Monoclonal antibodies to carbohydrate antigens in autologous bone marrow transplantation

Normal and malignant myeloid cells express a highly immunogenic oligosaccharide, lacto-n-fucopentaose-III (LNF-III), that has been identified by numerous monoclonal antibodies (MoAb). We have been interested in the use of a particular monoclonal antibody to LNF-III, PM-81, in the treatment of patients with acute myelogenous leukemia using the antibody to treat bone marrow in vitro. Following in vitro treatment of bone marrow with PM-81 and another MoAb, AML-2-23, the remaining cells are used as an autograft in a patient treated with high-dose chemotherapy and radiotherapy. In order to enhance the ability of the MoAb to lyse leukemic cells in the remission bone marrow, we have explored the effect of neuraminidase treatment on leukemia cells. In this paper we describe that myeloid leukemia cells expressing low levels of LNF-III by immunofluorescence can be shown to have high levels of LNF-III after neuraminidase treatment. In addition, we show that normal bone marrow progenitor cells do not have cryptic LNF-III antigen, thus allowing the application of this finding to the clinical setting. Moreover, we have shown that leukemia colony-forming cells from one patient with acute myelogenous leukemia express cryptic LNF-III and that after exposure to neuraminidase there was an increased ability of PM-81 in the presence of complement to eliminate these colony forming cells. These data indicate that the LNF-III moiety is almost universally expressed on myeloid leukemia cells and their progenitors but not expressed on normal progenitors. Thus, it may be possible to enhance leukemia cell kill in vitro by neuraminidase treatment of bone marrow.     

Human cells in suspension. 1 Human lymphoid and granulopoietic cells in primary and secondary cultures: effects of in vitro induction and prolonged methanol acetic acid fixation on metaphase chromosome structure.

Modifications of Hungerford's method (1965) for production of chromosomal slides from human lymphoid cells in culture have been developed. Modified in vitro induction of banding and uncoiling has been used to produce chromosomal slides from human neoplastic cells of granulopoietic origin. The chromosomes are well spread and appear either long, thin and segmented or uncoiled. It is suggested that it is the combined action of the prolonged fixation used, and the in vitro induction, which leads to the observed structural alteration of the chromosomes. A method for increasing the yield of metaphase cells when working with bone marrow has been developed on the basis of culturing the granulopoietic cells in medium containing colony stimulating factor (CSF). Comparative analysis of metaphases from primary and secondary cultures of bone marrow cells showed that the culturing conditions for the secondary cultures do not induce chromosome abnormalities in the cells during the growth period.     

Adoptive transfer of T-cell precursors enhances T-cell reconstitution after allogeneic hematopoietic stem cell transplantation

Immunoincompetence after allogeneic hematopoietic stem cell transplantation (HSCT) affects in particular the T-cell lineage and is associated with an increased risk for infections, graft failure and malignant relapse. To generate large numbers of T-cell precursors for adoptive therapy, we cultured mouse hematopoietic stem cells (HSCs) in vitro on OP9 mouse stromal cells expressing the Notch-1 ligand Delta-like-1 (OP9-DL1). We infused these cells, together with T-cell-depleted mouse bone marrow or purified HSCs, into lethally irradiated allogeneic recipients and determined their effect on T-cell reconstitution after transplantation. Recipients of OP9-DL1-derived T-cell precursors showed increased thymic cellularity and substantially improved donor T-cell chimerism (versus recipients of bone marrow or HSCs only). OP9-DL1-derived T-cell precursors gave rise to host-tolerant CD4+ and CD8+ populations with normal T-cell antigen receptor repertoires, cytokine secretion and proliferative responses to antigen. Administration of OP9-DL1-derived T-cell precursors increased resistance to infection with Listeria monocytogenes and mediated significant graft-versus-tumor (GVT) activity but not graft-versus-host disease (GVHD). We conclude that the adoptive transfer of OP9-DL1-derived T-cell precursors markedly enhances T-cell reconstitution after transplantation, resulting in GVT activity without GVHD.     

