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.

scholarly journals Deficiency of the c-JUN Inhibitor DACH1 in FA Macrophages Accounts for the Cytokine Overproduction Phenotype

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1002-1002
Author(s):  
Michael Garbati ◽  
Grover C. Bagby
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Inflammatory Cytokines ◽  
Fanconi Anemia ◽  
Molecular Mechanisms ◽  
Cytokine Production ◽  
Bone Marrow Failure ◽  
Clonal Evolution ◽  
Mononuclear Phagocytes ◽  
Normal Cells

Abstract The inflammatory cytokines TNFα and IL-1β contribute to the bone marrow failure phenotype in Fanconi anemia (FA) as well as to clonal evolution to MDS and AML. Mononuclear phagocytes deficient in FANCA or FANCC overproduce these cytokines in response to toll-like receptor agonists (TLRa), but the precise molecular mechanisms by which FA proteins suppress cytokine production remains enigmatic. We show here that pretreatment of control macrophages with interferon (IFN) α enhances (by 3-fold) TLRa-induced production of both TNFα and IL-1β. FANCC-deficient cells produce more of these proteins (by 3-fold) after exposure to TLRa alone and IFNα does not enhance production of these cytokines. We therefore hypothesized that TLR stimulation of FANCC-deficient cells activates an IFNα-like pathway, one constrained by FANCC in normal cells. To test this notion, we performed gene expression microarray analysis (Affymetrix HTA 2.0) using RNA from FANCC-deficient (T-shFC) and control (T-shNT) THP-1 human mononuclear phagocytes treated with IFNα, the TLR7/8 agonist R848, or a combination of IFNα plus R848. We found that treatment of T-shFC cells with R848 alone was sufficient to enhance expression of 49 genes that were activated by IFNα in control cells. Thirteen of these genes were not induced by R848 in control cells but were induced by R848 in T-shFC cells (one of which, IFNgamma-inducible protein 30 (IFI30), is known to be overexpressed in Fancd2 -/- progenitor cells). These results support our hypothesis that FANCC functions normally to constrain IFNα pathway activation in TLRa-activated macrophages. Of greater functional importance vis-à-vis cytokine production, DACH1 mRNA was suppressed by 11-fold and DACH1 protein was barely detectable in FANCC-deficient cells under all conditions when compared to control cells. DACH1 is known to bind to and suppress the activities of a variety of transcription factors, notably c-JUN in fibroblasts. We tested the hypothesis that DACH1 deficiency is sufficient to account for the TLRa hypersensitivity of FA macrophages using gain- and loss-of-function studies. Knockdown of DACH1 in control (T-shNT) macrophages resulted in a 7-fold enhancement of R848-induced TNFα production, increased c-JUN protein (1.6-fold) and c-JUN phosphorylation (2.6-fold). Reporter gene expression (secreted embryonic alkaline phosphatase [SEAP]) from constructs containing both AP-1 and NF-κB sites was activated by DACH1 knockdown but deletion of the AP-1 site completely abrogated activation. Results of gain-of-function experiments with wild-type and mutant DACH1 cDNA sequences now taking place will be reported. Taken together, these results suggest that FANCC directly or indirectly enhances ground-state expression of DACH1 and thereby suppresses the TLR pathway in normal cells specifically by inhibiting R848-dependent activation of c-JUN. Conversely, loss of DACH1 expression in FANCC-deficient cells leads to unconstrained c-JUN activity, resulting in overproduction inflammatory cytokines. In light of the contribution of such cytokines to both bone marrow failure and clonal evolution, activation of DACH1 gene expression is a rational therapeutic objective in the management of patients with Fanconi anemia and is worthy of investigating in preclinical (animal) models. Disclosures No relevant conflicts of interest to declare.

TNFα Overproduction in FANCC-Deficient Cells Is TLR8 Dependent.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 494-494
Author(s):  
Scott Vanderwerf ◽  
Johanna Svahn ◽  
Praveen Anur ◽  
Ricardo Pasquini ◽  
Grover C. Bagby
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Clonal Evolution ◽  
Hematopoietic Cells ◽  
Mononuclear Phagocytes ◽  
C Cells ◽  
Mutant Cells

