CCR5 and RANTES/CCL5 Gene Polymorphisms Affect the Risk of EBV Reactivations after Allogeneic HSCT.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1262-1262
Author(s):  
Katarzyna Bogunia-Kubik ◽  
Anna Kuzyk ◽  
Emilia Jaskula ◽  
Andrzej Lange
Keyword(s):  
Chemokine Receptor ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Detection Threshold ◽  
Deletion Mutation ◽  
Viral Reactivation ◽  
Control Group ◽  
Healthy Individuals ◽  
Hematopoietic Stem ◽  
Ebv Reactivation

Abstract Epstein-Barr virus (EBV) reactivation is a serious complication affecting the recipients of allogeneic hematopoietic stem cell transplants (HSCT). Chemokines and their receptors play a major role in the inflammatory and immune responses that mediate allograft outcome. CC-chemokine receptor-5 (CCR5) is known to be involved in perpetuation of HIV infection (as a co-receptor for HIV entry) and the clinical course of this infection is favored by the presence of the 32-nucleotide deletion within the CCR5 gene (CCR5Δ32 polymorphism). The CCR5 deletion mutation (CCR5Δ32) results in a non-functional chemokine receptor. In the present study the CCR5Δ32 polymorphism and two single nucleotide substitutions (−28 C/G; −403 G/A) within, one of the CCR5 ligands, the CCL5/RANTES gene were analyzed in 75 HSCT recipients, 75 donors and related with EBV load. The control group constituted 99 healthy individuals. DNA was extracted from peripheral blood taken into EDTA using silica membranes. Viral load were assessed 2-3 moths after transplantation in blood cells employing a real-time PCR technique. The detection threshold for viral reactivation equaled 10 EBV-DNA copies/105 peripheral blood cells. EBV reactivation was detected in 26 patients. The CCR5Δ32 and RANTES (−28 C/G; −403 G/A) polymorphisms were analyzed by PCR and PCR-RFLP technique, respectively. Distributions of CCR5 and RANTES −28 genotypes were similar in patients, donors and healthy individuals. RANTES −403 AA homozygosity was more frequent in donors than controls (0.61 vs 0.47, p=0.036). The higher number of EBV copies was detected in patients lacking CCR5Δ32 deletion (0.92 vs 0.71, p=0.031). No significant correlation between EBV reactivation and RANTES −28 C/G polymorphism. However, patients transplanted with donors homozygous for RANTES −403 AA (genotype associated with an increased expression of the RANTES gene) more frequently presented with EBV reactivation (0.85 vs 0.49, p=0.002). In conclusion, the presence of the CCR5 deletion-mutation in the recipient and RANTES-403 G in the donor of HSC appeared to lower the susceptibility for EBV reactivation.

Efficient lentiviral gene transfer to canine repopulating cells using an overnight transduction protocol

Blood ◽  
2004 ◽  
Vol 103 (10) ◽  
pp. 3710-3716 ◽  
Author(s):  
Peter A. Horn ◽  
Kirsten A. Keyser ◽  
Laura J. Peterson ◽  
Tobias Neff ◽  
Bobbie M. Thomasson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Gene Transfer ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Lentiviral Vectors ◽  
Large Animal ◽  
Hematopoietic Stem ◽  
Lentiviral Transduction ◽  
Mobilized Peripheral Blood

Abstract The use of lentiviral vectors for the transduction of hematopoietic stem cells has evoked much interest owing to their ability to stably integrate into the genome of nondividing cells. However, published large animal studies have reported highly variable gene transfer rates of typically less than 1%. Here we report the use of lentiviral vectors for the transduction of canine CD34+ hematopoietic repopulating cells using a very short, 18-hour transduction protocol. We compared lentiviral transduction of hematopoietic repopulating cells from either stem cell factor (SCF)– and granulocyte-colony stimulating factor (G-CSF)–primed marrow or mobilized peripheral blood in a competitive repopulation assay in 3 dogs. All dogs engrafted rapidly within 9 days. Transgene expression was detected in all lineages (B cells, T cells, granulocytes, and red blood cells as well as platelets) indicating multilineage engraftment of transduced cells, with overall long-term marking levels of up to 12%. Gene transfer levels in mobilized peripheral blood cells were slightly higher than in primed marrow cells. In conclusion, we show efficient lentiviral transduction of canine repopulating cells using an overnight transduction protocol. These results have important implications for the design of stem cell gene therapy protocols, especially for those diseases in which the maintenance of stem cells in culture is a major limitation.

