Immunological Prophile of FA. A Multicentric retrospective Analysis of 61 Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1347-1347
Author(s):  
Paola Terranova ◽  
Enrico Cappelli ◽  
Regis Peffault De Latour ◽  
Johanna Svahn ◽  
Jean Soulier ◽  
...  
Keyword(s):  
T Cells ◽  
Dna Repair ◽  
Fanconi Anemia ◽  
Peripheral Blood ◽  
Serum Levels ◽  
Tnf Alpha ◽  
Cd4 Cells ◽  
Normal Range ◽  
Ifn Gamma ◽  
Immunological Status

Abstract Abstract 1347 INTRODUCTION: Fanconi Anemia (FA) is an autosomal and X-linked recessive disease characterized by marrow failure, somatic malformations and cancer proneness primarily leading to AML and head and neck carcinomas. The disease is due to mutations in at least 15 genes responsible for a failure of DNA repair mechanisms that renders the cells sensitive to interstrand cross linkers leading to block of cell cycle in G2 phase. However there is evidence that FA proteins have multiple functions as they are also implicated in cytokine hypersensitivity, response to oxidative stress and immune response. Scanty information is available on immunological status in FA patients. We conducted a retrospective multi-centric analysis of the pre HSCT immunological status of 61 FA patients in aplastic phase by assessing peripheral blood immunophenotype and immunoglobulin serum level (46/61). RESULTS: Absolute lymphocyte number was within the normal range for age in 70% of patients (43/61). Only 10% of subjects (6/61) were severely lymphocytopenic ( <1.0×109/l). Absolute B cells were below the lower limit of the normal range for the age (10th percentile) in 75% of patients. Absolute CD3+ and CD8+ cells were within the normal distribution for age in virtually all cases (98% and 94% respectively) whereas CD4+ cells and NK cells were below the lower normal range in 45% and 49% of cases respectively. Within the T cell population, CD3+CD45RA+ (naive) cells were above the normal range in 68% of patients while CD3+CD45+RO+ (memory) cells were reduced in 60 % patients. In 70% (22/32) of the patients in whom we tested regulatory T cells (CD3+/CD4+/CD25bright+/FoxP3+) they were found below the lower normal range. Serum immunoglobulin A and G were within the age matched range of normal controls whereas IgMs were lower than normal range for age in 50% (23/46) of patients. CONCLUSIONS: This is the largest study ever conducted on the immunological status of FA patients suggesting that these subjects have an impaired immunity which is not directly reflected by the number of circulating lymphocytes. Reduction of B and also of CD4+cells is consistent with low IgM serum levels. Deficiency of regulatory T cells and the relative excess of CD3+and CD8+ lymphocytes are in keeping with the hypothesis that a dysregulated immunity might contribute to the development of marrow failure. In fact it is possible that the lack of inhibitory effect of T regulatory cells might facilitate CD3+ and CD8+ subsets to produce myelosuppressive cytokines TNF-alfa and IFN-gamma to which FA hematopoietic cells are hypersensitive (1–4). The reduction of T memory cells, that constitutionally have a more hampered DNA repair capability vs T naive cells (5), looks consistent with the classical DNA repair deficiency of FA. Overall these data suggest that specific immune function in FA: (a) is impaired in a rather independent fashion from lymphopenia, (b) might contribute to development of marrow failure and (c) might reflect, to some extent, multifunctionality of FA proteins. Next step is to assess cytokine serum levels, ongoing at writing, that may help to further comprehend the above data. -1- Fagerlie SR, Bagby GC. Immune defects in Fanconi anemia. Crit Rev Immunol. 2006;26(1):81–96 -2- Dufour C, Corcione A, Svahn J, Haupt R, Poggi V, Béka'ssy AN, Scimè R, Pistorio, Pistoia V. TNF-alpha and IFN-gamma are overexpressed in the bone marrow of Fanconi anemia patients and TNF-alpha suppresses erythropoiesis in vitro. Blood. 2003 Sep 15;102(6):2053–9. −3 - Briot D, Macé-Aimé G, Subra F, Rosselli F. Aberrant activation of stress-response pathways leads to TNF-alpha oversecretion in Fanconi anemia. Blood. 2008 Feb 15;111(4):1913–23. -4- Vanderwerf SM, Svahn J, Olson S, Rathbun RK, Harrington C, Yates J, Keeble W, Anderson DC, Anur P, Pereira NF, Pilonetto DV, Pasquini R, Bagby GC. TLR8-dependent TNF-(alpha) overexpression in Fanconi anemia group C cells. -5- Scarpaci S, Frasca D, Barattini P, Guidi L, Doria G. DNA damage recognition and repair capacities in human naïve and memory T cells from peripheral blood of young and elderly subjects. Mech Ageing Dev. 2003 Apr;124(4):517–24. Disclosures: No relevant conflicts of interest to declare.

