Effect of Campath-1H antibody on human hematopoietic progenitors in vitro

The humanized antibody CAMPATH-1H has been shown in pilot studies to be beneficial in the treatment of lymphoid malignancy and other lymphoproliferative diseases. The antigen recognized by this antibody is not confined to lymphoid cells, and work with rat antibodies of similar specificity has not eliminated the possibility of damage to human hematopoietic progenitors, particularly those capable of repopulating bone marrow and sustaining hematopoiesis. This study aimed to discover if hematopoietic progenitor cells were affected by treatment with CAMPATH-1H, with or without human complement. Bone marrow mononuclear cells from healthy volunteers were treated with saturating concentrations of CAMPATH-1H, human complement, or CAMPATH- 1H plus human complement. The CD34-positive fraction of the mononuclear cells was treated similarly. Residual progenitor activity was measured in the colony-forming unit-granulocyte, erythroid, monocyte, megakaryocyte assay and compared with untreated controls. There was no significant difference (at the 5% level) between treated and control cells. Mononuclear cells were divided into CAMPATH-1H-positive and CAMPATH-1H-negative fractions by fluorescein isothiocyanate-CAMPATH-1H labeling and fluorescence-activated cell sorter separation. Hematopoietic progenitors were predominantly found in the CAMPATH-1H- negative fraction. Furthermore, mononuclear cells treated with CAMPATH- 1H and complement were equivalent to controls in experiments that investigated the capacity of these cells to form hematopoietic foci in long-term cultures.

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Effect of Campath-1H antibody on human hematopoietic progenitors in vitro

Blood ◽

10.1182/blood.v82.3.807.807 ◽

1993 ◽

Vol 82 (3) ◽

pp. 807-812 ◽

Cited By ~ 160

Author(s):

MH Gilleece ◽

TM Dexter

Keyword(s):

Bone Marrow ◽

Mononuclear Cells ◽

Fluorescein Isothiocyanate ◽

Lymphoid Cells ◽

Human Complement ◽

Hematopoietic Progenitors ◽

Pilot Studies ◽

Significant Difference ◽

And Control

Abstract The humanized antibody CAMPATH-1H has been shown in pilot studies to be beneficial in the treatment of lymphoid malignancy and other lymphoproliferative diseases. The antigen recognized by this antibody is not confined to lymphoid cells, and work with rat antibodies of similar specificity has not eliminated the possibility of damage to human hematopoietic progenitors, particularly those capable of repopulating bone marrow and sustaining hematopoiesis. This study aimed to discover if hematopoietic progenitor cells were affected by treatment with CAMPATH-1H, with or without human complement. Bone marrow mononuclear cells from healthy volunteers were treated with saturating concentrations of CAMPATH-1H, human complement, or CAMPATH- 1H plus human complement. The CD34-positive fraction of the mononuclear cells was treated similarly. Residual progenitor activity was measured in the colony-forming unit-granulocyte, erythroid, monocyte, megakaryocyte assay and compared with untreated controls. There was no significant difference (at the 5% level) between treated and control cells. Mononuclear cells were divided into CAMPATH-1H-positive and CAMPATH-1H-negative fractions by fluorescein isothiocyanate-CAMPATH-1H labeling and fluorescence-activated cell sorter separation. Hematopoietic progenitors were predominantly found in the CAMPATH-1H- negative fraction. Furthermore, mononuclear cells treated with CAMPATH- 1H and complement were equivalent to controls in experiments that investigated the capacity of these cells to form hematopoietic foci in long-term cultures.

