A lyophilized form of xenogeneic total RNA stimulates hematopoiesis in post-radiation myelosuppression

2021 ◽  
pp. 42-46
Author(s):  
Н.В. Тишевская ◽  
Н.М. Геворкян ◽  
А.А. Позина
Keyword(s):  
Bone Marrow ◽  
Comparative Analysis ◽  
Peripheral Blood ◽  
Lymphoid Cells ◽  
Male Rats ◽  
Erythroid Cells ◽  
Total Rna ◽  
Class 1 ◽  
Post Radiation ◽  
60Co Gamma Radiation

Введение. Аллогенная суммарная РНК, выделенная из клеток лимфоидных органов, стимулирует регенерацию кроветворной ткани после острого и хронического нарушения кроветворной функции. Цель исследования: 1) доказательство отсутствия ксеногенных ограничений механизмов лимфоцитарного контроля регенеративных процессов на примере гемостимулирующего действия суммарной РНК лимфоцитов бычьей селезенки в отношении кроветворения крыс, подвергшихся гамма-облучению в сублетальной дозе; 2) сравнительный анализ эффективности нативной и лиофилизированной форм указанной РНК. Методика. Работа выполнена на белых нелинейных крысах-самцах массой 200-220 г. Cуммарную РНК выделяли методом фенол - хлороформной экстракции из лимфоидных клеток бычьей селезенки. Для создания исходной миелосупрессии 30 крыс подвергли однократному общему воздействию гамма-излучения с источником 60Co в дозе 6 Гр при мощности дозы 0,1 Гр/с, после чего разделили их на 3 равные группы. Через 2 ч после облучения крысам контрольной группы внутрибрюшинно ввели по 0,5 мл 0,9% NaCl; крысам 2-й группы - нативную суммарную РНК в дозе 30 мкг/100г массы, крысам 3-й группы - лиофилизированную суммарную РНК в аналогичной дозе. На 3-и, 7-е и 12-е сут в периферической крови определяли количество ретикулоцитов, лейкоцитов и тромбоцитов, после чего крысы были выведены из эксперимента с целью исследования костномозгового кроветворения. Через 12 сут в костном мозге определяли количество эритроидных, лимфоидных, мегакариоцитарных и миелоидных клеток. Из костного мозга выделяли эритробластические островки (ЭО) и дифференцировали их на пролиферирующие (ЭО 1,2 классов и реконструирующиеся ЭО) и зрелые (ЭО 3 класса и инволюциирующие ЭО) морфо-функциональные клеточные ассоциации. Результаты. Под влиянием ксеногенной суммарной РНК в периферической крови крыс в 2-3 раза увеличилось количество лейкоцитов и в 1,6-1,75 раза возросло число ретикулоцитов. В костном мозге увеличилось количество пролиферирующих миелоидных и лимфоидных элементов, а также общее число клеток эритроидного ряда. Ксеногенная суммарная РНК стимулировала образование ЭО как на основе контакта свободных костномозговых макрофагов с молодыми эритроидными клетками (ЭО 1 и 2 классов), так и на базе реконструкции (ЭО реконструирующиеся). Сравнительный анализ эффектов нативной и лиофилизированной суммарной РНК не выявил различий между гемопоэтическими показателями у крыс, получивших эти препаратов. Заключение. Суммарная РНК, выделенная из лимфоидных клеток бычьей селезенки, активирует гемопоэз у крыс с постлучевой миелосупрессией, что свидетельствует об отсутствии ксеногенных ограничений у млекопитающих в механизмах лимфоцитарного контроля восстановительных процессов. Лиофилизированная суммарная РНК активирует костномозговое кроветворение в те же сроки и в том же объеме, что и нативная форма. Introduction. Allogeneic total RNA isolated from cells of lymphoid organs stimulates regeneration of hematopoietic tissue after acute and chronic disturbance of hematopoietic function. Aim. 1) To prove the absence of xenogeneic limitation for the lymphocytic regulation of regenerative processes using an example of the hemo-stimulating effect of total RNA from bovine spleen lymphocytes on hematopoiesis in rats exposed to sublethal gamma-irradiation; 2) To perform a comparative analysis of the effectiveness of the native and lyophilized forms of the total RNA. Methods. Experiments were performed on white outbred male rats weighing 200-220 g. Total RNA was isolated from bovine spleen lymphoid cells by phenol-chloroform extraction. To create the initial myelosuppression, 30 rats were exposed to a single general 60Co gamma radiation (6 Gy at 0.1 Gy/s). The rats were then divided into 3 equal groups. Two hrs after irradiation, the rats of the control group were injected intraperitoneally with 0.5 ml of 0.9% NaCl; rats of the second group received native total RNA, 30 μg/100 g body weight, and rats of the third group received lyophilized total RNA at a similar dose. On days 3, 7, and 12, the number of peripheral blood reticulocytes, leukocytes, and platelets was measured. The rats were then sacrificed, and bone marrow hematopoiesis was studied. After 12 days, the number of bone marrow erythroid, lymphoid, megakaryocytic, and myeloid cells was measured. Erythroblastic islets (EIs) were isolated from the bone marrow and differentiated into proliferating (class 1 and 2 EIs and reconstructing EIs) and mature (class 3 EIs and involving EIs) morpho-functional cell associations. Results. Under the influence of xenogeneic total RNA, the number of peripheral blood leukocytes increased by 2-3 times, and the number of reticulocytes increased by 1.6-1.75 times. In the bone marrow, the number of proliferating myeloid and lymphoid cells increased, as did the total number of erythroid cells. Xenogeneic total RNA stimulated formation of EIs, based both on the contact of free bone marrow macrophages with young erythroid cells (class 1 and 2 EIs) and on reconstruction (reconstructing EIs). Comparative analysis of the effects of native and lyophilized total RNA did not reveal differences between hematopoietic parameters in rats that received these agents. Conclusion. Total RNA isolated from bovine spleen lymphoid cells activates hematopoiesis in rats with post-radiation myelosuppression. This indicates the absence of mammalian xenogenic limitation of lymphocytic control of recovery processes. Lyophilized total RNA activates bone marrow hematopoiesis at the same rate and to the same extent as the native form.