Peripheral Blood Stem Cell Grafts Mobilized by G-CSF and Plerixafor in Comparison to G-CSF Alone: Phenotypic and Functional Characterization,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4045-4045
Author(s):  
Béatrice Gaugler ◽  
J Arbez ◽  
J Frikeche ◽  
Steven Le Gouill ◽  
Thomas Gastinne ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Peripheral Blood ◽  
T Cell Subsets ◽  
Tnf Alpha ◽  
Functional Markers ◽  
Ifn Gamma ◽  
Functional Features

Abstract Abstract 4045 Background. Plerixafor is a CXCR4 antagonist that is currently approved for use in combination with G-CSF to mobilize autologous peripheral blood stem cells in patients with lymphoma and myeloma candidate to autologous transplantation. Previous studies showed that the drug can efficiently mobilize higher numbers of CD34+ stem cells and early hematopoietic progenitors. However, immune factors contained in the graft may also be important in terms of post-transplant outcome. Thus, this study aimed to characterize the phenotype and function of the different immune effectors contained in aphaeresis samples obtained from patients mobilized with Plerixafor and G-CSF (P+G group) in comparison to grafts mobilized with G-CSF alone (G group). Patients and Methods. Aliquots of aphaeresis products were obtained from 36 lymphoma and myeloma patients following treatment with 5 days of G-CSF (10 μg/kg/day; n=18) or following sequential mobilization with G-CSF and Plerixafor (240 μg/kg; n=18), according to Plerixafor label. The phenotype and cytokine secretion profile of the different T cell and dendritic cell (DC) subsets were characterized by multicolor flow cytometry including intracellular cytokine staining. Results. In samples derived from grafts collected after P+G, there was a significantly higher % of total CD3+ T cells (median, 81%) as compared to samples collected after G alone (median 71%; p=0.01). However, the CD4/CD8 ratio was comparable between both groups (p=0.56). The % of CD19+ B cells and CD3-CD56+ NK cells were not significantly different between both groups. When considering the different T cell subsets (naïve, central memory, terminally differentiated and memory), there was no significant differences in the distribution of T cells between the 2 groups. However, from a functional level, there was a significant increase of CD8+ IFN-gamma and TNF-alpha secreting T cells in the P+G group as compared to the G group (median, 12.3% vs. 5.3%, p=0.01; and 5.9% vs. 2.8%, p=0.02, respectively). IFN-gamma and TNF-alpha secreting CD4+ T cells were also increased, but to a lesser extent, in the P+G group (median, 4.4% vs. 2.5%, p=0.04; 8.6% vs. 5.3%, p=0.07; respectively). Grafts mobilized with P+G contained a similar percentage of CD4+ regulatory T cells (Treg; characterized by CD25 and Foxp3 expression; median 5.9% vs. 5.0%, p=0.21). Nevertheless, the Treg compartment in the P+G grafts contained a lower proportion of ICOS+Foxp3+ cells (36.6% vs. 55%, p=0.09). Also, Treg from the P+G grafts displayed a significantly higher expression of CD127 (median MFI, 387 vs. 240, p=0.002) suggesting that Treg mobilized with P+G likely exhibit different functional properties. In terms of DC subsets, grafts mobilized with P+G contained similar % of myeloid (MDC, Lin-CD11c+HLA-DR+CD123-) and BDCA3+ DCs. In contrast, the % of plasmacytoid DCs (PDC; CD123+BDCA2+HLADR+) was significantly increased in the P+G grafts (median, 0.87% vs. 0.30%; p=0.002), leading to a significantly higher PDC/MDC ratio in the P+G group, 2.08 vs. 1.01, p<0.0001). PDCs mobilized by P+G displayed different functional markers in comparison to PDCs mobilized with G alone: higher % of ILT7+ PDCs (65.5 vs. 44.4, p=0.03), and decreased expression of CD86 (5.8% vs. 11.4%, p=0.007; MFI, 787 vs. 944, p=0.01) suggesting a potential regulatory capacity of PDCs mobilized by P+G. Conclusion. This comprehensive comparative analysis showed that grafts mobilized with P+G exhibited major different phenotypic and functional features in comparison to grafts mobilized with G alone, highlighting that such grafts may have a significant impact on patients' outcome after autologous and allogeneic stem cell transplantation. We are currently testing the in vivo relevance of these differences in a mouse transplant model. Disclosures: Mohty: Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Type 17 Immune Response Facilitates Progression of Inflammation and Correlates with Cognition in Stable Schizophrenia