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Removal of T cells from bone marrow for transplantation: a monoclonal antilymphocyte antibody that fixes human complement

Blood ◽

10.1182/blood.v62.4.873.bloodjournal624873 ◽

1983 ◽

Vol 62 (4) ◽

pp. 873-882 ◽

Cited By ~ 5

Author(s):

G Hale ◽

S Bright ◽

G Chumbley ◽

T Hoang ◽

D Metcalf ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Mononuclear Cells ◽

Lymphoid Cells ◽

Human Complement ◽

Peripheral Blood Mononuclear ◽

Graft Versus Host ◽

Antilymphocyte Antibody ◽

In Vitro Treatment

Graft-versus-host disease is one of the major problems in clinical bone marrow transplantation. Many experiments in animals have shown that it could be greatly reduced if mature T lymphocytes were removed from the donor marrow. Here we describe a new rat monoclonal antibody, CAMPATH 1, which is suitable for depleting lymphocytes from human marrow grafts. CAMPATH 1 is an IgM that fixes human complement. It binds to both T and B lymphocytes and some monocytes but not to other hemopoietic cells. When peripheral blood mononuclear cells were treated with CAMPATH 1 and complement, more than 99% of lymphocytes were killed and viable T cells could no longer be detected. Under these conditions, in vitro multipotential erythroid and myeloid colony-forming cells were unaffected. As well as being used for in vitro treatment of bone marrow to remove T cells, CAMPATH 1 could potentially be applied to other experimental and clinical situations where depletion of lymphoid cells is required, including serotherapy to achieve immunosuppression for organ transplants or to treat lymphocytic leukemias.

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Removal of T cells from bone marrow for transplantation: a monoclonal antilymphocyte antibody that fixes human complement

Blood ◽

10.1182/blood.v62.4.873.873 ◽

1983 ◽

Vol 62 (4) ◽

pp. 873-882 ◽

Cited By ~ 271

Author(s):

G Hale ◽

S Bright ◽

G Chumbley ◽

T Hoang ◽

D Metcalf ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Mononuclear Cells ◽

Lymphoid Cells ◽

Human Complement ◽

Peripheral Blood Mononuclear ◽

Graft Versus Host ◽

Antilymphocyte Antibody ◽

In Vitro Treatment

Abstract Graft-versus-host disease is one of the major problems in clinical bone marrow transplantation. Many experiments in animals have shown that it could be greatly reduced if mature T lymphocytes were removed from the donor marrow. Here we describe a new rat monoclonal antibody, CAMPATH 1, which is suitable for depleting lymphocytes from human marrow grafts. CAMPATH 1 is an IgM that fixes human complement. It binds to both T and B lymphocytes and some monocytes but not to other hemopoietic cells. When peripheral blood mononuclear cells were treated with CAMPATH 1 and complement, more than 99% of lymphocytes were killed and viable T cells could no longer be detected. Under these conditions, in vitro multipotential erythroid and myeloid colony-forming cells were unaffected. As well as being used for in vitro treatment of bone marrow to remove T cells, CAMPATH 1 could potentially be applied to other experimental and clinical situations where depletion of lymphoid cells is required, including serotherapy to achieve immunosuppression for organ transplants or to treat lymphocytic leukemias.

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Identification and characterization of osteoclast progenitors by clonal analysis of hematopoietic cells

Blood ◽

10.1182/blood.v80.7.1710.1710 ◽

1992 ◽

Vol 80 (7) ◽

pp. 1710-1716 ◽

Cited By ~ 21

Author(s):

MY Lee ◽

JL Lottsfeldt ◽

KL Fevold

Keyword(s):