scholarly journals Circulating Natural IgM Antibodies against Angiogenin in the Peripheral Blood Sera of Patients with Osteosarcoma as Candidate Biomarkers and Reporters of Tumorigenesis

2010 ◽  
Vol 2 ◽  
pp. BIC.S6040 ◽  
Author(s):  
Yulia A. Savitskaya ◽  
Genaro Rico ◽  
Luis Linares ◽  
Roberto González ◽  
René Téllez ◽  
...  
Keyword(s):  
Early Diagnosis ◽  
Sensitivity And Specificity ◽  
Peripheral Blood ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Healthy Individuals ◽  
Igm Antibodies ◽  
Potential Biomarker ◽  
Increased Risk ◽  
Natural Igm

Background Tumor immunology research has led to the identification of a number of tumor-associated self antigens, suggesting that most tumors trigger an immunogenic response, as is the case in osteosarcoma, where the detection of natural serum IgM antibodies might achieve the diagnosis of osteosarcoma. Natural IgM antibodies to tumor-associated proteins may expand the number of available tumor biomarkers for osteosarcoma and may be used together in a serum profile to enhance test sensitivity and specificity. Natural IgM antibodies can be consistently detected in the peripheral blood sera months to years before the tumor is diagnosed clinically. The study of the level of a potential biomarker many months (or years) prior to diagnosis is fundamentally important. Integrated circulating and imaging markers in clinical practice treating osteosarcoma have potential applications for controlling tumor angiogenesis. Objectives To study the expression of natural IgM antibodies to the tumor antigens of angiogenesis in the peripheral blood sera of osteosarcoma patients and healthy individuals, and to develop serum-based predictive biomarkers. Methods Peripheral venous blood samples were collected from 117 osteosarcoma patients and 117 patients with other tumors. All diagnosis was histologically confirmed. Staging of patients was performed according to the Enneking Surgical Staging System. The control group consisted of 117 age- and sex- matched healthy individuals. In this study, novel immunoconjugates were designed, synthesized and then used to develop a rapid, specific and sensitive enzyme-linked immunosorbent assay (ELISA) method to detect angiogenin (ANG)–IgM directly in the peripheral blood sera of humans. Results Serum ANG–IgM levels are significantly higher in osteosarcoma patients than in healthy individuals ( P < 0.005). Serum ANG–IgM levels varied widely, but were highly dependent on the concentration of IgM (r = 0.85; P < 0.0005). We found ANG–IgM in the sera of 85% of newly diagnosed osteosarcoma patients and ANG–IgM levels were significantly higher in osteosarcoma patients compared to any other tumors ( P < 0.001). Conclusions These results demonstrated that the combined biomarker ANG–IgM has greater sensitivity and specificity in early diagnosis of osteosarcoma patients than the traditional biomarkers (ANG and vascular endothelial growth factor). Circulating ANG–IgM immune complexes can potentially serve as a biomarker for increased risk of osteosarcoma, because relatively high serum levels were also detected in otherwise healthy individuals with a first degree family history of osteosarcoma and in patients with a diagnosis of benign conditions. Immunological aspects of angiogenesis for managing osteosarcoma will have a practical value in early diagnosis, prognosis and monitoring response to antiangiogenic therapy.

scholarly journals The mechanism of adaptation of the organism of patients with chronic heart failure combined with vitamin D deficiency and the morphofunctional state of peripheral blood erythrocytes

2019 ◽  
Vol 10 (3) ◽  
pp. 352-357
Author(s):  
N. I. Baryla ◽  
I. P. Vakaliuk ◽  
S. L. Pоpеl’
Keyword(s):  
Heart Failure ◽  
Vitamin D ◽  
Chronic Heart Failure ◽  
Red Blood Cells ◽  
Vitamin D Deficiency ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Functional Class ◽  
Exercise Stress ◽  
Control Group