Diagnostics ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 926
Author(s):  
Milica M. Borovcanin ◽  
Slavica Minic Janicijevic ◽  
Ivan P. Jovanovic ◽  
Nevena M. Gajovic ◽  
Milena M. Jurisevic ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Serum Levels ◽  
Antipsychotic Therapy ◽  
Necrosis Factor Alpha ◽  
Positive Correlation ◽  
Tnf Α ◽  
Immune Pathway

Dysregulation of the type 17 immune pathway has already been considered in schizophrenia and we previously measured decreased sera values of interleukin (IL)-17 in early stages. We further explored the possible correlation of IL-17 systemic levels with proinflammatory cytokines and cognitive scores and additionally analyzed the percentage of IL-17 producing lymphocytes in peripheral blood of patients with stable schizophrenia. We included 27 patients diagnosed with schizophrenia (F20), after a three-month stable depot antipsychotic therapy (risperidone or paliperidone) and 18 healthy control subjects. Positive and Negative Syndrome Scale of Schizophrenia and the Montreal-Cognitive Assessment (MoCA) were conducted. Sera concentrations of IL-17, IL-6, tumor necrosis factor alpha (TNF-α) and soluble ST2 receptor (sST2) were measured. Flow cytometry and Natural Killer (NK) and T cell analyses were done in 10 patients and 10 healthy controls. Moderate positive correlation was established between IL-17 and TNF-α (r = 0.640; p = 0.001), IL-17 and IL-6 (r = 0.514; p = 0.006), IL-17 and sST2 (r = 0.394; p = 0.042). Furthermore, a positive correlation between the serum levels of IL-17 and MoCA scores was observed, especially with visuospatial and executive functioning, as well as language functioning and delayed recall (p < 0.05). Significantly higher percentage of IL-17 producing CD56+ NK cells was measured in peripheral blood of patients with schizophrenia in remission vs. healthy individuals (p = 0.001). The percentage of CD4+ T cells and CD4+ T cells that produce IL-17 was significantly increased in patients (p = 0.001). This study revealed the involvement of innate type 17 immune response in the progression of inflammation and this could be related to cognitive functioning in stable schizophrenia.

scholarly journals VitaminD3 Regulates T Cell Immune Responses in Allergen and Rhinovirus Induced Asthma

