CD44-Mediated Adhesiveness of Human Hematopoietic Progenitors to Hyaluronan Is Modulated by Cytokines

Abstract Adhesive interactions between CD34+ hematopoietic progenitor cells (HPC) and bone marrow stroma are crucial for normal hematopoiesis, yet their molecular bases are still poorly elucidated. We have investigated whether cell surface proteoglycan CD44 can mediate adhesion of human CD34+ HPC to immobilized hyaluronan (HA), an abundant glycosaminoglycan of the bone marrow extracellular matrix. Our data show that, although CD34+ cells strongly express CD44, only 13.3% ± 1.1% spontaneously adheres to HA. Short-term methylcellulose assay showed that HA-adherent CD34+ cells comprised granulo-monocytic and erythroid committed progenitors (19.6% ± 2.5% and 7.3% ± 1.0% of the input, respectively). More primitive progenitors, such as pre–colony-forming units, also adhered to HA. Moreover, we found that CD44-mediated adhesion of CD34+ cells to HA could be enhanced by phorbol 12-myristate 13-acetate (PMA), the function-activating anti-CD44 monoclonal antibody H90, and cytokines such as granulocyte-monocyte colony-stimulating factor, interleukin-3 (IL-3), and stem cell factor. Enhancement through PMA required several hours, was protein-synthesis–dependent, and was associated with an increase of CD44 cell surface expression, whereas stimulation of adhesion by H90 monoclonal antibody and cytokines was very rapid and without alteration of CD44 expression. H90-induced activation occurred at 4°C and lasted for at least 2 hours, whereas activation by cytokines required incubation at 37°C and was transient. These data, which show for the first time that CD34+ HPC can directly adhere to HA via CD44, point out that this adhesive interaction to HA is a process that may also be physiologically regulated by cytokines.

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Human CD34+CXCR4− sorted cells harbor intracellular CXCR4, which can be functionally expressed and provide NOD/SCID repopulation

Blood ◽

10.1182/blood-2002-02-0564 ◽

2002 ◽

Vol 100 (8) ◽

pp. 2778-2786 ◽

Cited By ~ 124

Author(s):

Orit Kollet ◽

Isabelle Petit ◽

Joy Kahn ◽

Sarit Samira ◽

Ayelet Dar ◽

...

Keyword(s):

Bone Marrow ◽

Cell Surface ◽

Cord Blood ◽

Surface Expression ◽

Scid Mice ◽

Cell Surface Expression ◽

Human Cord Blood ◽

Murine Bone Marrow ◽

Human Cd34 ◽

Cd34 Cells

Homing and repopulation of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice by enriched human CD34+stem cells from cord blood, bone marrow, or mobilized peripheral blood are dependent on stromal cell-derived factor 1 (SDF-1)/CXCR4 interactions. Recently, human cord and fetal blood CD34+CD38−CXCR4− and CXCR4+ cells, sorted with neutralizing anti-CXCR4 monoclonal antibody (mAb), were shown to have similar NOD/SCID repopulation potential. Herein we report that human cord blood CD34+CXCR4+ (R4+) and CD34+CXCR4− (R4−) subsets, sorted with neutralizing anti-CXCR4 mAb, engrafted NOD/SCID mice with significantly lower levels of human cells compared with nonsorted and SDF-1–migrated CD34+ cells. Coinjection of purified cells with 10 μg anti-CXCR4 mAb significantly reduced engraftment of all CD34+ subsets, and 50 μg completely abrogated engraftment by R4− and CD34+ cells. Importantly, R4− cells harbor intracellular CXCR4, which can be rapidly induced to cell surface expression within a few hours. Moreover, 48 hours of cytokine stimulation resulted in up-regulation of both cell surface and intracellular CXCR4, restoring migration capacities toward a gradient of SDF-1 and high-level NOD/SCID repopulation potential. In addition, homing of sorted R4− cells into the murine bone marrow and spleen was significantly slower and reduced compared to CD34+ cells but yet CXCR4 dependent. In conclusion, R4− cells express intracellular CXCR4, which can be functionally expressed on the cell membrane to mediate SDF-1–dependent homing and repopulation. Our results suggest dynamic CXCR4 expression on CD34+ stem and progenitor cells, regulating their motility and repopulation capacities.