scholarly journals Atypical hodgkin and reed-sternberg cells in peripheral blood of a patient with advanced stage of Hodgkin's disease - a case report

2003 ◽  
Vol 131 (9-10) ◽  
pp. 400-402 ◽  
Author(s):  
Rajko Milosevic ◽  
Milica Colovic ◽  
Vesna Cemerikic-Martinovic ◽  
Natasa Colovic ◽  
Marina Bogunovic
Keyword(s):  
Bone Marrow ◽  
Lymph Nodes ◽  
Peripheral Blood ◽  
Hodgkin's Disease ◽  
Mononuclear Cells ◽  
Hodgkin’S Disease ◽  
Lymphoid Cells ◽  
High Dose ◽  
Advanced Stage ◽  
Neck Lymph Nodes

The occurrence of abnormal Hodgkin's and Reed-Sternberg cells in the peripheral blood in a patient suffering from Hodgkin's disease has been noticed exceptionally rare in a previous period, and especially rare in last ten years primarily due to successfull treatment of this disease. The presence of atypical mononuclear cells in peripheral blood which cytomorphologically resembled Reed-Sternberg cells was registered in 8 patients till 1966. During the last decade, the presence of atypical mononuclear cells in the peripheral blood was used for their isolation cultivation, and detailed immunophenotypic and genetic analysis. The analysis of mononuclear cells in rare patients with Hodgkin's disease was established that they belong to the B-lymphoid cells with expression of CD30 and CD15 antigens. The examination of presence of Hodgkin's cells in the peripheral blood of patients with Hodgkin's disease is important for patients with advanced stage of the disease in which autologous stem cell transplantation and high dose chmeotherapy is planned. The authors present a 33-year-old patient, who noticed enlarged neck lymph nodes in September 2000, high temperature and loss in weight. On physical examination enlarged neck lymph nodes 5x8 cm and hepatosplenomegaly were found. There was anemia and thrombo-cytopenia, and normal WBC count with 24% of lymphoid elements in differential formula. On histologic examination of lymph nodes Hodgkin?s disease, type nodular sclerosis with mixed cellularity was found. Histology of bone marrow showed nodal lymphomatous infiltration. Immunohistochemistry with monoclonal antibodies of concentrate of peripheral blood cells showed expression of CD30+ and CD15+, immunophenotypically and morphologically matching Reed-Sternberg cells. Cytogentic analysis of mononuclear cells of the bone marrow showed normal karyotype. The patient was in clinical stage IV/V of the disease and chemotherapy with 9 cycles of ABVD+Mp protocol was applied. He is still in remission.