2021 ◽  
Author(s):  
Susetta Finotto ◽  
Patricia Haag ◽  
Darja Andreev ◽  
Nina Li ◽  
Alexander Kiefer ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Murine Model ◽  
Peripheral Blood ◽  
Serum Levels ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Asthmatic Children ◽  
Rhinovirus Infection

Abstract Background: Serum 25(OH)-Vitamin D3 (VitD3) deficiency during infancy has been associated with asthma. The potential therapeutic role of VitD3 given in the airways and its interference with the allergen and Rhinovirus was the objective of this study. Methods: In two cohorts of children with and without asthma, serum levels of the C-reactive protein (CRP) were correlated to Serum VitD3 and in peripheral blood T cell inhibitor marker Programmed cell death protein 1 (PD1) mRNA was analyzed. In a murine model, VitD3 was given intranasally in vivo and in vitro to lung cells with allergen and Rhinovirus. Results: In the cohorts of pre-school age children without (control) asthma, CRP and VitD3 levels inversely correlated. In preschool asthmatic children that did not receive VitD3 supplementation as infant had more episode of asthma exacerbation associated with high CRP serum level. In peripheral blood cells from control but not asthmatic children with higher serum levels of VitD3 had lower PD1 mRNA levels. In murine model, OVA intranasal challenge induced Innate Lymphoid Cells type 2 (ILC2)-associated markers and Eosinophils in BALF and VitD3 inhibited lung inflammation and ILC2 markers. Furthermore, VitD3 given intranasally, induced CD4+T cells and reduced PD1, T regulatory cells in the lung. Similarly, VitD3 had a suppressive role on CD4+PD1+ T cells involved in T cell exhaustion in the airways in the absence of ST2 after Rhinovirus infection. Conclusion: These data support an inhibitory role of VitD3 on T cell exhaustion after allergen and rhinovirus infection that is relevant for pediatric asthma.

scholarly journals Anti-CD19 Chimeric Antigen Receptor T Cell Treatment of Relapsed /Refractory Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma Resistant to Bruton's Tyrosine Kinase (BTK) Inhibitor

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 36-36
Author(s):  
Weihong Chen ◽  
Xin Du ◽  
Wenyujing Zhou ◽  
Changru Luo ◽  
Xiaoqing LI
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Lymph Nodes ◽  
Peripheral Blood ◽  
Serum Levels ◽  
Car T Cell ◽  
Car T ◽  
Btk Inhibitor