The problem of structural changes in peripheral blood erythrocytes in patients with chronic heart failure in combination with vitamin D deficiency during exercise stress remains insufficiently studied. Vitamin receptors are located on smooth myocytes, endothelial cells, cardiomyocytes and blood cells. It affects the state of the cell membrane, the contractile function of the myocardium, the regulation of blood pressure, cardiac remodeling and reduction of left ventricular hypertrophy. Therefore, it is important to assess the level of vitamin D in blood plasma in individuals with chronic heart failure and to identify the effect of its deficiency on the state of peripheral red blood cells when performing a 6-minute walk test. A total of 75 patients of the main group with chronic heart failure stage II A, I–II functional class with different levels of vitamin D deficiency were examined. The control group included 25 patients with chronic heart failure stage II A, functional class I–II without signs of vitamin D deficiency. The average age of patients was 57.5 ± 7.5 years. All patients were asked to undergo the 6 minutes walking test. The level of total vitamin D in plasma was determined by enzyme immunoassay. Morphological studies of erythrocytes were performed on the light-optical and electron-microscopic level. The obtained results showed that patients of the main group with chronic heart failure had a decrease in vitamin D by 2.2 times compared with the control group. Correlation analysis showed a directly proportional relationship between vitamin D deficiency and the number of red blood cells of a modified form and red blood cells with low osmotic resistance. Dosed exercise stress in patients with chronic heart failure against a background of vitamin D deficiency leads to an increase in the number of reversibly and irreversibly deformed erythrocytes and a decrease in their osmotic stability. This indicates a disorder in the structural integrity of their membrane and can have negative consequences for the somatic health of such patients.

scholarly journals Abnormal Expression of the B-Cell Homing Chemokine Receptor BLR1 During the Progression of Acquired Immunodeficiency Syndrome

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 520-525 ◽  
Author(s):  
Reinhold Förster ◽  
Georgina Schweigard ◽  
Sabine Johann ◽  
Thomas Emrich ◽  
Elisabeth Kremmer ◽  
...  
Keyword(s):  
B Cell ◽  
Chemokine Receptor ◽  
Acquired Immunodeficiency Syndrome ◽  
Peripheral Blood ◽  
Lymphoid Tissue ◽  
Healthy Individuals ◽  
T Helper ◽  
Cell Homing ◽  
Acquired Immunodeficiency ◽  
Immunodeficiency Syndrome

Abstract The putative chemokine receptor BLR1 has been identified as the first G-protein–coupled receptor involved in B-cell migration and in microenvironmental homing to B-cell follicles and to germinal centers. In healthy individuals, expression of BLR1 is restricted to all mature recirculating B cells and to a subpopulation of T-helper memory cells. In the present study, we analyzed the distribution of BLR1 on defined lymphocyte subsets during the progression of acquired immunodeficiency syndrome. It is shown that the proportion of T-helper memory cells coexpressing BLR1 continuously decreases during the infection, whereas a high proportion of γ/δ T cells expressing BLR1 can be found in peripheral blood. The latter subpopulation is restricted to lymphoid tissues in healthy individuals. Most interestingly, in 75% of all human immunodeficiency virus (HIV)+ individuals, peripheral blood B cells were identified as not expressing BLR1 and phenotypically resembling germinal center cells of lymphoid tissue. Using BLR1 as a marker molecule, this study identifies peripheral blood lymphocytes in HIV+ individuals that are usually restricted to lymphoid tissue in healthy individuals. Because HIV infection is active in lymphoid tissue even at the clinically latent stage, aberrant expression of the B-cell homing chemokine receptor BLR1 might be an early indicator for the onset of destruction of lymphoid tissue.

Parvovirus B19 and Aplastic Anemia: Case Control Study: Preliminary Report from the ITESM Hematology Study Group.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3765-3765
Author(s):  
Jose R. Borbolla Escoboza ◽  
Marcos E. Garza-Madrid ◽  
Luis Villela ◽  
Manuel A. Lopez-Hernandez ◽  
Jorge Vela-Ojeda
Keyword(s):  
Bone Marrow ◽  
Aplastic Anemia ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Parvovirus B19 ◽  
Bone Marrow Failure ◽  
Control Group ◽  
Number Of Patients ◽  
Two Samples ◽  
Sense Primer