Abstract Abstract 494 The Fanconi anemia (FA) proteins play a role in regulating genome stability but it is not clear that loss of genoprotection in FA hematopoietic cells accounts for the molecular pathogenesis of bone marrow failure so characteristic of this disease. Other factors are known to influence survival and replication of FA stem cells. For example, not only are FA progenitors and stem cells hypersensitive to the apoptotic effects of TNFα, FA cells over-produce TNFα. Most importantly over-production of and hypersensitivity to TNFα in hematopoietic cells of Fancc-/- mice results in bone marrow hypoplasia 1;2 and long-term ex-vivo exposure of murine Fancc -/- hematopoietic cells to both growth factors and TNFα results in the evolution of cytogenetically marked preleukemic clones.3 Therefore, the hematopoietic phenotype of FA is likely multifactorial and may evolve from the overproduction of precisely the cytokine to which FA stem cells are hypersensitive. Methods: We sought to clarify the molecular basis of aberrant TNFα-production. We conducted gene expression microarray experiments using RNA samples from low density marrow cells obtained from 11 normal volunteers and 22 Fanconi anemia patients with uncomplicated marrow hypoplasia without clonal cytogenetic defects. Because the FA complex is known to enhance ubiquitinylation of FANCD2, we reasoned that the ubiquitinylation state of proteins involved in the TNF pathways might also be influenced by core FA proteins. Therefore, we conducted in vitro ubiquitinylation assays using hexahistidine-tagged ubiquitin and an ATP-recycling system added to lysates of FANCC-deficient lymphoblasts (HSC536) and control cells (isogenic cells complemented with WT FANCC cDNA). Following the ubiquitinylation reaction, ubiquitinylated proteins were affinity purified, digested and analyzed by 2D capillary LC-MS/MS. Mass spectra were obtained and peptide precursor-MS/MS spectrum pairs were analyzed using SEQUEST and support vector machine learning.4 Peptides identified only in one or the other cell line were considered. Results: Initially we anticipated focusing on the set of proteins uniquely ubiquitinated in normal cells. However, the transcriptomal results indicated that genes encoding proteins in the ubiquitin pathway were over-represented in the list of genes that were over-expressed in FA samples. Consequently, we examined both differential ubiquitination lists and found that a major regulator of TNF-gene expression, TLR8, appeared in the ubiquitinylated fraction only in mutant cells. In co-immunoprecipitation studies we confirmed that TLR8 (or a TLR8-associated protein) is ubiquitinylated in mutant FA-C cells, and using RNAi determined that high level TNFα synthesis in mutant cells depended upon TLR8 and its downstream signaling intermediates IRAK-1 and IKK-alpha/beta. FANCC deficient THP1 blue cells were created using lentiviral shRNA targeting FANCC. These cells exhibited the MMC hypersensitive phenotype and over-expressed both TNFα and an NF-kappaB reporter gene (secreted embryonic alkaline phosphatase) in response to TLR8 agonists but not to other TLR agonists. Primary splenic macrophages from Fancc-/- mice were also hypersensitive to the TLR8 agonist R848. TNFα production in FA-C cells was suppressed by inhibitors of TLR8, p38 MAPK, IRAK, and IKK. Engineered point mutants of FANCC were capable of complementing the mitomycin C hypersensitivity phenotype of FANCC mutant cells but did not suppress TNFα overproduction in FANCC mutant cells. In conclusion, TNF over-expression in FANCC-deficient cells reflects the loss of FANCC function as a suppressor of TLR8 activation. In addition, FANCC suppresses TLR8 dependent production of TNFα in normal mononuclear phagocytes at least in part by suppressing either TLR8 ubiquitinylation or by inhibiting its association with an ubiquitinylated protein. Finally, this function of FANCC is independent of its function in protecting the genome from cross-linking agent-induced damage. In light of the role of TNFα in bone marrow failure and clonal evolution in this disease, control of TNF-production by targeting the TLR8 pathway might provide an opportunity to enhance hematopoietic activity and forestall clonal evolution in patients with this disorder. 1. Sejas DP, et al, J Immunol 2007;178:5277-5287. 2. Zhang × et al, J.Cell Sci. 2007;120:1572-1583. 3. Li J, et al, J.Clin.Invest. 2007;117:3283-3295, 4. Anderson DC, et al, J Proteome.Res 2003;2:137-146. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Differential tumor necrosis factor alpha expression by astrocytes from experimental allergic encephalomyelitis-susceptible and -resistant rat strains.

1991 ◽  
Vol 173 (4) ◽  
pp. 801-811 ◽  
Author(s):  
I Y Chung ◽  
J G Norris ◽  
E N Benveniste
Keyword(s):  
Gene Expression ◽  
Tnf Alpha ◽  
Allergic Encephalomyelitis ◽  
Necrosis Factor Alpha ◽  
Ifn Gamma ◽  
Alpha Production ◽  
Factor Alpha ◽  
Alpha Gene ◽  
Necrosis Factor ◽  
Experimental Allergic