CASE PRESENTATION: A 68-year-old male was diagnosed with CLL/SLL in November 2007. Bone marrow asp/bx: 36.5% lymphocytes, 78% CD19, 65% ATM (11q22 deleted) positive cells, 13.5% D13S25 (13q14.3 deleted). On December 10, 2009, the patient took FCR scheme for five cycles, followed by FR scheme for one cycle, and then a month of Chlorambucil. On September 5, 2013, the patient took BR scheme for four cycles with no effect. From March 2015 to Feb 2016, 420 mg of Ibrutinib was administered daily. On January 15, 2016, the patient developed swollen lymph nodes in his right neck with intermittent lumps, fever and nausea. He was admitted into the hospital at Feb 2, 2016. Test results: multiple swollen superficial lymph nodes over the body, with the biggest measuring 60×30mm on the right neck, with no tenderness. Supplementary tests: peripheral white blood cells (WBC) 11.94×10E9/L, lymphocyte 7.5×10E9/L, CD19 cells 6.73×10E9/L, bone marrow lymphocyte 62%, peripheral blood lymphocyte 52%. Immunophenotype: CD5, CD19, CD20dim, CD23, CD11b dim, HLA-DR expression, visible CD5+CD19+ cell clusters, and visible immunoglobulin cKappa with restricted expression. On March 10, 2016, peripheral blood platelet 60 × 10E9/L, CD19 cells 1.94×10E9/L, lactate dehydrogenase 460U/L, FER 115.6ng/ml, hepatitis B virus carrier. Diagnosis: CLL/SLL IV stage, ATM (11q22) deletion, D13S25 (13q14. 3) positive, CD19 positive. Relapse of CLL/SLL occurred again after four months and at this stage the patient was considered for therapy in a clinical trial of CD19-specific chimeric antigen receptor (CAR-) T cell therapy. Ethical approval and informed consent were obtained for anti-CD19 CAR T Cell treatment of ibrutinib resistance in relapsed/refractory CLL/SLL. We infused autologous T cells transduced with a CAR T 19 retroviral vector with CLL/SLL at doses of 3.3 × 10E8 CART19 cells on Mar. 16 2016. Patients were monitored for responses, toxic effects, and the expansion and persistence of circulating CART19 cells. After CART19 cells were infused, the patient experienced chills, fever, headache, weak, anorexia, nausea, shortness of breath, chest tightness, heart palpitation, hypotension and shock for 9 days. The serum levels of IFN-Υ were at their highest at day 7 after CAR T cells infusion. Serum interleukin 6 (IL-6) was at 680pg/ml and CD3+ cells were 97.5%, CD8+ cells 72.8% (18.7-32.8%), FER was 1529.5ng/ml (Normal No. 22-322ng/ml) 14 days after CAR-T cell infusion. The serum levels of IL-6 were at their highest at day14. The patient was diagnosed as having cytokine release syndrome. After the patient took the anti-IL-6R antibody and anti-TNF antibody, he began to recover gradually. Enlarge lymph nodes shrunk after being infused with CART19 cells for 7 days. The peripheral blood CD19 B lymphocytes were 0 on day 14 after infused with CAR T19 cells. Q-PCR was used to detect the amount of the peripheral blood CART19 cells, which stood at 5485 copies/μl, 924 copies/μl, 191 copies/μl respectively 2 weeks, 6 weeks and 3 months after infusing with CART19 cells. The peripheral blood CART 19 cells were not detectable 4 months after infusing with CART19 cells until present. The lymphadenopathy was decreased gradually after 14 days of infusion. The MRI test showed that lymphadenopathy reduced markedly or disappeared after 6 months of infusion. ATM (11q22 deleted) negative, D13S25 (13q14.3 deleted) negative. After treatment with CAR T 19 cell therapy for 53 months, the patient remained disease-free, the patient's lymph nodes, lymphocytes and I mmunoglobulins were normal. CONCLUSIONS : Cancer immunotherapy as a method of cancer treatment is the most effective after conventional treatments such as radiotherapy, chemotherapy, and surgery. For BTK Inhibitor resistance in relapsed and refractory CD19+ CLL/SLL, CD19 is a favorable target, because the expression of CD19 is limited to B cells and not present in other tissues or cells. Currently, the efficacy of this treatment in treating CLL/SLL remains to be seen. The effects of chemotherapy on the patient's B cell lymphoma are negligible, due to the fact that his CLL/SLL have become relapsed and refractory. As a result we chose the CAR T19 cell therapy genetic engineering technique as a method of treatment, to which the patient has responded well. Therefor, CAR T cell technology overcome the limitations of existing cancer therapies and has great potential for development and application. Disclosures No relevant conflicts of interest to declare.

scholarly journals Increased levels of circulating IL-10 in persons recovered from hepatitis C virus (HCV) infection compared with persons with active HCV infection

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Dorcas Ohui Owusu ◽  
Richard Phillips ◽  
Michael Owusu ◽  
Fred Stephen Sarfo ◽  
Margaret Frempong
Keyword(s):  
Hepatitis C ◽  
Spontaneous Recovery ◽  
Predictive Marker ◽  
Serum Levels ◽  
Hcv Infection ◽  
Tnf Alpha ◽  
Hepatitis C Infection ◽  
Ifn Gamma ◽  
The Relationship

