Proportion Of Peripheral Blood Research Articles

Widespread alterations in the peripheral blood innate immune cell profile in cystic fibrosis reflect lung pathology.

Cystic fibrosis (CF) is caused by mutations to the CF transmembrane conductance regulator (CFTR) gene. CFTR is known to be expressed on multiple immune cell subtypes, dendritic cells, monocytes/macrophages, neutrophils and lymphocytes. We hypothesized that the lack of CFTR expression on peripheral blood innate immune cells would result in an altered cell profile in the periphery and that this profile would reflect lung pathology. We performed a flow cytometric phenotypic investigation of innate immune cell proportions in peripheral blood collected from 17 CF patients and 15 age-matched healthy controls. We observed significant differences between CF patients and controls in the relative proportions of natural killer (NK) cells, monocytes and their subsets, with significant correlations observed between proportions of NK and monocyte cell subsets and lung function (forced expiratory volume in 1 sec, % predicted; FEV1% predicted) in CF patients. This study demonstrates the widespread nature of immune dysregulation in CF and provides a basis for identification of potential therapeutic targets. Modulation of the distinct CF-related immune cell phenotype identified could also be an important biomarker for evaluating CFTR-targeted drug efficacy.

Postnatal deficiency of ADAMTS1 ameliorates thoracic aortic aneurysm and dissection in mice.

What is the central question of this study? Thoracic aortic aneurysm and dissection (TAAD) is characterized by extracellular matrix remodelling and an inflammatory response. Evidence suggests that ADAMTS1 is closely associated with TAAD development, but whether it contributes to the pathophysiology of TAAD remains unknown. What is the main finding and its importance? We generated inducible postnatal ADAMTS1 knockout mice and found that ADAMTS1 deficiency attenuated β-aminopropionitrile-dependent TAAD formation and rupture. Furthermore, ADAMTS1 deficiency suppressed neutrophil and macrophage infiltration by inhibiting inflammatory cytokine levels and macrophage migration during the early stage of β-aminopropionitrile-induced TAAD. ADAMTS1 could be a new therapeutic target for TAAD. Thoracic aortic aneurysm and dissection (TAAD), as a life-threatening cardiovascular disease, is characterized by extracellular matrix remodelling and an inflammatory response. A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) is an inflammation-related protein that is able to degrade extracellular matrix proteins in arteries. Herein, we investigated whether ADAMTS1 contributes to the pathophysiology of TAAD in mice. Using the mouse model of β-aminopropionitrile (BAPN)-induced TAAD, we found that ADAMTS1 expression was upregulated beginning in the early stage of TAAD development and localized predominantly in the aortic adventitia. ADAMTS1-floxed mice and whole-body tamoxifen-inducible ADAMTS1 knockout mice (ADAMTS1flox/flox Ubc-CreERT2+ , ADAMTS1 KO) were generated to investigate the direct causal role of ADAMTS1 in TAAD development. The incidence and rupture rates of BAPN-induced TAAD in ADAMTS1 KO mice were significantly lower than those in ADAMTS1flox/flox mice (45.5versus 81.8% and 18.2versus 42.4%, respectively). Aortas from BAPN-treated ADAMTS1flox/flox mice displayed profound destruction of the elastic lamellae, abundant neutrophil and macrophage accumulation in the adventitia, obviously increased neutrophil proportions in peripheral blood and significantly increased expression of inflammatory factors in the early stage of TAAD induction, all of which were markedly suppressed in ADAMTS1 KO mice. Furthermore, ADAMTS1-deficient macrophages exhibited abrogated migration capacity both in vivo and in vitro. In conclusion, ADAMTS1 plays a crucial role in postnatal TAAD formation and rupture by regulating inflammatory responses, suggesting that ADAMTS1 might be a new therapeutic target for TAAD.

Abstract TP76: Increased Regulatory T Cells After Treatment of Poly (adp-ribose) Polymerase-1 Inhibitor in Patients With Acute Ischemic Stroke