Bone Marrow ◽

Tumor Cell ◽

Mononuclear Cells ◽

Fetal Liver ◽

Tumor Cell Line ◽

Adherent Cell ◽

Mouse Bone Marrow ◽

Hematopoietic Progenitors ◽

Adult Mice

Abstract We have identified a distinct population of colony-forming cells that give rise to mononuclear cells expressing an enzyme marker and other features of the osteoclast in bone marrow cultures stimulated by conditioned medium of a murine tumor cell line. These colony-forming cells were defined as osteoclast colony-forming units (CFU-O). The tumor cell-derived activity was recently isolated and was named osteoclast colony-stimulating factor (O-CSF). To understand the development of osteoclast progenitors and to clarify the relationship of osteoclast progenitors to other hematopoietic progenitors, we examined CFU-O in hematopoietic tissues obtained from normal adult mice, mouse fetuses, and mice with 5-fluorouracil (5FU) treatment. CFU- O were present in the adult mouse bone marrow, adherent cell-depleted marrow, in the spleen, and in the day 14 fetal liver, with an incidence similar to other hematopoietic progenitors. The culture period required for the development of CFU-O-derived colonies in vitro and the manner in which CFU-O responded to 5FU suggested that CFU-O belonged to a relatively primitive progenitor population; they are clearly more immature than macrophage progenitors that respond to macrophage-CSF, but more mature than multilineage progenitors that respond to stem cell factor. Our studies have defined and characterized an osteoclast progenitor and distinguished it from other hematopoietic progenitors for the first time.

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Three-Dimensional Reconstructed Bone Marrow Matrix Culture Improves the Viability of Primary Myeloma Cells In-Vitro via a STAT3-Dependent Mechanism

Current Issues in Molecular Biology ◽

10.3390/cimb43010026 ◽

2021 ◽

Vol 43 (1) ◽

pp. 313-323

Author(s):

Yung-Hsing Huang ◽

Meaad Almowaled ◽

Jing Li ◽

Christopher Venner ◽

Irwindeep Sandhu ◽

...

Keyword(s):

Bone Marrow ◽

Cell Viability ◽

Mononuclear Cells ◽

Three Dimensional ◽

3D Culture ◽

Trypan Blue Exclusion ◽

Conventional Culture ◽

Significant Difference ◽

Dependent Mechanism

Primary myeloma (PM) cells are short-lived in conventional culture, which limited their usefulness as a study model. Here, we evaluated if three-dimensional (3D) culture can significantly prolong the longevity of PM cells in-vitro. We employed a previously established 3D model for culture of bone marrow mononuclear cells isolated from 15 patients. We assessed the proportion of PM cells, viability and proliferation using CD38 staining, trypan blue exclusion assays and carboxy fluorescein succinimidyl ester (CFSE) staining, respectively. We observed significantly more CD38+ viable cells in 3D than in conventional culture (65% vs. 25%, p = 0.006) on day 3. CFSE staining showed no significant difference in cell proliferation between the two culture systems. Moreover, we found that PM cells in 3D culture are more STAT3 active by measure of pSTAT3 staining (66% vs. 10%, p = 0.008). Treatment of IL6, a STAT3 activator significantly increased CD38+ cell viability (41% to 68%, p = 0.021). In comparison, inhibition of STAT3 with Stattic significantly decreased PM cell viability in 3D culture (38% to 17% p = 0.010). Neither IL6 nor Stattic affected the PM cell viability in conventional culture. This study suggests that 3D culture can significantly improve the longevity of PM cells in-vitro, and STAT3 activation can further improve their viability.

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Lead Impairs the Development of Innate Lymphoid Cells by Impeding the Differentiation of Their Progenitors

Toxicological Sciences ◽

10.1093/toxsci/kfaa074 ◽

2020 ◽

Vol 176 (2) ◽

pp. 410-422 ◽

Cited By ~ 1

Author(s):

Tingting Zhu ◽

Yifan Zhao ◽

Peng Zhang ◽

Yiming Shao ◽

Jinyi He ◽

...

Keyword(s):