Abstract Aplastic anemia (AA) is a classic bone marrow failure syndrome simply defined as peripheral blood pancytopenia and a hypocelular bone marrow, yet the diagnosis must be made by excluding other causes of bone marrow failure. The incidence rate of AA reported by the International Aplastic Anemia and Agranulocytosis Study (IAAAS) in the 1980s was 2 cases per 1 million people. This disease is known to be caused by exposure to radiation, chemotherapy and some viral agents, yet most of the cases are idiopathic. Epstein Barr virus and non-A, non-B or non-C Hepatitis virus have classically been related to the development of some AA cases. Recently there have been some reports of AA following Parvovirus B19 (PvB19) infection. This virus, the only parvoviridae virus capable of infecting humans, attacks erythrocyte precursors attaching to the P antigen in their surface and requiring Beta1 integrin for viral entry. Although PvB19 seems to infect only erytroid precursors, it is widely recognized that the infection with this virus can cause not only anemia, but neutropenia and thrombocytopenia as well, producing aplastic crisis of varying intensity. A correlation has recently been found between PvB19 DNA in peripheral blood and AA in children. We pretend to corroborate this observation and include adult patients in order to improve our understanding of the relationship between PvB19 and AA. So far we have taken peripheral blood samples from 9 AA patients and 9 controls paired by age, sex and community; we plan to include 100 AA patients and their controls from several hospitals around Mexico. DNA was extracted using the PUREGENE DNA extraction kit (Gentra, Minneapolis MN). Nested PCR was performed using the sense primer (P1) 5-AATACACTGTGGTTTTATGGGCCG-3, antisense (P2) 5-CCATTGCTGGTTATAACCACAGGT-3 for the first round and the sense primer (P3) 5-AATGAAAACTTTCCATTTAATGATGTAG-3 and antisense primer (P4) 5-CTAAAATGGCTTTTGCAGCTTCTAC-3for the second round. A DNA sample from a patient with active infectious mononucleosis with positive IgG and IgM against PvB19 in serum was used as positive control. Two samples from the AA group (22%) and 1 from the control group (11%) have turned positive for PvB19 DNA. The reported incidence for the presence of this virusDNA in the peripheral blood of the population is 3%. We expect that, as the number of patients grows, the percentage of positive samples in the control group will decrease, while the percentage of positive samples in the AA group will rise or be sustained. Our partial results point towards a possible relationship between AA and the presence of PvB19 DNA in the peripheral blood cells. It is possible that this virus is one of many factors capable of precipitating the development of AA by limiting the bone marrows capacity to produce blood cells. We are in the process of gathering more samples to prove if a relationship really exists and, if so, future studies will likely shed light upon the mechanism by which PvB19 contributes to the development of AA and other marrow failure syndromes.

Hematopoietic Stem Cell Differentiation Pathway in Humans Deduced From the Lineage Diversity of PNH-Type Cells in Patients with Bone Marrow Failure.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1489-1489
Author(s):  
Takamasa Katagiri ◽  
Zhirong Qi ◽  
Yu Kiyu ◽  
Naomi Sugimori ◽  
J. Luis Espinoza ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Bone Marrow Failure ◽  
Stem Cell Differentiation ◽  
Refractory Anemia ◽  
Hematopoietic Stem