Background: Regulatory T cells (Tregs) have been suggested to have modulatory function of immune response and improve outcome after ischemic stroke. Thus, various strategies to increase Treg in animal model of ischemic stroke have been studied with successful results. The aim of this study is to demonstrate Treg (CD4 + CD25 + FoxP3 + ) proportion in peripheral blood of ischemic stroke patient and the potential effect of poly (ADP-ribose) polymerase-1 (PARP-1) inhibitor on Treg proportion. Methods: Peripheral blood samples were collected from 12 ischemic stroke patients (within 72 hours of stroke onset) and 5 control subjects. Demographics and clinical information were obtained from patients and control subjects. Fluorescence-activate cell sorting (FACS) analyses and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were performed with peripheral blood mononuclear cells (PBMCs) before and after PARP-1 inhibitor (3-AB, JPI-289 1 μm, JPI-289 10 μm) treatment for 24 hours. Results: Treg proportion was significantly higher in healthy control (3.5 ± 1.8 %) than ischemic stroke patients (1.6 ± 0.6 %) ( p < 0.001). In ischemic stroke patients, regulatory T cell proportion was positively correlated with age (r = 0.595, p = 0.041), but did not show association with infarct volume (r = 0.367, p = 0.241). After PARP-1 inhibitor treatment, the proportion of Treg was increased in high dose (10 μm) JPI-289 group (2.1 ± 0.7 %). However, 3-AB and low dose (1 μm) JPI-289 did not show significant changes of Treg proportion. RT-PCR showed increased expression of Treg associated transcription factors such as FoxP3 and CTLA-4 mRNA after PARP-1 inhibitor treatment. PARP-1 inhibitor treatment also decreased pro-inflammatory cytokines such as IFN-γ, TNF-α, and IL-17 and increased anti-inflammatory cytokines such as IL-4, IL-10, and TGF-β1. Conclusion: Treg proportion was decreased in patient with acute ischemic stroke. However, it was up-regulated after treatment of PARP-1 inhibitors, especially with high dose JPI-289. In addition, PARP-1 inhibitor showed possible anti-inflammatory effect by regulating cytokines. PARP-1 inhibitor may ameliorate outcome of ischemic stroke by immune modulation.

Ratios of T-helper 2 Cells to T-helper 1 Cells and Cytokine Levels in Patients with Hepatitis B

Background:Hepatitis B is an immune response-mediated disease. The aim of this study was to explore the differences of ratios of T-helper (Th) 2 cells to Th1 cells and cytokine levels in acute hepatitis B (AHB) patients and chronic hepatitis B virus (HBV)-infected patients in immune-tolerance and immune-active phases.Methods:Thirty chronic HBV-infected patients in the immune-tolerant phase (IT group) and 50 chronic hepatitis B patients in the immune-active (clearance) phase (IC group), 32 AHB patients (AHB group), and 13 healthy individuals (HI group) were enrolled in the study. The cell proportions in peripheral blood, cytokine levels in plasma, and serum levels of HBV DNA, hepatitis B surface antigen, and hepatitis B e antigen were detected.Results:The Th1 cell percentage and Th2/Th1 ratio in the HBV infection group (including IT, IC, and AHB groups) were significantly different from those in HI group (24.10% ± 8.66% and 1.72 ± 0.61 vs. 15.16% ± 4.34% and 2.40 ± 0.74, respectively; all P < 0.001). However, there were no differences in the Th1 cell percentages and Th2/Th1 ratios among the IT, IC, and AHB groups. In HBV infection group, the median levels of Flt3 ligand (Flt3L), interferon (IFN)-γ, and interleukin (IL)-17A were significantly lower than those in HI group (29.26 pg/ml, 33.72 pg/ml, and 12.27 pg/ml vs. 108.54 pg/ml, 66.48 pg/ml, and 35.96 pg/ml, respectively; all P < 0.05). IFN-α2, IL-10, and transforming growth factor (TGF)-β2 median levels in hepatitis group (including patients in AHB and IC groups) were significantly higher than those in IT group (40.14 pg/ml, 13.58 pg/ml, and 557.41 pg/ml vs. 16.74 pg/ml, 6.80 pg/ml, and 419.01 pg/ml, respectively; all P < 0.05), while patients in hepatitis group had significant lower Flt3L level than IT patients (30.77 vs. 59.96 pg/ml, P = 0.021). Compared with IC group, patients in AHB group had significant higher median levels of IL-10, TGF-β1, and TGF-β2 (22.77 pg/ml, 10,447.00 pg/ml, and 782.28 pg/ml vs. 8.66 pg/ml, 3755.50 pg/ml, and 482.87 pg/ml, respectively; all P < 0.05).Conclusions:Compared with chronic HBV-infected patients in immune-tolerance phase, chronic HBV-infected patients in immune-active phase and AHB patients had similar Th2/Th1 ratios, significantly higher levels of IFN-α2, IL-10, and TGF-β. AHB patients had significantly higher IL-10 and TGF-β levels than chronic HBV-infected patients in immune-active phase.