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Ku80 autoantigen as a cellular coreceptor for human parvovirus B19 infection

Blood ◽

10.1182/blood-2005-02-0536 ◽

2005 ◽

Vol 106 (10) ◽

pp. 3449-3456 ◽

Cited By ~ 109

Author(s):

Yasuhiko Munakata ◽

Takako Saito-Ito ◽

Keiko Kumura-Ishii ◽

Jie Huang ◽

Takao Kodera ◽

...

Keyword(s):

Bone Marrow ◽

Cell Surface ◽

Cell Lines ◽

Bone Marrow Cells ◽

Parvovirus B19 ◽

Surface Expression ◽

Cell Surface Expression ◽

Human Parvovirus B19 ◽

Erythroid Cells ◽

Interfering Rna

AbstractHuman parvovirus B19 (B19) infects human erythroid cells expressing P antigen. However, some cell lines that were positive for P antigen failed to bind B19, whereas some cell lines had an ability to bind B19 despite undetectable expression of P antigen. We here demonstrate that B19 specifically binds with Ku80 autoantigen on the cell surface. Furthermore, transfection of HeLa cells with the gene of Ku80 enabled the binding of B19 and allowed its entry into cells. Moreover, reduction of cell-surface expression of Ku80 in KU812Ep6 cells, which was a high-sensitive cell line for B19 infection, by short interfering RNA for Ku80 resulted in the marked inhibition of B19 binding in KU812Ep6 cells. Although Ku80 originally has been described as a nuclear protein, human bone marrow erythroid cells with glycophorin A or CD36, B cells with CD20, or T cells with CD3 were all positive for cell-surface expression of Ku80. B19 infection of KU812Ep6 cells and bone marrow cells was inhibited in the presence of anti-Ku80 antibody. Our data suggest that Ku80 functions as a novel coreceptor for B19 infection, and this finding may provide an explanation for the pathologic immunity associated with B19 infection.

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Identification of a Novel Auto-Antibody Highly Prevalent in Patients with Hepatitis-Associated and Idiopathic Aplastic Anemia.

Blood ◽

10.1182/blood.v114.22.3200.3200 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3200-3200

Author(s):

Hiroyuki Takamatsu ◽

Zhirong Qi ◽

Tomoyuki Sakurai ◽

Luis Espinoza ◽

Naomi Sugimori ◽

...

Keyword(s):