Abstract Abstract 1489 Poster Board I-512 The hematopoietic stem cell (HSC) differentiation pathway in humans remains largely unknown due to the lack of an appropriate in vivo assay allowing the growth of HSCs as well as of clonal markers that enable the tracing of their progenies. Small populations of blood cells deficient in glycosylphosphatidylinositol-anchored proteins (GPI-APs) such as CD55 and CD59 are detectable in approximately 50% of patients with aplastic anemia (AA) and 15% of patients with refractory anemia (RA) of myelodysplastic syndrome defined by the FAB classification. Such blood cells with the paroxysmal nocturnal hemoglobinuria (PNH) phenotype (PNH-type cells) are derived from single PIGA mutant HSCs and their fate depends on the proliferation and self-maintenance properties of the individual HSCs that undergo PIG-A mutation by chance (Blood 2008;112:2160, Br J Haematol 2009 in press) Analyses of the PNH-type cells from a large number of patients on the diversity of lineage combination may help clarify the HSC differentiation pathway in humans because PIG-A mutant HSCs in patients with bone marrow failure appear to reflect the kinetics of healthy HSCs. Therefore, different lineages of peripheral blood cells were examined including glycophorin A+ erythrocytes (E), CD11b+ granulocytes (G), CD33+ monocytes (M), CD3+ T cells (T), CD19+ B cells (B), and NKp46+ NK cells (Nk) from 527 patients with AA or RA for the presence of CD55−CD59− cells in E and G, and CD55−CD59−CD48− cells in M,T, B, Nk with high sensitivity flow cytometry. Two hundred and twenty-eight patients (43%) displayed 0.003% to 99.1% PNH-type cells in at least one lineage of cells. The lineage combination patterns of PNH-type cells in these patients included EGM in 71 patients (31%), EGMTBNk in 43 (19%), EG in 37 (16%), T alone 14 (6%), EGMBNk in 11 (5%), G alone in 10 (4%), GM in 10 (4%), EGMNk in 7 (3%), EGMT in 7 (3%), EGMB in 6 (3%), EM in 5 (2%), EGMTB in 3 (1%), EGNk in 1 (0.4%), EGMTNk in 1 (0.4%), GMTB in 1 (0.4%), and GT in 1 (0.4%) (Table). All patterns included G or M, except for 14 patients displaying PNH-type T cells alone. No patients showed TB or TBNk patterns suggestive of the presence of common lymphoid progenitor cells. Peripheral blood specimens from 123 patients of the 228 patients possessing PNH-type cells were examined again after 3 to 10 months and all patients showed the same combination patterns as those revealed by the first examination. PIG-A gene analyses using sorted PNH-type cells from 3 patients revealed the same mutation in G and Nk for 1 patient and in G and T for 2 patients. These findings indicate that human HSCs may take a similar differentiation pathway to that of murine HSCs, the ‘myeloid-based model’ that was recently proposed by Kawamoto et al. (Nature 2008; 10:452), though the cases with PNH-type T cells alone remain to be elucidated. Table. Lineages of cells containing PNH-type cells in patients with AA or RA. The number in the parenthesis denotes the proportion of patients showing each combination pattern in the total patients possessing PNH-type cells. (+ ; presence of PNH-type cells) Disclosures No relevant conflicts of interest to declare.

High Transgene Expression Rates After Extended Follow up Among Rhesus Macaque Recipients of Autologous Hematopoietic Stem Cells Transduced with a Modified HIV1-Based Lentiviral Vector

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3118-3118
Author(s):  
Naoya Uchida ◽  
Phillip W. Hargrove ◽  
Coen J. Lap ◽  
Oswald Phang ◽  
Aylin C. Bonifacino ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Blood Cells ◽  
Transgene Expression ◽  
Vector System ◽  
Large Animal ◽  
Peripheral Blood Cells ◽  
Hematopoietic Stem ◽  
Integration Pattern ◽  
Cd34 Cells ◽  
Integration Sites