Increased IL-10/IL-17 ratio is aggravated along with the prognosis of patients with chronic lymphocytic leukemia

This study is to investigate the association between the Treg/Th17 cells and prognosis of chronic lymphocytic leukemia (CLL). Totally 50 CLL patients and 20 Health controls were included in this study. Regulatory T (Treg) cells and the cell subset secreting IL-17 (Th17) in peripheral blood were detected with flow cytometry. Serum levels of IL-10 and IL-17 were determined with ELISA, and expression of Foxp3 and RORγt was assessed with quantitative real-time PCR. Treg and Th17 cell proportions in peripheral blood in the CLL patients were significantly higher than control. Serum levels of IL-10 and IL-17, and expression of Foxp3 and RORγt, were significantly increased in the CLL patients. Ratios of Treg/Th17 and IL-10/IL-17 were significantly elevated in the CLL patients. Compared with those before treatment, Treg/Th17 and IL-10/IL-17 ratios were declined in the CLL patients in remission. Compared with the non-remission group, Treg cells were significantly decreased, while Th17 cells were significantly increased, resulting in decreased Treg/Th17 ratio, in the remission group. Moreover, the serum IL-10 level was significantly decreased, while the serum IL-17 level was significantly increased, resulting in declined IL-10/IL-17 ratio, in the remission group. Correlation analysis showed that, Treg and Th17 cell counts were significantly associated with CD38 and ZAP-70 expression in the CLL patients. Moreover, the IL-10/IL-17 ratio was also significantly associated with CLL prognostic factors. Altered Treg/Th17 and IL-10/IL-17 ratios in CLL would be aggravated along with the disease progression, which might be used as indicators for the disease prognosis.

Decreased proportions of CD4 + IL17+/CD4 + CD25 + CD127− and CD4 + IL17+/CD4 + CD25 + CD127 − FoxP3+ T cells in children with autoimmune thyroid diseases

Until now, altered balance of Th1 and Th2 immune cells has been postulated to play an important role in the pathogenesis of autoimmune thyroid diseases (AITD). However, recent studies on thyroid diseases have suggested a new role for Th17 cells that have been classified as a new lineage, distinct from Th1, Th2 and Treg cells. Despite wide interest, the role of Th17 cells in the pathogenesis of inflammatory and autoimmune diseases is still debated. The aim of the study was to estimate the proportions of Th17/Treg T cells in peripheral blood from patients with Graves’ disease (GD; n = 29, mean age 15.4 ± 5.1 years), Hashimoto’s thyroiditis (HT; n = 39, mean age 15.2 ± 4.1 years) and in healthy controls (n = 49, mean age 14.8 ± 3 years). Polychromatic flow cytometry and several fluorochrome-conjugated monoclonal antibodies were applied to delineate Th17 and Treg cells. The analysis of Th17/Treg T cell proportions in peripheral blood from patients with Graves’ disease revealed significantly lower ratios of CD4 + IL17+/CD4 + CD25 + CD127 − (p < 0.0021) and CD4 + IL17+/CD4 + CD25 + CD127 − FoxP3 + (p < 0.0031) than in the control group. In addition, in the case of HT, we observed a significant decrease in the ratios of CD4 + IL17+/CD4 + CD25 + CD127 − (p < 0.0001) and CD4 + IL17+/CD4 + CD25 + CD127 − FoxP3 + (p < 0.0001) T cells in comparison to healthy children. In patients with untreated GD, a statistically significant positive correlation was found between the proportions of CD4 + IL17+/CD4 + CD25 + CD127−, CD4 + IL17+/CD4 + CD25 + CD127 − FoxP3+ T cells and the TRAbs (R = 0.71, p < 0.029; R = 0.72, p < 0.026, respectively) and a positive correlation was noted between the percentage of CD4 + CD − IL − 17 + T cells and the level of TSAbs (R = 0.66, p < 0.037). We conclude that the changes in the proportion of Th17/Treg T cells in peripheral blood and their significant relationship with the level of anti-thyroid antibodies indicate an involvement of these cells in the pathogenesis of AITD.

Levamisole stimulates proliferation of circulating and intestinal immune cell subsets, gut health and performance in weaned pigs