Bone Marrow ◽

Mononuclear Cells ◽

Human Subjects ◽

Innate Lymphoid Cells ◽

Lymphoid Cells ◽

In Vitro Assays ◽

Peripheral Blood Mononuclear ◽

Occupationally Exposed ◽

Pb Exposure

Abstract Lead (Pb) is a heavy metal toxic to the immune system, yet the influence of Pb on innate lymphoid cells (ILC) remains to be defined. In this study, we found that occupationally relevant level of Pb exposure impaired ILC development at the progenitor level by activating Janus Kinase1. C57BL/6 mice treated with 1250 ppm, but not 125 ppm Pb acetic via drinking water for 8 weeks had reduced number of mature ILC, which was not caused by increased apoptosis or suppressed proliferation. Conversely, Pb increased the number of innate lymphoid cell progenitors (ILCP) in the bone marrow. The discordant observation indicated that an obstruction of ILCP differentiation into mature ILC during Pb exposure existed. Pb directly acted on ILCP to suppress their proliferation, indicating that ILCP were less activated during Pb exposure. Reciprocal ILCP transplantation assay confirmed that Pb impeded the differentiation of ILCP into mature ILC, as ILCP gave rise to fewer mature ILC in Pb-treated recipients compared with control recipients. In vitro assays suggested that the obstruction of ILCP differentiation by Pb exposure was due to increased activation of Janus Kinase1. Thus, Pb impeded ILCP differentiation into mature ILC to result in an accumulation of ILCP in the bone marrow and the resultant decreased number of mature ILC in lymphoid and nonlymphoid tissues in mice. Moreover, by analyses of ILC and ILCP in peripheral blood mononuclear cells of human subjects occupationally exposed to Pb, we revealed that Pb might also impede the development of ILC in human.

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Tumor-Activated Human NK Cells Specificall Ylyse Autologous and Allogeneic NK-Resistant Tumor Targets.

Blood ◽

10.1182/blood.v108.11.3707.3707 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3707-3707

Author(s):

Mark W. Lowdell ◽

Ismail Bakhsh ◽

Janet North ◽

Chloe Marden ◽

Elena Addison ◽

...

Keyword(s):

Breast Cancer ◽

Bone Marrow ◽

Nk Cells ◽

Tumor Cells ◽

Mononuclear Cells ◽

Leukemia Cell ◽

Lymphokine Activated Killer Cells ◽

Normal Peripheral Blood ◽

Significant Difference

Abstract Pre-incubation of resting human NK cells with the leukemia cell CTV-1 primes NK cells to lyse NK-resistant cell lines, primary leukemias and solid tumors, even when HLA-matched; allogeneic or autologous. The primed NK cells remained non-responsive to HLA-C matched and mismatched normal peripheral blood or bone marrow mononuclear cells from multiple donors and did not affect in vitro colony forming unit assays. NK cells isolated from normal healthy donors and co-incubated with irradiated CTV-1 tumor cells synthesized CD69 within 60 min with maximal expression achieved within 6 hr. The tumor-activated NK cells (T-ANKs) lysed NK resistant RAJI and Daudi cells in a 4-hour assay (Fig 1). The degree of lysis was equivalent to that of matched lymphokine activated killer cells (LAK) after non-specific activation with IL-2. CTV-1 cells are HLA-C type 2 homozygous and express HLA-Bw4 alleles. They thus can ligate KIR2DL1 (CD158a) and KIR3DL1 (CD158e1) on NK cells. Co-cultures of HLA-matched or mis-matched NK with CTV-1 showed there was no significant difference in the generation of T-ANK activity in the presence or absence of KIR ligation. T-ANK cells from allogeneic donors were capable of lysis of primary AML cells of all FAB types, the relatively NK-resistant breast cancer cell line, MCF-7, and primary tumor cells isolated from resected tissue from patients with breast cancer and ascities from patients with ovarian cancer. Autologous T-ANK cells were generated from five AML patients in clinical remission; all of whom had low level or undetectable NK activity to their presentation disease. T-ANK cells from all five donors consistently induced significantly greater lysis of autologous AML blasts than the matched NK cells (p<0.05) (Fig 2). To investigate the tumor-restriction of allogeneic T-ANK cells we isolated NK cells from normal donors and either activated them with CTV-1 cells overnight or maintained them in media. These T-ANK cells were then compared with NK cells from the same donor with respect to lysis of normal autologous and allogeneic PBMC. Neither NK nor T-ANK cells lysed autologous PBMC nor did they lyse PBMC from HLA-C mismatched normal donors. To determine the likelihood of bone marrow suppression by T-ANK cells we established hematopoietic colony forming assays with bone marrow from 5 normal donors and added T-ANK from HLA-C mismatched donors at increasing ratios. CFU-GM, BFU-E and CFU-GEMM were not affected by co-incubation with HLA-mismatched T-ANK even at effector:target ratios of 20:1. We believe that this demonstrates a two stage process for NK activation; priming and triggering, the ligands for which are distinct. Tumor priming of NK primes cells capable of lysing a broad spectrum of NK-resistant tumors which may irrespective of KIR ligation which may represent an “off-the-shelf” immunotherapy product. Fig 1 Fig 1. Fig 2 Fig 2.