Abstract Abstract 3118 Hematopoietic stem cell (HSC)-targeted gene therapy is potentially curative for the hemoglobin disorders; however, highly efficient, lineage specific globin expression remains elusive, and large animal models thus remain important for further development toward clinical application. We previously constructed a chimeric HIV1 vector (χHIV vector) system to circumvent a species specific restriction to HIV1-based vectors wherein the HIV1 vector genome is packaged in the context of the simian immunodeficiency virus (SIV) capsid for efficient transduction of rhesus CD34+ cells in vitro (J Virol. 2009) and in vivo (ASH 2009). In this study, we sought to evaluate transduction efficiency and vector integration pattern among long-term repopulating cells in the rhesus HSC transplantation model. We followed up transgene expression rates among peripheral blood cells of three animals for 1.5–2 years. For two animals (RQ7307 and RQ7280), half of the CD34+ cells were transduced with a standard SIV vector and the other half with the χHIV vector using the same protocol. Transduced cells were transplanted into lethally irradiated rhesus macaques, as previously described (J Virol. 2009). The transgene expression rates in peripheral blood cells plateaued 3–4 months after transplantation and similar transgene expression rates continued in all cell lineages for at least 1.5 years (Figure). The χHIV vector demonstrated that 2–3 fold higher transgene expression rates were seen in granulocytes (RQ7307: 8.6±0.2% vs. 3.1±0.1%, RQ7280: 27.9±0.7% vs. 18.4±0.2%) and RBCs (RQ7307: 3.3±0.1% vs. 0.9±0.0%, RQ7280: 10.0±0.1% vs. 4.0±0.1%), and equivalent transgene expression rates in lymphocytes (RQ7307: 7.8±0.2% vs. 4.5±0.1%, RQ7280: 22.4±0.5% vs. 17.6±0.3%) and platelets (RQ7307: 3.1±0.1% vs. 2.7±0.1%, RQ7280: 12.3±0.2% vs. 16.8±0.2%), compared to the SIV vector. The average vector copy numbers in transduced cells were 4.6–5.7 for the χHIV vector and 1.5–2.0 for the SIV vector in both transplanted animals, evaluated by Southern blot analysis. We then performed transplantation of rhesus CD34+ cells which were transduced with the χHIV vector alone to evaluate transgene expression and vector integration pattern. Transgene expression rates among peripheral blood cells in this animal (RQ7387) plateaued 1–3 months after transplantation, with stable high transgene expression rates of 51.7±1.2% in granulocytes, 54.7±0.1% in lymphocytes, 22.1±0.2% in RBCs, and 19.1±0.1% platelets for 2 years after transplantation. Multi-lineage marking was observed by flow cytometric analysis. We then evaluated integration sites for the χHIV vector in the recipient of χHIV vector alone transduced cells by linear amplification mediated-PCR, using peripheral blood cells of RQ7387 in 0.5–1.5 years after transplantation. We found a total of 344 integration sites for the χHIV vector, and our data demonstrated that the χHIV vector integrated into gene regions, especially introns, when compared to the integration pattern of computer-generated random controls (p<0.001). On the other hand, our data revealed fewer integrations of the χHIV vector into ≤30kb upstream of genes (p<0.001) and into the upstream regions of transcription start sites. Most of the integration sites had low gene density (0–10 genes within 1 Mb upstream or downstream of integration sites, p<0.01), compared to that of random controls. No specific trend was noted for the number of integration sites around CpG islands and the number of CISs around integration sites. These data suggest that the χHIV vector has integration patterns comparable to HIV1 and SIV vectors. In summary, our χHIV vector shows efficient transduction for rhesus long-term repopulating cells, achieving sufficient levels for therapeutic effects in gene therapy trials for globin disorders. This χHIV vector system should allow preclinical testing of HIV1-based therapeutic vectors in large animal models. Disclosures: No relevant conflicts of interest to declare.

Neutropenia Does Not Delay Lymphopoietic Regeneration in NODSCIDcγ−/− Mice

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4831-4831
Author(s):  
Stefanie Bugl ◽  
Stefan Wirths ◽  
R Müller Martin ◽  
Märklin Melanie ◽  
Tina Wiesner ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Control Group ◽  
Early Event ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Lymphoid Progenitors ◽  
Fate Decision

Abstract Abstract 4831 Introduction: Previously it was demonstrated that lymphopoiesis is rapidly established after transplantation of wild type stem cells into lymphopenic NODSCIDcγ−/− mice. These data were interpreted as evidence for an “empty” preformed lymphopoietic niche being replenished by lymphoid progenitors. We hypothesized that antibody-induced neutropenia might influence early post transplant fate decision to myeloid rather than lymphoid differentiation resulting in delayed lymphoid reconstitution. Materials and Methods: 25,000 flow sorted CD45.2-expressing wild type Lin-/Sca1+/c-Kit+ (LSK) cells from C57BL/6 mice were transplanted into sublethally irradiated B-/T-/NK-cell deficient NODSCIDcγ−/− mice (CD45.1). Three groups of n = 7 mice received anti-Gr1 or anti-1A8 i.p. every 48 h to induce continuous antibody-mediated neutropenia vs. PBS as control. Blood was harvested at regular intervals to monitor the engraftment. After 16, 22, and 34 days, animals were sacrificed and underwent blood and bone marrow analysis. Results: Hematopoietic regeneration started with the emergence of donor-derived monocytes in all groups as well as neutrophils in the control group as early as 9 days after transplantation. On day 14, B cells were to be detected for the first time, followed by T lymphocytes approximately 20 days after transplantation. Besides the fact that neutrophils were undetectable in the antibody treated groups, the peripheral blood revealed no significant changes between the neutropenic mice and the control group at any point of time. At the bone marrow level, an increase of LSK and granulocyte-macrophage progenitors (GMPs) at the expense of megakaryocyte erythrocyte progenitor cells (MEPs) was found in neutropenic mice. Common lymphoid progenitors (CLPs), however, were not significantly different. Conclusions: The engraftment of wild type donor cells after hematopoietic stem cell transplantation into NODSCIDcγ−/− mice started with the production of monocytes and neutrophils. B-lymphocytes were detectable by day 14 after transplantation. The production of T-cells started around day 20. Continuous antibody-mediated neutropenia did not significantly delay lymphoid regeneration. Although the marrow of neutropenic mice displayed increased proliferation of granulocyte progenitors, CLPs were unchanged. We conclude that the detection of donor-derived lymphocytes in the host peripheral blood is a relatively early event after LSK transplantation. Moreover, antibody induced neutropenia is not sufficient to induce sustainable changes in early hematopoietic fate decisions on the bone marrow level. Disclosures: No relevant conflicts of interest to declare.