Valpotić, H., Šperanda, M., Kovšca-Janjatović, A., Ðidara, M., Lacković, G., Božić, F., Habrun, B., Srečec, S., Mataušić-Pišl, M. and Valpotić, I. 2014. Levamisole stimulates proliferation of circulating and intestinal immune cell subsets, gut health and performance in weaned pigs. Can. J. Anim. Sci. 94: 43–53. With the growing knowledge of the porcine immune system and its endogenous modulation, it has been clearly stated that exogenous modulation through the use of substances able to modulate immune functions represents an important prophylactic/therapeutic approach in prevention/treatment of both stress- and F4+ and F18+ enterotoxigenic E. coli (ETEC)-induced infections accompanied weaning. The aim of this study was to evaluate the effectiveness of levamisole (LEVA; 2.5 mg kg−1 BW in 10 mL) applied per os to weaned pigs in proliferation of circulating and intestinal immune cell subsets throughout a period of 5 wk. Changes in proportion or number of peripheral blood and ileal mucosal leukocytes tested were studied either weekly by flow cytometry or at the end of the experiment (day 35) by immunohistology/histomorphometry, respectively. Pigs treated with LEVA had increased proportions of peripheral blood CD45+ lymphoid cells, CD4+ and CD8+ T cells, and CD21+ B cells (P&lt;0.01) between days 14 and 35 following the treatment. Also, LEVA stimulated the proliferation of CD45RA+ naïve lymphoid cells in interfollicular (P&lt;0.001) and follicular areas (P&lt;0.05) of ileal Peyer’s patches at day 35 of the experiment. These pigs had a significantly higher (P&lt;0.05) average body weight (19.7 vs. 17.1 kg) and weight gain at the end of experiment compared with the control pigs (for 15%). We conclude that LEVA stimulated the proliferation of circulating and intestinal lymphoid cell subsets tested and improved performance in weaned pigs, and thus, the drug may nonspecifically enhance their immunity/resistance to F4+ and F18+ ETEC strains.

Regulatory T Cells Are Not Predictive of Outcomes in a Nonhuman Primate Model of Vascularized Composite Allotransplantation

T regulatory cells (Tregs) have been associated with prolonged allograft survival and tolerance across a wide variety of species and organ types. We used our nonhuman primate model of facial vascularized composite allotransplantation (VCA) to study the association of Tregs with graft outcomes. We quantified Tregs in peripheral blood and allograft biopsies from nonhuman primates after heterotopic partial facial segment allotransplantation from major histocompatibility complex class I-mismatched donors using flow cytometry and immunohistochemistry. Immunosuppression consisted of tacrolimus and mycophenolate mofetil without induction or depletional therapies. Circulating and graft skin Treg values were compared with graft outcomes and with histologic grade from concurrent biopsies. Treg proportion in peripheral blood ranged from 0.156% to 9.00% with a mean of 3.34%±0.22%. FoxP3 staining was observed in 3 of 29 graft biopsies. Median circulating Treg value did not predict time to Banff grade II rejection (hazard ratio, 0.9; confidence interval, 0.4-2.2) or graft loss (hazard ratio, 0.5; confidence interval, 0.01-5.3). Animals that experienced rejection did not have significantly different peripheral blood or graft skin Treg values from those that did not. Biopsy specimens with grade I or II rejection were more likely to contain Tregs (25% vs. 0%; P=0.044) despite no difference in concurrent circulating Tregs (3.56% vs. 3.36%; P=0.704). These findings in a clinically relevant model suggest that Tregs may have limited prognostic value with standard immunosuppressive protocols used in VCA. Further studies are necessary to determine the specific role of Tregs in VCA and any role of Treg monitoring in clinical practice.

AB0192 Altered T cell compartment could contribute to radiographic changes in AS

BackgroundIn terms of the presence of chronic inflammation in AS, direct and indirect evidence suggests that T cells play a role in its pathogenesis. Although the proportions of peripheral blood...

Hematopoietic SCT in Europe: data and trends in 2011.

In all, 651 from 680 centers in 48 countries reported 35 660 hematopoietic SCT (HSCT) in 32 075 patients (13 470 allogeneic (42%), 18 605 autologous (58%)) to the 2011 survey. Main indications were: leukemias; 10 113 (32% 94% allogeneic); lymphoid neoplasias; non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasma cell disorders; 18 433 (57% 12% allogeneic); solid tumours; 1573 (5% 5% allogeneic); and non-malignant disorders; 1830 (6% 92% allogeneic). There were more unrelated donors than HLA identical sibling donors (54% versus 39%); proportion of peripheral blood as stem cell source was 99% for autologous and 73% for allogeneic HSCT. Cord blood was only used in allogeneic transplants (6% of total). In the past 10 years, the overall number of transplants has increased by 53%. Allogeneic HSCT have doubled (from 7272 to 14 549) while, autologous have increased by 32% and continue to increase by about 1100 HSCT per year since 2001. In the past 2 years, an increase of >2000 HSCT per year was seen. Transplant activity is shown by team size. For allogeneic HSCT, we show use of reduced-intensity conditioning versus myeloablative conditioning across Europe and use of post-transplant donor lymphocyte infusions with considerable variation across different countries.