scholarly journals Administration of recombinant human interleukin-7 alters the frequency and number of myeloid progenitor cells in the bone marrow and spleen of mice

Blood ◽  
1992 ◽  
Vol 79 (5) ◽  
pp. 1121-1129 ◽  
Author(s):  
G Damia ◽  
KL Komschlies ◽  
CR Faltynek ◽  
FW Ruscetti ◽  
RH Wiltrout
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Fold Increase ◽  
Peripheral Blood Lymphocytes ◽  
Lymphoid Cells ◽  
Blood Leukocytes ◽  
Interleukin 7 ◽  
Immature B Cells ◽  
Myeloid Progenitor Cells ◽  
Phenotype Analysis

Abstract The administration of greater than or equal to 5 micrograms interleukin- 7 (IL-7) twice a day to mice for 4 to 7 days increased by twofold to fivefold the total number of splenic and peripheral blood leukocytes, but did not appreciably increase bone marrow (BM) cellularity. This regimen of IL-7 administration also resulted in a greater than 90% reduction in the frequency and total number of single lineage colony- forming unit-culture (CFU-c) and multilineage CFU-granulocyte, erythroid, monocyte, megakaryocyte colonies that could be cultured from the BM, but a fivefold to 15-fold increase in the number of these progenitors that could be cultured from the spleen. All of these effects were reversible with progenitor and white blood cell numbers returning to near normal by day 6. Morphologic analysis of cells obtained from the BM of IL-7-treated mice showed an increase in lymphoid cells. Surface phenotype analysis showed that most of this IL- 7-induced increase in lymphocytes was attributable to an increase in immature B cells (B220+, sIg-), while cells expressing the myelomonocytic markers 8C5 and MAC-1 decreased by twofold to threefold. Further studies showed that the administration of IL-7 to mice that had been rendered leukopenic by the injection of cyclophosphamide (Cy) or 5- fluorouracil (5FU) exhibited a more rapid recovery and/or overshoot in their peripheral blood lymphocytes when compared with mice treated with Cy or 5FU alone. These results show that IL-7 can differentially regulate myelopoiesis in the BM and spleen, while stimulating lymphopoiesis.

Most of the Short Term Repopulating Cells in Human Mobilized Peripheral Blood Do Not Have a Side Population (SP) Phenotype.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2867-2867
Author(s):  
M. Fischer ◽  
M. Schmidt ◽  
S. Klingenberg ◽  
C. Eaves ◽  
C. von Kalle4 ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Side Population ◽  
Isolation Procedure ◽  
Lymphoid Cells ◽  
Short Term ◽  
Hoechst 33342 ◽  
Hoechst Staining ◽  
Mobilized Peripheral Blood

Abstract The multidrug resistance transporter, ABCG2, is expressed in primitive hematopoietic stem cells from a variety of sources. These cells are detected in dual wave-length fluorescent FACS profiles as a “side population” (SP cells) on the basis of their ability to efflux the fluorescent dye, Hoechst 33342. We have previously shown that 2 types of human short term repopulating cells (STRC) can be enumerated by limiting dilution analysis of their efficient ability to regenerate exclusively myeloid cells after 3 weeks (STRC-Ms), or both myeloid and lymphoid cells after 6–12 weeks (STRC-MLs) in NOD/SCID-b2microglobulin-/- (b2m-/-) mice. Previous findings also implicated these STRCs as determinants of the rapidity of early hematologic recovery in patients transplanted with cultured mobilized peripheral blood (mPB) cells. Here we asked whether any human STRCs have an SP phenotype and hence whether the isolation of SP cells would retain the rapid repopulating activity of a clinical transplant. CD3- SP and non-SP cells were isolated by FACS from low-density (LD) mPB cells after Hoechst staining and transplanted at limiting dilutions into 117 sublethally irradiated b2m-/- mice. The numbers and types of human hematopoietic cells present in the bone marrow of these mice were subsequently monitored by FACS analysis of bone marrow cells aspirated serially, 3, 8 and 12 wks post-transplant. A verapamil-sensitive SP population was reproducibly detected in all 5 patients’ samples studied (0.039 ± 0.012% of the CD3- LD cells). The in vivo assays failed to detect either STRC-Ms or STRC-MLs in the SP fraction and all these activities were obtained from the non-SP cells. If even a single recipient of the largest dose of SP cells transplanted had been positive, this would have detected 10% of the STRCs present. Thus, >90% of all STRC-M and STRC-ML in mPB are non-SP cells. However, 4 of 40 mice transplanted with SP mPB cells produced some B-lymphoid cells only starting 12 wks post-transplant. However, this result is difficult to interpret since subjecting the STRC-Ms to the Hoechst 33342 staining and FACS isolation procedure alone eliminated their ability to generate megakaryocytic progeny in vivo, although this did not occur when these cells were just stained for CD34 and then isolated by FACS. In addition, the differentiation behaviour of STRC-MLs was not affected by the Hoechst staining and subsequent FACS isolation procedure. In summary, we demonstrate that purification of SP cells depletes human mPB transplants of STRCs, thereby raising serious concerns about the safety of any clinical use of SP cell-enriched transplants as stem cell support after myeloablation. Our results also suggest that the staining and enrichment procedure for isolating SP human cells may differentially affect the lineage potential of some types of STRCs, including those whose activity may be indispensable for rapid and multi-lineage hematologic recovery.