Helicobacter pylori (H. pylori) molecular signature in conjunctival mucosa-associated lymphoid tissue (MALT) lymphoma

Conjunctival mucosa-associated lymphoid tissue (MALT) lymphoma is an extranodal marginal zone B-cell lymphoma that is characterized by an exaggerated clonal expansion of B cells, which implicate a pathological proliferative response to antigen(s) including bacteria. Helicobacter pylori (H. pylori) infection is recognized as one of the causative agents of gastric MALT lymphoma; however, it has not been reported in extra gastric MALT lymphoma. We studied 5 patients (4 adults and 1 child) with salmon-colored conjunctival lesions. One patient also had a history of abnormal bone marrow biopsy a year earlier with lymphoid aggregates involving 5% of the overall bone marrow. The conjunctival lesions of the 5 patients were biopsied. Histopathological diagnoses were consistent with conjunctival MALT lymphoma. Lymphoma and normal conjunctival cells were microdissected using laser capture microscopy or manual techniques. DNA was extracted and subjected to PCR amplification using H. pylori gene-specific primers from the urease B and vac/m2 gene. Cells from chronic conjunctivitis (normal lymphocytes), conjunctival human T-cell lymphotropic virus type-1/adult T-cell leukemia/lymphoma (HTLV-1/ATL), and orbital B-cell lymphoma were also microdissected, processed and analyzed. PCR amplification and Southern blot hybridization demonstrated H. pylori DNA in the conjunctival MALT lymphoma cells of 4/5 cases. The negative case was the one with a history of abnormal bone marrow. In contrast, H. pylori gene was not detected in normal conjunctival cells from the cases of MALT lymphoma or the lymphocytes, ATL and orbital B-lymphoma cells from the controls. These data suggest that H. pylori may play a role in conjunctival MALT lymphoma.     

In vivo overexpression of Core2 N-Acetylglucosaminyltransferase prevents repopulation of the bone marrow with colony forming cells but fails to affect normal T cell development

UDP-GlcNAc:Galβ1 → 3GalNAc-R β1 → 6N-acetylglucosaminyltransferase (Core2 N-acetyl-glucosaminyltransferase, C2GnT; EC 2.4.1.102) forms β1 → 6N-acetyl-glucosaminyl linkages in O-glycoproteins and creates branches for the addition of N-acetyl-lactosamine antennae. Changes in C2GnT activity have been associated with immune disorders, malignancies, and T-cell ontogeny. In this study, we used SCID (severe combined immune deficiency) mice to determine the effects of C2GnT overexpression on hemopoiesis, and in particular, on thymocyte development. BALB/c bone marrow cells transfected with C2GnT using the retroviral murine stem cell vector were used to repopulate SCID mice. Mice were analysed 3 weeks to 3 months after bone marrow transfer. Elevated levels of C2GnT activity in bone marrow, spleen, and thymus from mice repopulated with C2GnT transfected bone marrow cells indicated that C2GnT was overexpressed in recipient mice. In C2GnT repopulated mice, up to 50% of T cells showed an increase in CD43 130-kDa expression, compared with T cells from control animals, indicative of an elevated C2GnT activity in these cells. Furthermore, T-cell subset numbers appeared to be normal, suggesting that C2GnT overexpression did not alter T-cell ontogeny. Interestingly, C2GnT overexpression negatively affected the repopulation of myeloid cells. Only insignificant numbers of interleukin-3/granulocyte-macrophage colony stimulating factor (IL-3/GM-CSF) responsive bone marrow cells were found to be retrovirally transfected in C2GnT repopulated mice, whereas up to 50% of IL-3/GM-CSF responsive bone marrow cells were found to be retrovirally transfected in corresponding controls. These data indicate that in vivo overexpression of C2GnT negatively interferes with the myeloid differentiation pathway but does not affect T-cell development. J. Cell. Physiol. 176:350–358, 1998. © 1998 Wiley-Liss, Inc.     