Virus dose-dependent neutrophil and lymphocyte proportions in peripheral blood during influenza A infection of mice

Virus dose-dependent neutrophil and lymphocyte proportions in peripheral blood during influenza A infection of mice

174O - VEGF-a-induced Treg Proliferation, A Novel Mechanism of Tumor Immune Escape in Colorectal Cancer: Effects of Anti-VEGF/VEGFR Therapies

ABSTRACT Background Regulatory T cells (Treg) are suspected of hindering an effective antitumor immune response in cancer. Multi-target anti-angiogenic tyrosine kinase inhibitors (TKI) that are routinely used as first or second line treatment of cancer patients, have been shown to decrease Treg proportion in tumor-bearing mice and metastatic renal cancer patients. However, the role of VEGF/VEGFR blockade in this effect is still debatable, and the direct impact of VEGF-A on Treg has not been studied. Methods Treg proportion, number were analyzed by flow cytometry in peripheral blood of metastatic colorectal cancer (mCRC) patients treated with bevacizumab, and in CT26 tumor-bearing mice treated with drugs targeting the VEGF axis. The direct impact of VEGF on Treg increase in cancer was also studied. Results Sunitinib (a TKI targeting VEGFR, PDGFR, c-kit), and anti-VEGF-A antibody both decreased Treg in CT26 tumor-bearing mice. Masitinib, a TKI that does not target VEGFR, did not reduce Treg proportion in CT26 bearing mice. Bevacizumab, an anti-VEGF-A monoclonal antibody, reduced Treg proportion in peripheral blood of mCRC patients. Proliferation of Treg was enhanced in CT26 tumor-bearing mice compared to tumor-free mice and was decreased after anti-VEGF-A treatment. Furthermore, in vitro experiments have shown that VEGF-A could directly induce Treg proliferation. VEGFR1 and 2 were expressed on Treg in the presence of a tumor. Anti-VEGFR2 antibody administration reduces Treg proportion and also proliferation in CT26 bearing mice, but anti-VEGFR1 antibody did not, suggesting that VEGF-A-induced Treg proliferation was dependent on VEGFR2 expression. In metastatic CRC patients, Treg proliferation was also enhanced compared to healthy volunteers and was blocked by bevacizumab treatment. Conclusions We identified a new mechanism by which VEGF-A induced by the tumor could stimulate Treg proliferation. VEGF-A/VEGFR2 blockade reduced Treg proportion and proliferation in tumor-bearing mice and metastatic CRC patients suggesting that combination of anti-VEGF-A/VEGFR2 therapies with immunotherapeutic approaches in the future might be particularly relevant in CRC patients. Disclosure J. Taieb: advisory role, Roche; research grant, Roche. All other authors have declared no conflicts of interest.

The EBMT activity survey: 1990–2010

A total of 654 centers from 48 countries were contacted for the 2010 survey. In all, 634 centers reported a total of 33 362 hematopoietic SCT (HSCT) with 30 012 patients receiving their first transplant (12 276 allogeneic (41%) and 17 736 autologous (59%)). Main indications were leukemias: 9355 (31%; 93% allogeneic), lymphoid neoplasias specifically Non Hodgkin's lymphoma, Hodgkin's lymphoma and plasma cell disorders: 17 362 (58%; 12% allogeneic), solid tumors: 1585 (5%; 6% allogeneic) and non-malignant disorders: 1609 (6%; 88% allogeneic). There were more unrelated donors than HLA-identical sibling donors (53% versus 41%); the proportion of peripheral blood as stem cell source was 99% for autologous and 71% for allogeneic HSCT. Cord blood was primarily used in allogeneic transplants (6% of total) with three autologous cord blood HSCT being reported. The number of transplants has increased by 19% since 2005 (allogeneic 37% and autologous 9%) and continued to increase by about 1100 HSCT per year since 2000. Patterns of increase were distinct and different. The data show the development of transplantation in Europe since 1990, with the number of patients receiving a HSCT increasing from 4200 to over 30 000 annually. The most impressive trend seen is the steady increase of unrelated donor transplantation, in parallel to the availability of unrelated donors through donor registries.

The EBMT activity survey 2009: trends over the past 5 years

Six hundred and twenty-four centers from 43 countries reported a total of 31,322 hematopoietic SCT (HSCT) to this 2009 European Group for Blood and Marrow Transplantation (EBMT) survey with 28,033 first transplants (41% allogeneic, 59% autologous). The main indications were leukemias (31%; 92% allogeneic), lymphomas (58%; 12% allogeneic), solid tumors (5%; 6% allogeneic) and non-malignant disorders (6%; 88% allogeneic). There were more unrelated than HLA-identical sibling donors (51 vs 43%) for allogeneic HSCT; the proportion of peripheral blood as stem cell source was 99% for autologous and 71% for allogeneic HSCT. Allogeneic and autologous HSCT continued to increase by about 1000 HSCT per year since 2004. Patterns of increase were distinct and different. In a trend analysis, allogeneic HSCT increased in all World Bank Categories (P=0.01, two sided; all categories), autologous HSCT increased in middle- (P=0.01, two sided) and low-income (P=0.01, two sided) countries. EBMT practice guidelines appeared to have an impact on trend, with a clear increase in absolute numbers within the categories 'standard' and 'clinical option' for both allogeneic and autologous HSCT (P=0.01, two sided; for both allogeneic and autologous HSCT) and a clear decrease in autologous HSCT for the 'developmental' and 'generally not recommended' indications (P=0.01, two sided). These data illustrate the status and trends of HST in Europe.