An In Vitro Model of Proliferating CLL Cells for Drug Sensitivity Analysis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2956-2956
Author(s):  
K. Ganeshaguru ◽  
N. I. Folarin ◽  
R. J. Baker ◽  
A. M. Casanova ◽  
A. Bhimjiyani ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Lymph Nodes ◽  
Peripheral Blood ◽  
Drug Sensitivity ◽  
In Vitro Model ◽  
Survivin Expression ◽  
Lymphoid Cells ◽  
Malignant Cells ◽  
Vitro Model

Abstract B-cell chronic lymphocytic leukaemia (CLL) is a heterogeneous disease with a variable clinical course. The disease is characterised by the proliferation in the bone marrow and lymph node of a clonal population of CD5+ve cells that accumulates in the peripheral blood. Therefore, the characteristics of the proliferative compartment are important in determining the kinetics of disease progression in CLL and the sensitivity of the malignant cells to cytotoxic drugs. However, laboratory studies on drug sensitivity of CLL have been performed exclusively on resting circulating peripheral blood cells since it is not feasible to obtain cells from the proliferating pool in sufficient numbers for in vitro analysis. CLL cells can be stimulated to proliferate in vitro using CpG oligonucleotides (ODN) and other factors. The aim of the present study was to generate and validate an in vitro model using malignant cells from the peripheral blood of patients with CLL. The expression pattern of proteins eg., survivin in this model should mimic that in proliferating CLL cells in the bone marrow and lymph nodes. Survivin is a member of the family of inhibitor of apoptosis (IAP) proteins with an additional role in cell cycle progression. Survivin has been shown to be expressed in proliferating bone marrow and lymphoid cells. Cells from patients with CLL were activated for 72h with a combination of ODN (1μM), IL-2 (100u/ml) and CD40L (0.5μg/ml) (ODN*). Activated cells retained their characteristic CLL immunophenotype as determined by the continued expression of CD5, CD19, CD23 and CD25 (n=5). Cell proliferation was confirmed by increased incorporation of 3H-thymidine into DNA in activated cells (n=12). Novel findings in the ODN* activated CLL cells were significant increases in expression of CD38 (n=7, p=0.0001) and of T-cell zeta associated protein (ZAP-70) tyrosine kinase (n=14, p=0.0005). The increased expression of both these proteins in circulating peripheral blood CLL cells has been associated with poor prognosis. All six ODN* activated CLL isolates analysed by western blotting showed increased survivin expression with no constitutive expression in the controls. Drug sensitivity was studied in cells from eight patients using the MTT assay. Activated cells showed significantly greater resistance to chlorambucil (median IC50=164.4±28.18μM) compared to control cells (median IC50=93.63±14.96μM, p=0.044). Figure 1 shows representative IC50 curves. The increased resistance of the activated cells to chlorambucil may be a consequence of the upregulation of survivin. In summary, the in vitro model replicates several key features of authentic proliferating CLL cells found in bone marrow and lymph nodes. It also shows increased resistance to the conventional drug chlorambucil. This model may be of value in evaluating novel drugs and drug combinations which may be more effective in killing the proliferating population that maintain the malignant cell population in CLL. Figure Figure

scholarly journals Subpopulations of normal peripheral blood and bone marrow cells express a functional multidrug resistant phenotype [see comments]