Sparc Is Dispensable for Murine Hematopoiesis, Despite Its Suspected Role in 5q- Myelodysplastic Syndrome

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4822-4822
Author(s):  
Kavitha Siva ◽  
Pekka Jaako ◽  
Kenichi Miharada ◽  
Emma Rörby ◽  
Mats Ehinger ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Knockout Mice ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Lethal Dose ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Normal Peripheral Blood ◽  
Expression Levels

Abstract Abstract 4822 Hematopoiesis is a complex process where a limited number of stem cells give rise to all mature blood cells. It involves interplay of several factors, many of which are yet to be identified. In a search for novel regulators of hematopoiesis, we chose to study SPARC (Secreted Protein Acidic and Rich in Cysteine, also known as Osteonection and BM40) because it is downregulated upon hematopoietic differentiation (Bruno et al., Mol Cell Biol, 2004) and might therefore play a role in the regulation of hematopoietic stem cells (HSC). SPARC is a matricellular protein that forms a major component of bone and is ubiquitously expressed in a variety of tissues. It is the founding member of a family of SPARC-like proteins. Several publications have indicated an important role for SPARC in hematopoiesis. In particular – knockdown of SPARC in zebrafish embryos resulted in an altered number of circulating blood cells, and a knockout mouse model showed thrombocytopenia and reduced erythroid colony formation. We carried out an in depth phenotypic and functional analysis of the hematopoietic system of SPARC knockout mice; using it as a model to gain insight into the role of SPARC in hematopoiesis. These mice are viable and fertile but show severe osteopenia and age-onset cataract at about six months of age. They also show an altered response to tumour growth and wound healing. We used mice (129SVJ background) (Gilmour et al. EMBO, 1998) that were less than six months old. These mice had normal peripheral blood counts and the bone marrow and spleen showed no alterations in morphology or cellularity. A detailed phenotypic analysis of precursors within the bone marrow showed no significant differences in myelo-erythroid precursors as compared to wild types (n=6). Though in vitro, the precursors showed lower ability to form BFU-E (n=5, p=0.048). In transplantations of lethally irradiated recipient mice, SPARC knockout cells gave rise to multi-lineage long-term reconstitution. Also, when competed with wild type cells, they provided reconstitution as well as their wild type counterparts. When SPARC knockout mice (n=8) were transplanted with wild type cells, there was normal reconstitution, indicating that a SPARC deficient niche can fully support normal hematopoiesis. We also tested if SPARC deficient mice respond differently to hematopoietic stress. We subjected mice (n=7) to sub lethal dose of irradiation and to experimentally induced anemia (n=7) and followed recovery by analyzing peripheral blood counts. In both SPARC knockouts and wild type mice, the blood counts recovered in a similar fashion. In conclusion, we find that SPARC is dispensable for murine hematopoiesis. It is possible that there are compensatory mechanisms involving other members of the SPARC family that ultimately lead to normal hematopoiesis in the murine model. In humans, SPARC maps to the deleted region in 5q MDS and has been reported to be 71 % down regulated in patient samples (Lehmann et al. Leukemia, 2007). It is the most prominent gene that is up regulated in response to lenalidomide, a drug that inhibits the malignant clone (Pellagatti et al. PNAS, 2007). SPARC is thus increasingly speculated to be involved in the pathophysiology of this hematopoetic disease. We analysed the expression levels of SPARC mRNA in the hematopoietic stem/progenitor cell compartment and found high expression levels in the CD34+ fraction of human cord blood cells. In contrast, there is very low level of SPARC expression in all compartments of murine HSCs. Therefore SPARC function may play a more important role in human hematopoiesis than in murine blood cell regulation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Of Mice and Men – or a Lack of Positive Effect of Enhanced Vegetative Nervous System Tonus on Mobilization of Hematopoietic Stem and Progenitor Cells – Results in Patients with Elevated Catecholamine Level in Peripheral Blood Suffering from Acute Psychotic and Anxiety Disorders