Antioxidant Treatment Rescues An Accelerated Aging Phenotype In Dyskerin Mutated Bone Marrow Stem Cells.

Abstract Abstract 2614 X-linked Dyskeratosis Congenita (DC) is due to mutations in the DKC1 gene, which encodes the protein dyskerin. Dyskerin is a highly conserved nucleolar protein that, as part of a specialized nucleolar RNP, catalyzes the pseudouridylation of specific residues in newly synthesized ribosomal RNAs and spliceosomal snRNAs. Dyskerin also associates with telomerase and is involved in telomere maintenance. In addition to the well known effect of telomere homeostasis on cancer, it is evident that telomere maintenance may also be important in replicative aging because of telomere shortening due to the limited expression of telomerase activity in dividing somatic cells. Accumulating evidence suggests that dysfunctional telomeres resulting in premature cellular senescence is the primary cause of bone marrow failure in dyskeratosis congenita. It is important to determine the mechanism whereby Dkc1 mutations lead to premature cellular senescence in bone marrow. We have produced a line of mice containing a mutation, Dkc1Δ15, which is a copy of a pathogenic human mutation. Male Dkc1Δ15 mice showed a decrease in the proportion of B and T lymphocytes in peripheral blood and reduced body weight with age but no overt bone marrow failure syndrome phenotypes. Our previous competitive bone marrow transplantation experiments showed that the Dkc1Δ15 mutation caused decay of stem cell function with age. Bone marrow from older Dkc1Δ15 mice was markedly inefficient in repopulation studies compared with bone marrow from age matched wild type mice. We also found that N-acetyl cysteine (NAC) could at least partially rescue the growth disadvantage of dyskerin mutant spleen cells or fibroblasts which was associated with accumulation of DNA damage and reactive oxygen species. To determine if NAC, or other antioxidants might be useful therapeutically it is important to determine their effects on stem cell function, which is defective in DC. To this end we established a cohort of mice that were given NAC in their drinking water (1mg/ml) from 3-weeks of age and maintained on NAC for 1 year. We found that long term NAC treatment did not show significant side effects on the mice. They had slightly increased neutrophils, but no difference in life span and body weight compared with the untreated group. Impressively, old male Dkc1Δ15 mice showed corrected B and T cell proportions in peripheral blood after treatment with NAC. Competitive bone marrow transplantation experiments were carried out in which a 1:1 mixture of BM cells from mutant and WT mice was used to repopulated lethally irradiated recipient mice. These experiments showed that, when taken from NAC treated animals, old Dkc1Δ15 BM cells could compete with age matched WT cells with 40–45% of Dkc1Δ15 cells in primary recipients compared with only 20% for the untreated group. Moreover, after secondary transplantation, cells from the NAC treated group still represent 15–20% of Dkc1Δ15 cells in recipients while those from the untreated group could not be detected. These results strongly suggest that NAC treatment can partially restore the bone marrow repopulating ability of Dkc1Δ15 stem cells. Together with our previous results these data suggest that a pathogenic Dkc1 mutation, through its effect on telomerase, initiates stem cell aging before telomeres are short and that increased oxidative stress might play a role in this process. Moreover the effects of the mutation may be prevented or delayed by antioxidant treatment, although the precise mechanism will be the subject of future investigation. Disclosures: Bessler: Alexion Pharmaceutical Inc: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; Taligen: Consultancy.

FTY720 attenuates lesional interleukin‐17+ cell accumulation in rat experimental autoimmune neuritis

Experimental autoimmune neuritis (EAN) is a well-known animal model of human demyelinating polyneuropathies. Here we have studied the spatiotemporal accumulation of interleukin (IL)-17(+) cells in sciatic nerves of EAN rats and effects of FTY720, an agonist of sphingosine-1-phosphate (S1P) receptors. In this study, we examined the spatiotemporal expression of IL-17 using immunohistochemistry and RT-PCR, and analysed the IL-17(+) cell proportion in blood and lymph nodes using flow cytometry. In sciatic nerves of EAN rats, IL-17(+) cells were mainly found to concentrate around blood vessels and IL-17(+) cell accumulation was temporally correlated with severity of neurological signs. FTY720, which has been shown to reduce severity of EAN, attenuated accumulation of IL-17(+) cells in sciatic nerves, decreased IL-17(+) cell proportion in peripheral blood, but increased IL-17(+) cell proportion in lymph nodes, suggesting the involvement of S1P signal pathway in regulating IL-17(+) cell trafficking. our data are consistent with the possibility that IL-17(+) cells might contribute to the pathogenesis of EAN and the S1P signal pathway may be involved in the in vivo trafficking of IL-17(+) cells.