Blood ◽  
1992 ◽  
Vol 80 (11) ◽  
pp. 2729-2734 ◽  
Author(s):  
D Drach ◽  
S Zhao ◽  
J Drach ◽  
R Mahadevia ◽  
C Gattringer ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Flow Cytometric Analysis ◽  
Myeloid Cells ◽  
Mdr1 Gene ◽  
Glycophorin A ◽  
Erythroid Cells ◽  
Normal Peripheral Blood ◽  
Mdr1 Mrna ◽  
Mdr1 Expression

The multidrug-resistance gene, MDR1 is expressed in many normal tissues, but little is known about its expression in normal hematopoietic cells. Using the monoclonal antibody C219 and flow cytometric analysis, P-glycoprotein (P-gp) was found to be expressed in all peripheral blood (PB) subpopulations (CD4, CD8, CD14, CD19, CD56) except granulocytes. To specifically determine MDR1 gene expression, these PB subpopulations were isolated by fluorescence-activated cell sorting (FACS) and analyzed for MDR1 mRNA by polymerase chain reaction (PCR). All subsets were positive by PCR, but only minimal MDR1 mRNA was detected in monocytes and granulocytes. Significant efflux of Rhodamine- 123 (Rh-123), a measure of P-gp function, was detected in CD4+, CD8+, CD14+, CD19+, and CD56+ cells but not in granulocytes. Next, PCR- analysis was performed on FACS-sorted bone marrow (BM) cells to assess MDR1 expression in different maturational stages. Precursors (CD34+), early and late myeloid cells (CD33+/CD34+, CD33+/CD34-) as well as lymphocytes of the B-cell lineage (CD19+/CD10+, CD19+/CD10-) expressed the MDR1 gene. BM monocytic cells (CD33++/CD34-) were negative, and a very weak signal was detected in erythroid cells (glycophorin A+). Significant Rh-123 efflux was found in CD34+, CD10+, CD33+, and CD33++ BM cells, but not in glycophorin A+ cells. We conclude that PB and BM lymphocytes, PB monocytes, BM progenitors, and immature myeloid cells, but not late BM monocytes, erythroid cells, and PB granulocytes, express MDR1 mRNA and a functional P-gp. These results have to be taken into account when MDR1 expression is determined in tumor samples containing normal blood cells.

scholarly journals A special report: bone marrow transplants using volunteer donors-- recommendations and requirements for a standardized practice throughout the world--1994 update. The WMDA Executive Committee

Blood ◽  
1994 ◽  
Vol 84 (9) ◽  
pp. 2833-2839
Author(s):  
JM Goldman
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Executive Committee ◽  
Peripheral Blood ◽  
Lymphoid Cells ◽  
Bone Marrow Transplants ◽  
Special Report ◽  
The World ◽  
International Donor ◽  
Standardized Practice

A primary function of the World Marrow Donor Association is to establish general guidelines covering collaboration between international donor registries and practice in regard to bone marrow transplants (BMTs) in which the donor and recipient reside in different countries. To this end, a special report proposing specific recommendations and requirements was published in 1992. This paper amplifies the previous publication and gives special attention to accreditation of national “hubs” (defined as coordinating centers for each country) and donor, harvest, and transplant centers, details of the marrow harvest procedure, use of peripheral blood (PB) stem cells for allografting, and use of PB lymphoid cells for treatment of leukemia relapsing after BMT.

scholarly journals Expression of the YB5.B8 antigen (c-kit proto-oncogene product) in normal human bone marrow

Blood ◽  
1991 ◽  
Vol 78 (1) ◽  
pp. 30-37 ◽  
Author(s):  
LK Ashman ◽  
AC Cambareri ◽  
LB To ◽  
RJ Levinsky ◽  
CA Juttner
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Lymphoid Cells ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Hematopoietic Stem ◽  
Normal Peripheral Blood ◽  
Normal Human ◽  
Normal Human Bone Marrow