There is evidence that the cytokine tumor necrosis factor alpha (TNF-alpha) contributes to the pathogenesis of neurological autoimmune diseases such as multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). TNF-alpha exerts damaging effects on oligodendrocytes, the myelin-producing cell of the central nervous system (CNS), and myelin itself. We have recently demonstrated TNF-alpha expression from astrocytes induced by lipopolysaccharide (LPS), interferon gamma (IFN-gamma), and interleukin 1 beta (IL-1 beta). Astrocytes secrete TNF-alpha in response to LPS alone, and can be primed by IFN-gamma to enhance LPS-induced TNF-alpha production. IFN-gamma and IL-1 beta, cytokines known to be present in the CNS during neurological disease states, do not induce TNF-alpha production alone, but act synergistically to stimulate astrocyte TNF-alpha expression. Inbred Lewis and Brown-Norway (BN) rats differ in genetic susceptibility to EAE, which is controlled in part by major histocompatibility complex (MHC) genes. We examined TNF-alpha gene expression by astrocytes derived from BN rats (resistant to EAE) and Lewis rats (highly susceptible). Astrocytes from BN rats express TNF-alpha mRNA and protein in response to LPS alone, yet IFN-gamma does not significantly enhance LPS-induced TNF-alpha expression, nor do they express appreciable TNF-alpha in response to the combined stimuli of IFN-gamma/IL-1 beta. In contrast, astrocytes from Lewis rats express low levels of TNF-alpha mRNA and protein in response to LPS, and are extremely responsive to the priming effect of IFN-gamma for subsequent TNF-alpha gene expression. Also, Lewis astrocytes produce TNF-alpha in response to IFN-gamma/IL-1 beta. The differential TNF-alpha production by astrocytes from BN and Lewis strains is not due to the suppressive effect of prostaglandins, because the addition of indomethacin does not alter the differential pattern of TNF-alpha expression. Furthermore, Lewis and BN astrocytes produce another cytokine, IL-6, in response to LPS, IFN-gamma, and IL-1 beta in a comparable fashion. Peritoneal macrophages and neonatal microglia from Lewis and BN rats are responsive to both LPS and IFN-gamma priming signals for subsequent TNF-alpha production, suggesting that differential TNF-alpha expression by the astrocyte is cell type specific. Taken together, these results suggest that differential TNF-alpha gene expression in response to LPS and IFN-gamma is strain and cell specific, and reflects both transcriptional and post-transcriptional control mechanisms.(ABSTRACT TRUNCATED AT 400 WORDS)

TNF-alpha Production by Peripheral Blood Monocytes in Multiple Sclerosis Patients and Healthy Controls

2015 ◽  
Vol 44 (6) ◽  
pp. 590-601 ◽  
Author(s):  
Mehrdad Farrokhi ◽  
Masoud Etemadifar ◽  
Maryam Sadat Jafary Alavi ◽  
Sayyed Hamid Zarkesh-Esfahani ◽  
Mohaddeseh Behjati ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Peripheral Blood ◽  
Tnf Alpha ◽  
Healthy Controls ◽  
Blood Monocytes ◽  
Peripheral Blood Monocytes ◽  
Alpha Production ◽  
Multiple Sclerosis Patients

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.

scholarly journals Polymorphisms in the tumor necrosis factor alpha (TNF-alpha) gene correlate with murine resistance to development of toxoplasmic encephalitis and with levels of TNF-alpha mRNA in infected brain tissue.

1992 ◽  
Vol 175 (3) ◽  
pp. 683-688 ◽  
Author(s):  
Y R Freund ◽  
G Sgarlato ◽  
C O Jacob ◽  
Y Suzuki ◽  
J S Remington
Keyword(s):  
Brain Tissue ◽  
Tumor Necrosis ◽  
Toxoplasmic Encephalitis ◽  
Tnf Alpha ◽  
Necrosis Factor Alpha ◽  
Alpha Production ◽  
D Region ◽  
Factor Alpha ◽  
Alpha Gene ◽  
Necrosis Factor

Murine resistance to development of toxoplasmic encephalitis (TE) has recently been mapped to the D region of the major histocompatibility complex (H-2). Since the gene for tumor necrosis factor alpha (TNF-alpha) is located 5' of the D region and TNF-alpha has been implicated as playing a role in neurological diseases, we were interested in determining the relationship of TNF-alpha production to TE resistance. We have demonstrated that resistance to TE in inbred mice can be correlated with specific restriction fragment length polymorphisms and microsatellite variants in the TNF-alpha gene. Mice that are susceptible to TE express elevated levels of TNF-alpha mRNA in brain tissue 6 wk after infection with the ME49 strain of Toxoplasma gondii. Resistant mice and all mice that are uninfected show no detectable TNF-alpha mRNA expression in brain tissue. Differences in the TNF-alpha gene between susceptible and resistant mice have been localized to the first intron, the promoter, and the 3' end of the TNF-alpha gene. These data implicate differences in regulation of TNF-alpha production in brain tissue as contributing to differences in susceptibility to development of TE.

scholarly journals Induction of TNF-.ALPHA. Gene Expression by Heat Inducible Promoter gadd 153.