Abstract Objective Approximately 70% of all hepatitis C (HCV) infections develop chronic disease. Active or exacerbated chronic hepatitis C infection subsequently progress to liver disease. The role of T-cells secretions in achieving viral clearance is still not well understood. Thus, the current study was set to determine the relationship between the T cell cytokine profiles, biochemical parameters and persistent HCV infection or spontaneous recovery. Results Twenty-five percent (41/163) of the anti-HCV positive participants had recovered from HCV and had significantly higher concentration of IL-10 compared to those with active HCV infection (P < 0.012). Other circulating cytokines measured; IL-2, IFN gamma, TNF alpha, IL-5 and IL-17 were similar in both groups. Participants with active HCV infection had significantly higher aspartate transaminase (AST) (35 units) and alanine transaminase (46 units) compared to those in the recovered state (P < 0.001). Thus, serum levels of IL10 could be explored in larger prospective cohort study as a predictive marker of recovering from an active HCV infection.

scholarly journals Inhibition of CD4 cross-linking-induced lymphocytes apoptosis by vesnarinone as a novel immunomodulating agent: vesnarinone inhibits Fas expression and apoptosis by blocking cytokine secretion

Blood ◽  
1996 ◽  
Vol 87 (6) ◽  
pp. 2361-2368 ◽  
Author(s):  
N Oyaizu ◽  
TW McCloskey ◽  
S Than ◽  
S Pahwa
Keyword(s):  
T Cells ◽  
Mononuclear Cells ◽  
Interleukin 2 ◽  
Cytokine Secretion ◽  
Tnf Alpha ◽  
Cross Linking ◽  
Apoptosis Induction ◽  
Peripheral Blood Mononuclear ◽  
Cell Surface Molecule ◽  
Ifn Gamma

Evidence is accumulating that T cells from human immunodeficiency virus type 1 (HIV-1)-infected individuals show accelerated cell death through apoptosis. We have recently demonstrated that the cross-linking of CD4 molecules (CD4XL) results in death of normal peripheral T cells through apoptosis and imbalanced cytokine secretion (ie, induction of tumor necrosis factor-alpha [TNF-alpha] and interferon-gamma [IFN-gamma] in the absence of interleukin-2 [IL-2] or IL-4 secretion). These upregulated cytokines (TNF-alpha/IFN-gamma) largely contributed to upregulation of the apoptosis-inducing cell surface molecule, Fas (APO- 1/CD95) and apoptosis induction. The present study investigated the effect of vesnarinone as a novel immunomodulating agent on CD4XL- induced T-cell apoptosis. The addition of vesnarinone to peripheral blood mononuclear cells (PBMC) significantly inhibited CD4XL-induced lymphocyte apoptosis. This apoptosis-inhibitory effect of vesnarinone was associated with the blocking of CD4XL-induced TNF-alpha IFN-gamma secretion and of Fas antigen upregulation. However, vesnarinone did not block effects of exogenously supplemented TNF-alpha/IFN-gamma on Fas induction. These data suggest that vesnarinone inhibits CD4XL-induced TNF-alpha/IFN-gamma secretion, thereby blocking subsequent Fas upregulation and apoptosis induction. Given the potent pathogenic role of imbalanced cytokine secretion observed in HIV-infection, an agent such as vesnarinone may be of therapeutic value in slowing disease progression.

Different Profiles of IL-10+IFN-γ–IL-4–CD4+ T Cells in the Peripheral Blood in Atopic and Non-Atopic Asthmatics

Respiration ◽  
2008 ◽  
Vol 75 (3) ◽  
pp. 281-287
Author(s):  
K. Matsumoto ◽  
H. Inoue ◽  
M. Tsuda ◽  
T. Nakano ◽  
M. Komori ◽  
...  
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Ifn Gamma

Kinase Inhibitors Reduce TNF-Alpha Over-Production in Monocytes From Fanconi Anemia Group A Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2409-2409
Author(s):  
Enrico Cappelli ◽  
Johanna Svahn ◽  
Praveen Anur ◽  
Fabio Corsolini ◽  
Piero Farruggia ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fanconi Anemia ◽  
Peripheral Blood ◽  
Kinase Inhibitors ◽  
Bone Marrow Failure ◽  
Mononuclear Phagocytes ◽  
Tnf Alpha ◽  
Regulatory Pathways ◽  
Alpha Production ◽  
Alpha Gene