Cell Surface ◽

Cell Lines ◽

Leukemia Cell ◽

Peptide Mass Fingerprinting ◽

Surface Expression ◽

Cell Surface Expression ◽

Healthy Individuals ◽

Peptide Mass ◽

Leukemia Cell Lines ◽

Cd34 Cells

Abstract Abstract 3200 Poster Board III-137 Hepatitis-associated aplastic anemia (HAA) is a subset of acquired AA that is highly responsive to immunosuppressive therapy. The target antigens of the immune system attack in HAA are thought to be a protein shared by both liver and hematopoietic stem cells, since it is usually associated with severe hepatitis of unknown etiology. Screening sera from patients with HAA for the presence of antibodies (Abs) recognizing liver cell-derived proteins may be useful in identifying novel auto-antigens in AA. To test this hypothesis, sera from HAA patients were examined using immunoblotting with a lysate of a hepatocellular carcinoma cell line Huh7 and subsequent peptide mass fingerprinting. Methods and Results The serum of a patient with typical HAA (a 23 year-old male) possessing a small population of paroxysmal nocturnal hemoglobinuria (PNH)-type cells was used for Western blotting (WB) with the lysates of Huh7. A distinct band of 70 kDa protein was revealed. The same band was revealed when the culture supernatant of Huh7 cells was subjected to WB. The peptide mass fingerprinting of the 70 kDa band identified this protein to be heat shock protein (HSP) 72. HSP72 is a stress-inducible protein and extracellular HSP72 enhances the cytotoxicity of CD4+ T cells and NK cells. An examination of the sera from HAA patients, idiopathic acquired AA (IAA) patients and healthy individuals with WB revealed the anti-HSP72 Abs to be detected in 10 of 12 (83%) HAA patients and in 57 of 80 (71%) IAA patients while it was detected only in 8 of 59 (14%) healthy individuals. The prevalence of anti-HSP72 Abs in AA was markedly higher than that of anti-kinectin Abs (39%), anti-PMS1 Abs (10%), anti-DRS-1 Abs (38%) or anti-moesin Abs (37%) reported previously. Anti-HSP72 Abs were frequently detectable both in patients with IAA possessing PNH-type cells (63%) and in patients without PNH-type cells (86%), a finding contrasting to the higher prevalence of anti-DRS-1 Abs and anti-moesin Abs in patients with PNH-type cells than in those without PNH-type cells reported previously. Although anti-HSP72 Abs were detectable in the sera of patients with rheumatoid arthritis and systemic lupus erythematosus, the prevalence was 15% (4 of 27) and 20% (1 of 5), respectively. In contrast to a previous report that detected anti-HSP72 Abs in 24% of patients with chronic hepatitis C, WB failed to detect the Abs in the sera of 4 patients with autoimmune hepatitis and 5 with hepatitis B or C. Ten patients with HAA were treated with immunosuppressive therapy, and 7 of the 8 responders expressed anti-HSP72 Abs. The quantification of the gene expression level of HSP72 by blood cells using real-time PCR demonstrated that the HSP72 mRNA levels were markedly higher in myeloid leukemia cell lines as well as CD34+ cells isolated from 3 healthy individuals in comparison to that in lymphoid or monocytoid leukemia cell lines. HSP72/GAPDH ratios of PBMCs and CD34+ cells from 3 healthy individuals, K562, KH88, OUN-1 were 0.51, 1.31, 1.02, 0.07 and 0.09 respectively. Other leukemia cell lines such as Daudi, Molt-4 and THP-1 did not display detectable levels of HSP72 mRNA. The cell surface expression of HSP72 was examined in various kinds of leukemia cell lines and CD34+ bone marrow (BM) cells derived from 3 healthy individuals using Ab to HSP72 (Clone C92F3A-5) because previous studies demonstrated heat-inducible expression of HSP72 by K562. Flow cytometry detected cell surface HSP72 on immature CML cell lines such as K562 but not on CD34+ BM cells, acute promyelocytic leukemia cell lines such as NB-4 and HL-60, and lymphoid leukemia cell lines such as Molt-4 and Daudi. Exposure to 42°C for 2 h increased the HSP72 expression on K562 cells and Molt-4 cells but not on CD34+ cells. Conclusion Anti-HSP72 Ab is the most prevalent auto-Ab in AA among the auto-Abs previously detected. Given the increased expression of HSP72 by immature myeloid cells as well as stress-inducible cell surface expression of the molecule, immune responses to HSP72 may thus play an essential role in the pathogenesis of HAA and IAA. Disclosures No relevant conflicts of interest to declare.

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Cell Surface Expression of ICAM-1 (CD54) and LFA-3 (CD58), Two Adhesion Molecules, Is Up-Regulated on Bone Marrow Leukemic Blasts After In Vivo Administration of High-Dose Recombinant Interleukin-2

Journal of Immunotherapy ◽

10.1097/00002371-199112000-00004 ◽

1991 ◽

Vol 10 (6) ◽

pp. 412-417 ◽

Cited By ~ 11

Author(s):

Daniel Olive ◽

Marc Lopez ◽

Didier Blaise ◽

Patrice Viens ◽

Anne-Marie Stoppa ◽

...

Keyword(s):

Bone Marrow ◽

Cell Surface ◽

Adhesion Molecules ◽

Interleukin 2 ◽

Surface Expression ◽

Cell Surface Expression ◽

High Dose ◽

Recombinant Interleukin 2 ◽

In Vivo Administration

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Cholangiocyte expression of α2β1-integrin confers susceptibility to rotavirus-induced experimental biliary atresia

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00442.2007 ◽

2008 ◽

Vol 295 (1) ◽

pp. G16-G26 ◽

Cited By ~ 30

Author(s):

Mubeen Jafri ◽

Bryan Donnelly ◽

Steven Allen ◽

Alex Bondoc ◽

Monica McNeal ◽

...