Abstract The c-kit proto-oncogene product is a member of the family of growth factor receptors with intrinsic tyrosine kinase activity. In the mouse c-kit maps to the W locus, which is known to be of central importance in hematopoiesis. Monoclonal antibody (MoAb) YB5.B8, which was raised against peripheral blood blast cells from a patient with acute myeloid leukemia (AML), was recently shown to bind to the extracellular domain of the c-kit product. This antibody does not bind detectably to normal peripheral blood cells and identifies a sub-group of AML patients with poor prognosis. We have used MoAb YB5.B8 to study the expression of c- kit by normal human bone marrow cells by immunofluorescence and flow cytometry, and to isolate multipotential and erythroid colony-forming cells. In a series of 11 normal adult bone marrow specimens, MoAb YB5.B8 bound to 4.0% +/- 1.8% of the cells in the low-density fraction. Dual-labeling experiments were performed with YB5.B8, and CD33, CD34, and CD10 MoAbs. Three populations of cells binding YB5.B8 could be identified based on their pattern of coexpression of the other markers; ie, YB5.B8+/CD34+/CD33-, YB5.B8+/CD34+/CD33+ and YB5.B8+/CD34+/CD33+. These populations had distinctive two-dimensional light scatter characteristics and are likely to correspond to precursor colony- forming cells, colony-forming cells, and maturing mast cells, respectively. No cells binding both YB5.B8 and an MoAb to the early lymphoid marker CD10 were found, implying that most early lymphoid cells do not express c-kit. MoAbs to the c-kit protein should prove valuable in multimarker studies of human hematopoietic stem and progenitor cells. Definition of a reference range of c-kit expression in normal human bone marrow will provide a sound basis for further studies of this marker in diagnosis and prognosis in AML.

The Labile Iron Pool in Normal and Pathological Erythroid Cells - Analysis by Flow Cytometry.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3597-3597
Author(s):  
Eitan Fibach ◽  
Eugenia Prus
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Peripheral Blood ◽  
Erythroid Cell ◽  
Second Phase ◽  
Erythroid Cells ◽  
Labile Iron Pool ◽  
High Affinity ◽  
Labile Iron ◽  
Iron Pool

Abstract The cellular "labile iron pool" (LIP) is made up of iron ions bound to low affinity ligands varying in composition and quantity under different physiological settings. It is localized primarily, but not exclusively, in the cytosol and, as such, is regarded as the crossroad of cellular iron traffic. The level of the pool is regulated and maintained within a restricted range that meets the cell requirements for iron but prevents excess from developing and triggering cellular damage. The LIP can be quantified due to its ability to bind to cell-permeable chelators, such as calcein-AM. Upon entering viable cells, calcein undergoes hydrolysis by esterases and becomes fluorescent. Its fluorescence is quenched upon binding to cellular LIP, the extent of which is correlated with the amount of LIP. The addition of a non-fluorescent, high affinity chelator, such as salicylaldehyde isonicotinoyl hydrazone (SIH), which removes the iron from the iron-calcein complex, increases the fluorescence emitted by the cells. The difference in the cellular fluorescence before and after incubation with the high affinity chelator reflects the amount of LIP. We adapted this procedure to multi-parameter flow cytometry for measuring LIP in erythroid cells derived from the peripheral blood, bone marrow and primary cultures. The validity of the technique was determined using K562 cells - a human erythroid cell line. Cellular fluorescence increased following incubation with calcein in a concentration- and time-dependent manner. It was further augmented by cell- permeable, high affinity iron chelators such as SIH and Deferiprone (L1), but not by desferrioxamine - an impermeable chelator. Using this method, we showed that pre-incubation of the cells with iron sources such as ferrous ammonium sulfate increased their LIP level. We then studied the LIP content in peripheral blood erythroid cells. Cells were simultaneously stained with calcein and thiazol-orange, a nucleic acid specific dye, which stains reticulocytes according to their RNA content, i.e., degree of maturation. The results indicate that the LIP content decreased (69-fold) with maturation, reaching its lowest level in mature RBC. A comparison of RBC from normal donors (N=5) and patients with β-thalassemia (N=5) indicated higher a (2.4-fold) LIP in the latter. For analysis of bone marrow samples, cells were stained with calcein and fluorochrome-conjugated antibodies to surface antigens (CD45, CD71 and glycophorin A). The results indicated that the LIP content was the highest in basophilic erythroblasts and was reversely correlated with erythroid cell maturation. Finally, we studied erythroid cells in two-phase cultures of peripheral blood-derived erythroid progenitors. Following one week in the absence of erythropoietin, the cells were re-cultured in erythropoietin-supplemented medium. Analysis of the cells on different days of the second phase showed that the LIP content decreased as the cells matured and accumulated hemoglobin. The LIP content could be modulated by changing the culture conditions: increasing by supplementing normal cultures with extra iron (in the form of hollo-transferrin) and decreasing in thalassemic cultures grown in the presence of L1 or SIH. The present findings indicate that the LIP content of erythroid cells is altered under different physiological (e.g., maturation) and pathological (e.g., iron overload, e.g., in thalassemia) conditions. The results also show that flow cytometry, a standard methodology in most hematological labs, could be useful for evaluating the LIP in various diseases and for studying the efficacy of various chelators.