Lymphocyte subpopulations in bronchoalveolar lavage fluid in AIDS

Lymphocyte subpopulations in peripheral blood and bronchoalveolar lavage fluid (BAL) were examined in 29 patients with AIDS (26 men, three women; median age 32 [16-55] years). Patients in group 1 (n = 12) had no lung disease, in group 2 (n = 11) had Pneumocystis carinii pneumonia, in group 3 (n = 6) had other lung disease. There were 13 men and two women (median age 48 [21-80]) in the control group (bronchoscoped for mild pulmonary symptoms: no abnormal findings in the BAL). Compared with the control group, patients with AIDS had a significant deficiency in helper cells, both in blood (7-23% vs 50%; P < 0.01) as well as in the BAL (7-24% vs 52%; P < 0.001). There was a correlation of the percentage helper cell proportion in peripheral blood and BAL (for both group 1 and 2, rs = 0.75; P < 0.05). The proportion of helper cells in peripheral blood and BAL in AIDS patients was significantly lower in those with than without lung disease (group 1: 23% blood, 24% BAL vs group 2: 9% blood, 7% BAL; group 3: 7% blood, 7% BAL; P < 0.02 blood, P < 0.004 BAL). The percentage proportion of suppressor cells was greater in both blood and BAL in AIDS patients (group 1: blood 47%, BAL 63%; group 2: blood 44%, BAL 76%; group 3: blood 52%, BAL 75%) than in the controls (blood 28%, BAL 33% [P < 0.01], but there was no correlation between peripheral blood and BAL. In addition, the absolute number of suppressor cells in the lavage (30 cells/microliters in group 1 and 3, 65 cells/microliters in group 2) was significantly higher than in the controls (8 cells/microliters).--In AIDS patients there occurs a lymphocytosis in the BAL, while in blood there is a lymphopenia. The concordant decrease in helper cells in blood and BAL is decisive for the severity of any pulmonary infections.

Proportion of peripheral blood and decidual CD4+ CD25bright regulatory T cells in pre-eclampsia

CD4(+) CD25(bright) regulatory T (T(reg)) cells have been identified as a principle regulator of tolerance during pregnancy. In the setting of pre-eclampsia, however, little is known about the dynamics of these cells. In the current study, we determined CD4(+) CD25(bright) T(reg) cells in the peripheral blood using flow cytometry and forkhead box P3 (FoxP3(+)) cells at the placental bed using immunohistochemical staining. Peripheral blood mononuclear cells (PBMC) of 38 pre-eclamptic cases (17 cases Japanese, 21 cases Polish), 40 normal late pregnancy subjects (20 subjects Japanese, 20 subjects Polish), and 21 non-pregnant healthy controls (10 subjects Japanese, 11 subjects Polish) were included. We found the percentage of CD25(bright) cells within the CD4(+) T cell population in PBMC was reduced significantly in both Japanese and Polish pre-eclamptic cases than in normal pregnancy subjects (P < 0.001) and non-pregnant healthy controls (P < 0.001). Also, the percentage of FoxP3(+) cells within CD3(+) T cells in the placental bed biopsy samples of pre-eclamptic cases were decreased compared to those in normal pregnancy subjects. These findings suggest that a decreased number of T(reg) cells was present in pre-eclampsia, and these changes might break the maternal tolerance to the fetus.

Minimal Residual Disease (MRD) Analysis in Non-MRD Based ALL IC-BFM 2002 Protocol for Childhood ALL: Is It Possible To Omit Minimal Residual Disease in Risk Stratification?.