Keyword(s):

Monoclonal Antibody ◽

Cell Surface ◽

Biliary Atresia ◽

Cell Lines ◽

Surface Expression ◽

Cell Surface Expression ◽

Newborn Mice ◽

A Cell

Inoculation of BALB/c mice with rhesus rotavirus (RRV) in the newborn period results in biliary epithelial cell (cholangiocyte) infection and the murine model of biliary atresia. Rotavirus infection of a cell requires attachment, which is governed in part by cell-surface expression of integrins such as α2β1. We hypothesized that cholangiocytes were susceptible to RRV infection because they express α2β1. RRV attachment and replication was measured in cell lines derived from cholangiocytes and hepatocytes. Flow cytometry was performed on these cell lines to determine whether α2β1 was present. Cholangiocytes were blocked with natural ligands, a monoclonal antibody, or small interfering RNA against the α2-subunit and were infected with RRV. The extrahepatic biliary tract of newborn mice was screened for the expression of the α2β1-integrin. Newborn mice were pretreated with a monoclonal antibody against the α2-subunit and were inoculated with RRV. RRV attached and replicated significantly better in cholangiocytes than in hepatocytes. Cholangiocytes, but not hepatocytes, expressed α2β1 in vitro and in vivo. Blocking assays led to a significant reduction in attachment and yield of virus in RRV-infected cholangiocytes. Pretreatment of newborn pups with an anti-α2 monoclonal antibody reduced the ability of RRV to cause biliary atresia in mice. Cell-surface expression of the α2β1-integrin plays a role in the mechanism that confers cholangiocyte susceptibility to RRV infection.

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Neutrophils express CD52 and exhibit complement-mediated lysis in the presence of alemtuzumab

Blood ◽

10.1182/blood-2009-02-203075 ◽

2009 ◽

Vol 114 (14) ◽

pp. 3052-3055 ◽

Cited By ~ 37

Author(s):

Lyn R. Ambrose ◽

Anne-Sophie Morel ◽

Anthony N. Warrens

Keyword(s):

Monoclonal Antibody ◽

Adverse Event ◽

Cell Surface ◽

Surface Expression ◽

Cell Surface Expression ◽

Disease States ◽

Dose Dependent

Neutropenia is a recognized adverse event in patients treated with the humanized anti-CD52 monoclonal antibody alemtuzumab. However, as it is widely believed that neutrophils do not express CD52, the etiology of alemtuzumab-associated neutropenia is unclear. We have found that neutrophils express both mRNA coding for CD52 and the protein itself on the cell surface. We confirmed cell-surface expression using 3 different anti-CD52 antibodies, and note that neutrophils express lower levels of CD52 than lymphocytes and eosinophils. Further, incubation of alemtuzumab with neutrophils results in dose-dependent, complement-mediated lysis in the presence of both heterologous and autologous complement. These data offer an explanation for the etiology of alemtuzumab-associated neutropenia. In a climate of increased use of alemtuzumab in leukemia and other disease states, as well as in transplantation, these data highlight the need for increased vigilance of emerging neutropenia in patients treated with alemtuzumab.

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Reversibility of CD34 expression on human hematopoietic stem cells that retain the capacity for secondary reconstitution

Blood ◽

10.1182/blood-2002-01-0025 ◽

2003 ◽

Vol 101 (1) ◽

pp. 112-118 ◽

Cited By ~ 71

Author(s):

Mo A. Dao ◽

Jesusa Arevalo ◽

Jan A. Nolta

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cell Surface ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cd34 Cells ◽