Cul4A Is Required for Cell Viability and Its Deficiency in Hematopoietic Cells Causes Apoptosis and Is Fatal.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 639-639
Author(s):  
Kristin T. Chun ◽  
David L. Waning ◽  
Binghui Li ◽  
Nan Jia ◽  
Yahaira M. Naaldijk ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Small Intestine ◽  
Peripheral Blood ◽  
Hematopoietic Cells ◽  
Lymphoid Cells ◽  
Conditional Knockout ◽  
Hematopoietic Stem ◽  
Mutant Cells ◽  
Total Cellularity

Abstract Critical regulators of hematopoiesis are controlled by ubiquitin-mediated proteolysis. Cul4A encodes a core subunit of one ubiquitin ligase, and previous results with hematopoietic cell lines and with Cul4A haploinsufficient mice indicate that Cul4A is required for hematopoietic stem cell function and to maintain the homeostasis of progenitors, precursors, and mature hematopoietic cells. Because Cul4A-deficiency is embryonic lethal, we generated Cul4A conditional knockout mice to examine the requirement of Cul4A for hematopoiesis in adult mice. A mutant Cul4A allele (Cul4Aflox) was constructed where its first coding exon was flanked by loxP sites. Transgenic mice with this mutant allele and the interferon-inducible Cre transgene, Mx1-Cre, were derived. When deletion of Cul4A was induced in Cul4Aflox/flox Mx1-Cre mice, the animals died within 3–10 days of the beginning of induction. Necropsies performed four days after the beginning of induction showed that all of the tissues where Mx1-Cre was reported to be expressed appeared normal, except the bone marrow, spleen, and small intestine. The red pulp in the spleen was diminished, there were many fewer nucleated cells in the bone marrow, and the microvilli of the small intestine (duodenum) were dramatically shortened. The mass and total cellularity of mutant spleens were half of controls (Cul4Aflox/flox mice without Mx1-Cre), and bone marrow total cellularity was one-tenth of controls. The frequency of mutant hematopoietic progenitors was reduced 3800-fold in the bone marrow and 80-fold in the spleen. Peripheral blood counts of mature myeloid and lymphoid cells were also dramatically reduced. To separate the in vivo effects of Cul4A-deficiency in hematopoietic cells from those in other cell types, conditional mutant bone marrow was transplanted into wild type recipients, these cells were allowed to engraft for 2 months, and then Cul4A deletion was induced. Mutant animals died within 9–11 days of the beginning of induction with bone marrow nearly empty of cells, spleens only 29% the mass of controls, myeloid and lymphoid counts in the peripheral blood reduced to nearly zero, hematocrits at only 21% of controls, and platelet counts at only 10% of controls. The small intestine, however appeared normal, indicating that Cul4A-deficiency in hematopoietic cells is sufficient to cause death. To examine the fate of Cul4A-deficient hematopoietic cells, deletion was induced in vivo in Cul4Aflox/flox Mx1-Cre and control mice, and then bone marrow was harvested and cultured in vitro. Apoptotic cells were detected (either Annexin V positive, 7-AAD negative or TUNEL positive cells) 2–5 days after induction. At 4 and 5 days after induction, the frequency of apoptotic mutant cells was significantly greater than controls (P=0.01 and 0.03, respectively), and at 5 days the frequency of TUNEL positive cells was 4.5-fold greater in the mutant cells. Together, these results indicate that Cul4A-deficiency in hematopoietic cells results in apoptosis, a failure of the hematopoietic system, and death. Analyses of how the expression levels of Cul4A target proteins are altered by Cul4A-deficiency will be presented.

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