Abstract Abstract 2409 Fanconi Anemia (FA) is a chromosomal instability syndrome with hypersensitivity to alkylating agents as principal diagnostic feature. Many laboratories have demonstrated the involvement of FA proteins in DNA repair mechanisms. Recent work has also demonstrated other functions of some FA proteins suggesting that they play alternative roles in other regulatory pathways, particularly those that influence hematopoiesis. Eighty percent of FA patients develop bone marrow failure with a high incidence of evolution in myelodysplasia and/or acute leukemia. Each of these abnormalities has been related to TNF-alpha hypersensitivity in the stem and progenitor cell pools and the toll-like receptor dependent overproduction of TNF-alpha by FA macrophages. The TNF-hypersensitive phenotype involves at least two kinases, PKR and ASK1, each of which is hyperactivated in FA cells and induce apoptotic responses both in ground state and after TNF-alpha and IFN-gamma stimulation. Recent evidence showed that R848 (a TLR8 ligand) and endotoxin (LPS, a TLR4 ligand)-induced TNF-alpha gene expression in Fancc-deficient mononuclear phagocytes cells is inhibited by kinase inhibitors dasatinib and BIRB796 We sought to evaluate the activity of these agents in primary mononuclear phagocytes obtained from children with FA-A. Objectives: To determine whether primary monocytes from the peripheral blood of FANCA-deficient patients: (a) exhibit the TNF-overproduction phenotype in response to LPS and the TLR8 ligand R848, and (b) respond to dasatinib and BIRB796 by suppressing TNF-production. Methods: Six FA patients with mild to severe marrow failure on no treatment were included in this study. Healthy subjects were recruited as normal controls that were run in parallel in each case. CD14+ monocytes freshly isolated from peripheral blood were cultured for 24 hours with LPS and R848 with or without dasatinib or BIRB796. Supernatant media were collected and frozen at −80 degrees. After thawing the samples, TNF-alpha content was quantified by ELISA. Results: Baseline TNF-alpha concentration (without any TLR stimulation) was higher in FA patients than control. After LPS or R848 stimulation FA-A monocytes produced substantially more TNF-alpha than did the control samples. Both dasatinib and BIRB796 suppressed TLR-induced (both LPS and R848) TNF-alpha production. Specifically, with R848 as the agonist, BIRB 769 suppressed TNF-alpha production by 60% and dasatinib by 42%. Both inhibitors were even more potent in suppressing LPS induced TNF-alpha expression as both reduced TNF-alpha by 75%. In the absence of TLR stimulation, the presence of BIRB or dasatinib in culture reduced TNF-alpha by >50% compared to baseline in patient samples. The inhibitory effect of kinase inhibitors was observed also in the normal control. Conclusions: These findings: (a) demonstrate for the first time that TLR-induced TNF-alpha gene expression in primary FANCA deficient mononuclear phagocytes is aberrantly regulated and (b) that in FANCA-deficient macrophages the TNF-alpha overproduction phenotype can be controlled by therapeutically achievable doses of BIRB796 and dasatinib. In addition (c), since both agents function in large part to suppress p38 MAPK activation future, our data point to the biochemical roles played by FANCA in modulating upstream pathways that govern p38 activation. Moreover (d), given that in FA patients, TNF hypersensitive stem cells are over-exposed to TNF-alpha, particularly during inflammatory events and that exposure to TNF was shown not only to suppress hematopoiesis in FA but also to favor the emergence of neoplastic clones, these results point to these two agents as potential candidates for preclinical trials seeking to enhance hematopoiesis and suppress clonal evolution. Disclosures: No relevant conflicts of interest to declare.

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