Cd34 Expression

Abstract The cell surface protein CD34 is frequently used as a marker for positive selection of human hematopoietic stem/progenitor cells in research and in transplantation. However, populations of reconstituting human and murine stem cells that lack cell surface CD34 protein have been identified. In the current studies, we demonstrate that CD34 expression is reversible on human hematopoietic stem/progenitor cells. We identified and functionally characterized a population of human CD45+/CD34− cells that was recovered from the bone marrow of immunodeficient beige/nude/xid (bnx) mice 8 to 12 months after transplantation of highly purified human bone marrow–derived CD34+/CD38− stem/progenitor cells. The human CD45+ cells were devoid of CD34 protein and mRNA when isolated from the mice. However, significantly higher numbers of human colony-forming units and long-term culture-initiating cells per engrafted human CD45+ cell were recovered from the marrow of bnx mice than from the marrow of human stem cell–engrafted nonobese diabetic/severe combined immunodeficient mice, where 24% of the human graft maintained CD34 expression. In addition to their capacity for extensive in vitro generative capacity, the human CD45+/CD34− cells recovered from thebnx bone marrow were determined to have secondary reconstitution capacity and to produce CD34+ progeny following retransplantation. These studies demonstrate that the human CD34+ population can act as a reservoir for generation of CD34− cells. In the current studies we demonstrate that human CD34+/CD38− cells can generate CD45+/CD34− progeny in a long-term xenograft model and that those CD45+/CD34− cells can regenerate CD34+ progeny following secondary transplantation. Therefore, expression of CD34 can be reversible on reconstituting human hematopoietic stem cells.

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Immunotherapeutic Targeting of TSLPR with Chimeric Antigen Receptor T Cells in AML

Blood ◽

10.1182/blood-2021-153815 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2789-2789

Author(s):

Lindsey F Call ◽

Sommer Castro ◽

Thao T. Tang ◽

Cynthia Nourigat-Mckay ◽

LaKeisha Perkins ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Cell Surface ◽

Peripheral Blood ◽

Surface Expression ◽

Cell Surface Expression ◽

Car T Cells ◽

Car T

Abstract Adoptive transfer of T cells engineered to express chimeric antigen receptors (CARs) has achieved impressive outcomes in the treatment of refractory/relapsed B-ALL, providing potentially curative treatment options for these patients. The use of CAR T in AML, however, is still in its infancy with limitations due to the innate heterogeneity associated with AML and the lack of AML-specific targets for therapeutic development. The CRLF2 gene encodes for thymic stromal lymphopoietin receptor (TSLPR) and has previously been shown to be highly upregulated in a subset of children and adults with B-ALL. Targeting TSLPR with CAR T cells demonstrates potent anti-leukemia activity against TSLPR-positive B-ALL (PMID 26041741). Through Target Pediatric AML (TpAML), we profiled the transcriptome of nearly 3000 children and young adults with AML and identified CRLF2 (TSLPR) to be highly expressed in a subset of AML, including the majority of AML harboring KM2TA (aka MLL) fusions. TSLPR cell surface expression was validated in primary patient samples using flow cytometry, which showed uniform expression of TSLPR on AML blasts. Given that TSLPR is expressed in AML with confirmed cell surface expression, we developed TSLPR-directed CAR T for preclinical evaluation in AML. We generated a TSLPR-directed CAR using the single-chain variable fragment (scFv) derived from an anti-TSLPR binder (clone 3G1, MD Anderson), IgG4 spacer and 41-BB/CD3zeta signaling domains. The in vitro cytotoxicity of TSLPR CAR T cells was evaluated against the REH-1 cell line and primary AML specimens. TSLPR CAR T cells demonstrated anti-leukemia activity against REH-1 as well as against primary AML specimens. To evaluate the in vivo efficacy of TSLPR CAR T cells, we developed a patient-derived xenograft (PDX) model using bone marrow cells from a TSLPR-positive patient. These cells provided a robust model system to evaluate the in vivo activity of TSLPR CAR T cells, as they produced an aggressive leukemia in humanized NSG-SGM3 mice. The PDX generated from these cells died within 2 months of transplant with significant leukemia infiltration into the bone marrow, liver, and spleen. In the in vivo study, the leukemia burden was assessed by flow cytometric analysis of AML cells in the peripheral blood and bone marrow aspirates following treatment with unmodified control or TSLPR CAR T cells given at 10x10 6 T cells per mouse. After CAR T treatment, we detected a significant decrease in leukemia infiltration into the peripheral blood and bone marrow in the CAR T-treated mice compared to mice that received unmodified T cells. In this study, we report that similar to B-ALL, CRLF2 (TSLPR) is overexpressed in a subset of AML, providing a strategy to eliminate AML cells with CAR T cell therapy. We validated the cell surface expression of TSLPR and showed that the expression is uniform across AML specimens. We further demonstrate that CAR T cells targeting TSLPR were effective in eliminating AML cells in vitro and in vivo. Given that TSLPR is highly expressed in the KMT2A-rearranged AML, a subtype that is associated with poor outcomes, TSLPR-directed CAR T cells represent a promising immunotherapy for this high-risk AML subset. Disclosures Pardo: Hematologics, Inc.: Current Employment.