Abstract More than 800 children with acute lymphoblastic leukaemia (ALL) are treated every year according to ALL IC-BFM 2002 protocol, which was designed by the International-BFM Group as a parallel to MRD-based ALL/AIEOP BFM 2000 study. The ALL-IC BFM 2002 risk group stratification comprises blast proportion in peripheral blood (PB) after 7 days of prednisone and one IT-MTX (prednisone response) and bone marrow (BM) morphology evaluation at days 15 and 33 of therapy together with age, initial WBC and presence of BCR/ABL and MLL/AF4 fusion. One of the aims of the ALL IC-BFM 2002 study is the comparison of this risk group assessment to the MRD-based criteria used in ALL-BFM 2000. We analyzed a total of 203 patients treated according to the ALL IC-BFM 2002 in the Czech Republic, Israel, Hong Kong and Uruguay for the presence of clonal antigen receptor rearrangements. MRD was evaluated in 175 patients at several time-points of therapy including mandatory points at week 5 and 12, which are used in the ALL/AIEOP BFM 2000 stratification. In total, 654 follow-up BM specimens and 80 PB samples were tested. In the univariate analysis, a good molecular response defined as MRD negativity at both week 5 and 12 was associated with the age of 1–6 years (p=0.0001), WBC&lt;20,000/mm3 (p=0.0002), non-T immunophenotype (p&lt;0.0001), good prednisone response (p=0.0006), presence of TEL/AML1 fusion (p=0.003) and non-M3 morphology (≤25% blasts in BM) at day 15 (p=0.02). There was no significant association of MRD negativity with sex and hyperdiploidy; non-M3 BM morphology at day 8 was significant only when analyzing non-T ALL (p=0.02). Patients with BCP ALL had significantly lower MRD levels at day 15 (p=0.03) and at day 33 (p=0.001) than T-ALL patients; the difference was no more significant at week 12. Patients stratified to standard risk group (SRG) according to the ALL IC-BFM 2002 criteria had a significantly better molecular response defined as MRD negativity at week 5 and 12 than intermediate risk group (IRG) patients (p=0.009). However, in 24 of 69 SRG patients (34.7%), MRD positivity at week 5 and/or at week 12 was observed (ranging from borderline positivity to 1.5x10(-2)), thus identifying patients who would not qualify to MRD-based SRG in ALL/AIEOP BFM 2000. Within ALL IC SRG, patients with slow molecular response did not differ significantly from those with good MRD response in age, sex, WBC, BM morphology at day 15 and presence of hyperdiploidy or TEL/AML1 fusion. The only difference was in a higher proportion of M3 BM at day 8 in MRD slow-responders (p=0.04). Our findings revealed a significant divergence between the stratification results of ALL IC-BFM 2002 and ALL-BFM 2000. A fast morphological response to treatment (M1 or M2 bone marrow at day 15) together with other low-risk features does not necessarily correspond with rapid MRD clearance. Supported by MSM0021620813, Israel Cancer Association and Children’s Cancer Foundation Hong Kong.

Minimal Residual Disease Detection Identifies Differences between the Risk Groups Defined by the ALL IC-BFM 2002 and the ALL-BFM 2000 Protocols.

Abstract Minimal residual disease (MRD) testing based on a unique Ig/TCR gene rearrangement pattern of each patient’s leukaemia turned out to be an independent tool to determine treatment response and the risk of relapse in paediatric acute lymphoblastic leukaemia (ALL). Since 07/2000, MRD information at week 5 and 12 of therapy has been used for stratification in ALL-BFM 2000 trial. In parallel, ALL IC-BFM 2002 has been designed by the International-BFM Group to test the morphological assessment of the early treatment response. Patients are stratified according to the blast proportion in peripheral blood (PB) at day 8 and in bone marrow (BM) at day 15 and 33 of therapy, age, initial WBC and the presence of BCR/ABL and MLL/AF4 fusion. One of the goals of the study is the comparison of this risk group assessment to the MRD-based criteria used in ALL-BFM 2000. In the Czech Republic, 73 patients were treated according to ALL IC-BFM 2002 protocol from 11/2002 to 12/2003, 29 in the standard-risk (SR), 35 in the intermediate-risk (IR) and 9 in the high-risk (HR) group. The SR, HR and all T-cell ALL patients were examined for clonal Ig/TCR rearrangements. RQ-PCR patient-specific systems were designed for each of these patients according to the ESG-MRD-ALL criteria. For 39 of the 40 patients tested (97.5%) at least one target with minimal sensitivity of 10(−4) was identified. MRD was evaluated in BM samples from 34 patients at several time points inclusive of the mandatory 5 and 12 week ones. Simultaneously the PB specimens of the T-ALL patients were tested. In total, 205 BM and 64 PB specimens were included. In 7 patients of 24 in the SR group, MRD positivity at week 5 and/or at week 12 was observed (ranging between 9.7x10(−4) and 1.5x10(−2)), thus identifying patients who would not qualify to the MRD-based SR group in ALL-BFM 2000 despite the identical induction regimen. In T-ALL patients, PB-MRD levels paralleled those in BM. MRD results showed no separation of MRD levels between IR- and HR-stratified T-ALL patients. These preliminary findings reveal a significant difference between the stratification results of ALL IC-BFM 2002 and ALL-BFM 2000. A fast response as measured by the morphology criterion (M1 or M2 bone marrow at day 15) together with other low-risk features does not necessarily correspond with rapid MRD clearance. The complete analysis of MRD is planned for the international consortium participating in the ALL IC-BFM 2002 protocol.