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2454-2454
Author(s):  
Daniel Pedziwiatr ◽  
Jolanta Kucharska-Mazur ◽  
Ewa Suszynska ◽  
Marta Tkacz ◽  
Agata Poniewierska ◽  
...  
Keyword(s):  
Nervous System ◽  
Anxiety Disorders ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Control Group ◽  
Catecholamine Level ◽  
Vegetative Nervous System ◽  
Hematopoietic Stem ◽  
Peripheral Tissues ◽  
Normal Controls

Abstract Background. It is well known that hematopoietic stem/progenitor cells (HSPCs) circulate under steady-state conditions at detectable levels in peripheral blood (PB), with their numbers increasing in response to stress, inflammation, and tissue and organ injury. Moreover, it has been demonstrated in mice that enhanced tonus of vegetative nervous system regulates mobilization of HSPCs into PB. Moreover, UDP-galactose:ceramide galactosyltransferase-deficient mice, which exhibit aberrant nerve conduction and do not release norepinephrine (NE) into the BM microenvironment, do not mobilize HSPCs in response to G-CSF. However, as recently reported modification of sympathetic output does not affect G-CSF-induced mobilization in humans, as would be predicted. Specifically, normal human HSPC volunteer donors who were receiving NE reuptake inhibitors (NRI) for depression or β2-blockers because of hypertension mobilize in a similar manner as normal controls (Leukemia 2013; 27:24-31). Mobilization in these patients was neither enhanced by NRI administration nor suppressed by β2-blockers, as one would expect based on murine data reported in the literature. Aim of the study. To address this intriguing issue and discrepancy between human and mice, we analyzed levels of circulating HSPCs in patients suffering from acute psychosis and anxiety disorders – clinical situations with elevated level of catecholamine in PB. Namely, these patients are under the influence of several neural mediators, and it is well known that the levels of NE and dopamine are elevated in peripheral tissues and blood. Material and Metods. Enrolled in this study were 30 unrelated individuals with a diagnosis of the first-episode psychosis and 30 patients suffering from acute anxiety disorders. The patients were compared with an ethnic- and gender-matched control group of 35 healthy volunteers without psychiatric disorders, which were excluded according to an examination by a specialist psychiatrist. Patients with a history of serious lifetime medical events, organic brain injuries, or drug/alcohol dependence were excluded from the study. Mobilization of HSPCs was evaluated by i) FACS to enumerate the number of CD34+, CD133+, CD34+CD45+Lin–, and CD133+CD45+Lin– cells circulating in PB, which are enriched for HSPCs, as well as by ii) functional in vitro assays to detect the number of CFU-GM and BFU-E clonogenic progenitors circulating in PB. In parallel we measured level of adrenaline, norepinephrine (NE) and dopamine in PB serum. Both cells and catecholamine levels were enumerated in acute psychotic and anxiety disorders patients before and after treatment and compared with age- and sex-matched controls. Results. We did not observe any significant differences in the numbers of circulating CD34+, CD133+, CD34+CD45+Lin–, and CD133+CD45+Lin– cells as well as clonogenic BFU-E and CFU-GM between normal controls and psychotic patients and patients with anxiety disorders. In particular number of circulating in PB HSPCs was not affected by increased level of adrenaline, norepinephrine and dopamine in PB of patients suffering from acute psychotic syndromes. Conclusions. Our data argue against an effect of enhanced vegetative nervous system tone on the number of HSPCs circulating in PB in humans. Our negative data performed on patients suffering from acute psychoses and anxiety disorders somewhat corroborate data reported for normal HSPC volunteer donors that were previously treated with NRI because of depression or with β2-blockers because of high blood pressure and mobilized with G-CSF (Leukemia 2013; 27:24-31). This finding suggests that there are some clear differences between rodents and humans in the effect of the vegetative nervous system on HSPCs mobilization. Disclosures No relevant conflicts of interest to declare.

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