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Defects of the Tpo-Receptor, Mpl, Caused by Mutations Found in CAMT Patients.

Blood ◽

10.1182/blood.v106.11.2181.2181 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2181-2181

Author(s):

Marloes R. Tijssen ◽

Franca di Summa ◽

Sonja Van den Oudenrijn ◽

Carlijn Voermans ◽

C.Ellen Van der Schoot ◽

...

Keyword(s):

Bone Marrow ◽

Amino Acid ◽

Cell Surface ◽

Cell Line ◽

Mrna Level ◽

K562 Cells ◽

Surface Expression ◽

Cell Surface Expression ◽

Amino Acid Substitutions ◽

Bhk Cells

Abstract Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare disorder that presents with severe thrombocytopenia and absence of megakaryocytes in the bone marrow. The disease may develop into bone marrow aplasia. In vitro, CD34-positive hematopoietic progenitor cells from CAMT patients did not show any megakaryocyte formation in a Tpo-driven expansion culture. We and others found genetic defects in the gene encoding the Tpo receptor, c-mpl (Van den Oudenrijn et al., Br J Haematol.2002, 117: 390–398 and Ballmaier et al., Ann N Y Acad Sci.2003, 996: 17–25). In our patients, we found four mutations that predicted amino-acid substitutions, of which three in the extracellular domain; Arg102Pro, Pro136His and Arg257Cys, and one in the intracellular signaling domain (Pro635Leu), which may result in either defective Tpo-binding and/or signaling. To investigate this, we transfected full-length Mpl (wt and mutants) into the erythroleukemic cell line K562 and truncated Mpl (encompassing the extracellular domain; wt and mutants) into Baby Hamster Kidney (BHK) cells. In the K562 cells, the mRNA level (RQ-PCR) of the Pro136His mutant was severely decreased compared to the wt transfectant, while the mRNA level of the other mutants was comparable to that of wt. On Western blot, wt Mpl migrated as two, presumably differently glycosylated, bands of 75 kD and 72 kD. The mutants showed an altered migration pattern, which might result from differences in glycosylation. With the Pro635Leu mutant lower signals were obtained when equal amounts of total protein were loaded. Since the Mpl mRNA level was comparable to that of wt, this suggests a higher level of protein degradation. Upon transfection of the Arg102Pro and the Arg257Cys mutants in BHK cells, we observed that these mutants did not gain endo-H resistency, which suggests an aberrant processing of these mutant Mpls through the Golgi apparatus and retention in the ER. However, in cell fractionation experiments with surface-biotinylated K562 cells, biotinylated wt Mpl and mutant Mpl (except Pro136His) could be detected. Apparently, in K562 cells, the amino-acid substitutions do not impair membrane expression completely. To examine whether the mutant receptors were still able to signal after Tpo incubation, K562 cells were serum-starved and subsequently stimulated with 50 ng/ml rhTpo for 5 to 30 minutes. All mutants, including Pro136His, showed increased ERK phosphorylation after 5 minutes. To summarize, the Pro136His mutant is hardly expressed in the K562 expression model, presumably because of instability of the mRNA, but is still able to induce signaling. In contrast to the results obtained in the BHK model, the Arg102Pro and Arg257Cys mutants, showed cell-surface expression in the K562 cell line. The obtained cell-surface expression in the K562 model may have been significantly increased compared to the in vivo situation on hematopoietic stem cells, because of artificially induced efficient expression. Finally, with a super-physiological concentration of rhTpo, we obtained evidence that all Mpl mutants were able to signal upon Tpo binding. Whether impaired signaling by the Mpl mutants in the presence of physiological levels of Tpo may contribute to the development of CAMT, will be investigated.

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