of infection 44.8% of morphine treated, infected mice survived. Morphine treatment also caused deaths such that the survival in morphine treated, retrovirally infected was higher than would have been expected if the death rate in virally infected, and morphine injected animals were combined during combined treatment. Thus these drugs of abuse can modulate peripheral blood lymphoid subsets, suppression caused by retroviral infection, and survival.

Enhanced survival by vitamin A supplementation during a retrovirus infection causing murine aids

Infection by LP-BM5 murine leukemia virus (MuLV) produces and AIDS-like condition in mice. The viral infection suppressed the percentage of peripheral blood cells showing surface markers for macrophages, activated macrophages, T lymphocytes and activated lymphoid cells. High diatary vitamin A (retinyl palmitate) caused increased numbers of activated macrophages. It also increased the percentage of cells with markers for Ia + cells and macrophages in the retrovirally infected mice compared to infected controls. In uninfected mice retinyl palmitate stimulated the percentage of cells with activated lymphocytes bearing IL-2R, and T cytotoxic cells. These were associated with a retarded death rate during infection with LP-BM5 murine leukemia in C57BL/6 mice. By 25 weeks of infection and 20 weeks of retinyl palmitate supplementation 71.3% survived, while 45.0% virally infected controls survived. The mice also had elevated numbers of B cells measured in the blood after 4 and 8 weeks of dietary treatment. Vitamin A stimulation may play a role in the slower death rate for retrovirally infected mice.