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Rituximab Sensitizes B-CLL Cells with Inactivated p53 to Fludarabine.

Blood ◽

10.1182/blood.v108.11.4973.4973 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4973-4973

Author(s):

Martin Trbusek ◽

Sona Cejkova ◽

Jitka Chumchalova ◽

Zdenek Pospisil ◽

Jana Smardova ◽

...

Keyword(s):

Human Plasma ◽

Caspase 3 ◽

Mononuclear Cells ◽

Statistical Significance ◽

P53 Gene ◽

Antagonistic Effect ◽

Significant Difference ◽

P53 Inactivation ◽

And Control

Abstract Background: Defects in the p53 gene predispose B-CLL patients for an inferior outcome, particularly a resistance to treatment by conventional chemotherapeutics. Very little data exist, however, about the efficacy of monoclonal antibody rituximab on B-CLL cells bearing p53 abnormalities. One reason for that might be a methodological - rituximab alone does not have virtually any effect on the viability of B-CLL cells when cultivated in vitro, unless an active human plasma is added. After that, however, the cells are quickly lysed by complement, what is a process independent on p53. Aims: We used an in vitro system (not containing the active human plasma) to monitor a rituximab activity on B-CLL cells with p53 inactivation in relation to subsequently used nucleoside analog fludarabine, which demonstrably acts through the p53 in B-CLL cells. Methods: The p53 abnormalities in blood samples of B-CLL patients were detected by FISH and by functional yeast analysis coupled to sequencing, as described previously (Trbusek et al., Leukemia2006, 20: 1159–1161). Vitally frozen samples were used in all cases, after 24h pre-cultivation. Mononuclear cells were cultivated for 72h with or without 20μg/ml of rituximab and subsequently for another 48h with four different concentrations of fludarabine (40μg/ml–0,625μg/ml). The cell viability was determined by a WST-1 assay. A sensitization effect of rituximab pretreatment was determined by an ANOVA analysis, with the value p=0,05 being a threshold for a statistical significance. To monitor an apoptosis (a suppossed mechanism of fludarabine action), the Western blotting was used for the caspase-3 cleavage, which was proved previously to occur in drug-treated B-CLL cells. Results: In the subgroup of eleven p53-wt samples the three cases manifested sensitization by rituximab for fludarabine activity, one case showed an oposite (antagonistic) effect, while there was no significant difference for another seven samples. Among ten p53-mutated samples there was just one case exhibiting no influence of rituximab pretreatment (with the p53 alleles being deletion / 281 Asp→Glu), one sample manifested with antagonistic effect (del / 220 Tyr→Cys), while the remaining eight cases showed a statistically significant sensitization by rituximab (del / truncated protein aa 314, del / no protein, del/ wt protein, del / 248 Arg→Gln, del / 249 Arg→Gly, del / del aa 252, del / del aa 252–254, and a composed mutant 281Asp→Asn / 254 Ile→Thr). We noticed a statistically significant potentiation also in three out of four ATM-deleted samples (ATM is the p53-regulatory kinase). An apoptosis occured after fludarabine addition both in pretreated and control cells, as evidenced by the caspase-3 cleavage in some (but not all) samples. Conclusions: We show, to our konwledge for the first time, that rituximab can significantly sensitize the B-CLL cells bearing different types of p53 mutations to fludarabine. This result warrants further investigation of the mechanism behind.