2000 ◽  
Vol 16 (2) ◽  
pp. 91-98 ◽  
Author(s):  
AKIRA ITO ◽  
MASASHIGE SHINKAI ◽  
ISABELLE ANNE BOUHON ◽  
HIROYUKI HONDA ◽  
TAKESHI KOBAYASHI
Keyword(s):  
Gene Expression ◽  
Inducible Promoter ◽  
Tnf Alpha ◽  
Alpha Gene

scholarly journals Prevalence of Inflammatory Pathways Over Immuno-Tolerance in Peripheral Blood Mononuclear Cells of Recent-Onset Type 1 Diabetes

2022 ◽  
Vol 12 ◽  
Author(s):  
Aritania Sousa Santos ◽  
Edécio Cunha-Neto ◽  
Nelson Vinicius Gonfinetti ◽  
Fernanda Bernardi Bertonha ◽  
Pauline Brochet ◽  
...  
Keyword(s):  
Gene Expression ◽  
Peripheral Blood Mononuclear Cells ◽  
Immune Tolerance ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Regulatory Pathways ◽  
Recent Onset ◽  
Inflammatory Pathways ◽  
Blood Mononuclear Cells

BackgroundChanges in innate and adaptive immunity occurring in/around pancreatic islets had been observed in peripheral blood mononuclear cells (PBMC) of Caucasian T1D patients by some, but not all researchers. The aim of our study was to investigate whether gene expression patterns of PBMC of the highly admixed Brazilian population could add knowledge about T1D pathogenic mechanisms.MethodsWe assessed global gene expression in PBMC from two groups matched for age, sex and BMI: 20 patients with recent-onset T1D (≤ 6 months from diagnosis, in a time when the autoimmune process is still highly active), testing positive for one or more islet autoantibodies and 20 islet autoantibody-negative healthy controls.ResultsWe identified 474 differentially expressed genes between groups. The most expressed genes in T1D group favored host defense, inflammatory and anti-bacterial/antiviral effects (LFT, DEFA4, DEFA1, CTSG, KCNMA1) and cell cycle progression. Several of the downregulated genes in T1D target cellular repair, control of inflammation and immune tolerance. They were related to T helper 2 pathway, induction of FOXP3 expression (AREG) and immune tolerance (SMAD6). SMAD6 expression correlated negatively with islet ZnT8 antibody. The expression of PDE12, that offers resistance to viral pathogens was decreased and negatively related to ZnT8A and GADA levels. The increased expression of long non coding RNAs MALAT1 and NEAT1, related to inflammatory mediators, autoimmune diseases and innate immune response against viral infections reinforced these dataConclusionsOur analysis suggested the activation of cell development, anti-infectious and inflammatory pathways, indicating immune activation, whereas immune-regulatory pathways were downregulated in PBMC from recent-onset T1D patients with a differential genetic profile.

Mobilization and collection of peripheral blood CD34+ cells from patients with Fanconi anemia

Blood ◽  
2001 ◽  
Vol 98 (10) ◽  
pp. 2917-2921 ◽  
Author(s):  
James M. Croop ◽  
Ryan Cooper ◽  
Christine Fernandez ◽  
Vicki Graves ◽  
Susan Kreissman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Fanconi Anemia ◽  
Peripheral Blood ◽  
Bone Marrow Failure ◽  
Peripheral Blood Stem Cells ◽  
Target Weight ◽  
Peripheral Blood Cd34 ◽  
Cd34 Cells ◽  
Blood Stem Cells

Abstract A potential therapeutic option for patients with Fanconi anemia is collection of peripheral blood stem cells prior to the development of severe pancytopenia. These hematopoietic cells potentially could be infused when symptomatic bone marrow failure develops, as autologous rescue after chemotherapy in the event of leukemic transformation, or as targets for gene therapy. Eight patients with Fanconi anemia were mobilized with 10 μg/kg per day of granulocyte colony-stimulating factor (median, 10 ± 4 days) to determine the feasibility of collecting peripheral blood stem cells for future use. Six patients achieved a peripheral blood CD34+ count of ≥ 6/μL and underwent apheresis. The collection goal was 2 × 106 CD34+ cells/kg based on a predicted weight 5 years from the date of collection. A mean of 2.6 ± 0.9 × 106 CD34+ cells/kg of the weight at the time of collection were collected, which corresponded to 1.9 ± 0.4 × 106 CD34+cells/kg of the target weight. The collections required a mean of 4 ± 3 days (range, 2-8 days) of apheresis. Six of the 8 subjects had ≥ 1 × 106 CD34+ cells/kg cryopreserved based on both actual and target weights, and 4 subjects had ≥ 2 × 106 CD34+ cells/kg cryopreserved based on the target weight. These results suggest that some patients with Fanconi anemia can have adequate numbers of CD34+ cells mobilized and collected from the peripheral blood prior to the onset of severe bone marrow failure, but they may require an extended mobilization and multiple days of collection.

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