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Identification and characterization of osteoclast progenitors by clonal analysis of hematopoietic cells

Blood ◽

10.1182/blood.v80.7.1710.bloodjournal8071710 ◽

1992 ◽

Vol 80 (7) ◽

pp. 1710-1716 ◽

Cited By ~ 1

Author(s):

MY Lee ◽

JL Lottsfeldt ◽

KL Fevold

Keyword(s):

Bone Marrow ◽

Tumor Cell ◽

Mononuclear Cells ◽

Fetal Liver ◽

Tumor Cell Line ◽

Adherent Cell ◽

Mouse Bone Marrow ◽

Hematopoietic Progenitors ◽

Adult Mice

We have identified a distinct population of colony-forming cells that give rise to mononuclear cells expressing an enzyme marker and other features of the osteoclast in bone marrow cultures stimulated by conditioned medium of a murine tumor cell line. These colony-forming cells were defined as osteoclast colony-forming units (CFU-O). The tumor cell-derived activity was recently isolated and was named osteoclast colony-stimulating factor (O-CSF). To understand the development of osteoclast progenitors and to clarify the relationship of osteoclast progenitors to other hematopoietic progenitors, we examined CFU-O in hematopoietic tissues obtained from normal adult mice, mouse fetuses, and mice with 5-fluorouracil (5FU) treatment. CFU- O were present in the adult mouse bone marrow, adherent cell-depleted marrow, in the spleen, and in the day 14 fetal liver, with an incidence similar to other hematopoietic progenitors. The culture period required for the development of CFU-O-derived colonies in vitro and the manner in which CFU-O responded to 5FU suggested that CFU-O belonged to a relatively primitive progenitor population; they are clearly more immature than macrophage progenitors that respond to macrophage-CSF, but more mature than multilineage progenitors that respond to stem cell factor. Our studies have defined and characterized an osteoclast progenitor and distinguished it from other hematopoietic progenitors for the first time.

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Does Needle Design Affect the Regenerative Potential of Bone Marrow Aspirate? An In Vitro Study

Life ◽

10.3390/life11080748 ◽

2021 ◽

Vol 11 (8) ◽

pp. 748

Author(s):

Nadia Feddahi ◽

Monika Herten ◽

Tjark Tassemeier ◽

Heike Rekasi ◽

Alexander Hackel ◽

...

Keyword(s):

Bone Marrow ◽

Iliac Crest ◽

Mononuclear Cells ◽

Bone Marrow Aspirate ◽

In Vitro Study ◽

Contralateral Side ◽

Disc Disease ◽

Significant Difference ◽

Spinal Disc

While autologous bone is still the gold standard for treatment of bone defects, its availability is limited. Sufficient numbers of mesenchymal stroma cells (MSC) may be an alternative. Small volumes of bone marrow aspirate (BMA) were harvested with two different needle systems comparing the yield and regenerative potency of the MSCs. BMA (10 mL) was aspirated from the posterior iliac crest of 12 patients with degenerative spinal disc disease using both needle systems in each patient: the Jamshidi needle (JAM) and on the contralateral side the Marrow Cellution® Needle (AMC). Number of mononuclear cells (MNCs) and regeneration capacity (colony-forming unit/CFU) were determined. MSCs were characterized for surface markers and their differentiation into trilineages. There was no significant difference between the two harvesting needles regarding the quantity of MNCs in BMA: 5.2 ± 1.8 × 109 MNC/mL for AMC vs. 4.8 ± 2.5 × 109 MNC/mL for JAM, p = 0.182. The quantity of CFUs per ml BMA was similar for both groups: 3717 ± 5556 for AMC and 4305 ± 5507 for JAM (p = 0.695). The potency of MSCs expressed as colony-forming potential per 106 MNC resulted in 0.98 ± 1.51 for AMC and 1.00 ± 0.96 for JAM (p = 0.666). Regardless of the needle design, 10 mL bone marrow aspirate contains a sufficient number of about 40,000 MSCs that can be used to enhance bone healing.

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