Nature of T-cells resident in spleens of thymectomized,lethally irradiated,bone marrow-reconstituted mice

Adult thymectomized, lethally irradiated, bone marrow-reconstituted (ATxXB) mice that had been weakly primed with SRBC or HRBC between thymectomy and irradiation were shown to retain antigen-specific immunological memories for at least 1–5 months after bone marrow reconstitution. This could be shown by anamnestic antibody response in vivo as well as by proliferative response of the spleen cells to the test antigens in vitro. Spleen cells taken from ATxXB mice showed a reduced but significant proliferative response to nonspecific T-cell mitogens, in particular to Con A, in vitro. Treatment of the donor bone marrow cells used for reconstitution of ATxXB mice with anti-Thy 1.2 sera + C′ did not affect the generation of immunological memories nor the magnitude of the proliferative response of spleen cells to nonspecific T-cell mitogens in vitro, indicating that the cells responsible for such functions were host derived. Finally, the antibody-forming capacity of spleen cells derived from SRBC-primed ATxXB mice to the test antigen in vitro was completely abrogated by exposure to 450 R, whereas the helper function of the same cell suspension remained unaffected even after exposure to 1000 R. Implication of these findings on the nature of T cells resident in spleens of ATxXB mice was discussed.     

Colony-forming cells with high proliferative potential (HPP-CFC)

Colony-forming cells with a high proliferative potential (HPP-CFC) have been defined by their ability to form large colonies in vitro (diameters greater than 0.5 mm and containing approximately 50,000 cells) in bone marrow cell cultures. The HPP-CFC have been characterized by: 1) a relative resistance to treatment in vivo with the cytotoxic drug 5-fluorouracil, 2) a high correlation with cells capable of repopulating the bone marrow of lethally irradiated mice, 3) their multipotential ability to generate cells of the macrophage, granulocyte, megakaryocyte and erythroid lineages, and 4) their multifactor responsiveness. The HPP-CFC have been described in both mouse and human bone marrow. These properties suggest that the HPP-CFC represent an important cell type in hematopoiesis and provide a model system, particularly in the human, for studying the properties of primitive progenitor cells in vitro.     

In vivo osteoinductive effect and in vitro isolation and cultivation bone marrow mesenchymal stem cells

Bone marrow contains cell type termed mesenchymal stem cells (MSC), first recognized in bone marrow by a German pathologist, Julius Cohnheim in 1867. That MSCs have potential to differentiate in vitro in to the various cells lines as osteoblast, chondroblast, myoblast and adipoblast cells lines. Aims of our study were to show in vivo capacity of bone marrow MSC to produce bone in surgically created non critical size mandible defects New Zeland Rabbits, and then in second part of study to isolate in vitro MSC from bone marrow, as potential cell transplantation model in bone regeneration. In vivo study showed new bone detected on 3D CT reconstruction day 30, on all 3 animals non critical size defects, treated with bone marrow MSC exposed to the human Bone Morphogenetic Protein 7 (rhBMP-7). Average values of bone mineral density (BMD), was 530 mg/cm3, on MSC treated animals, and 553 mg/cm3 on control group of 3 animals where non critical size defects were treated with iliac crest autologue bone graft. Activity of the Alkaline Phosphatase enzyme were measurement on 0.5, 14, 21, 30 day and increased activity were detected day 14 on animals treated with bone marrow MSCs compared with day 30 on iliac crest treated animals. That results indicates strong osteoinduction activity of the experimental bone marrow MSCs models exposed to the rhBMP-7 factor Comparing ALP activity, that model showed superiorly results than control group. That result initiates us in opinion that MSCs alone should be alternative for the autolologue bone transplantation and in vitro study we isolated singles MSCs from the bone marrow of rat's tibia and femora and cultivated according to the method of Maniatopoulos et all. The small initial colonies of fibroblast like cells were photo-documented after 2 days of primary culture. Such isolated and cultivated MSCs in future studies will be exposed to the growth factors to differentiate in osteoblast and indicate their clinically potential as alternative for conventional medicine and autologue bone transplantation. That new horizons have potential to minimize surgery and patient donor morbidity, with more success treatment in bone regenerative and metabolism diseases.     

Colony-forming units of the bone marrow and spleen of immunodeficient mice after stimulation of the cells with thymalin

It has been previously demonstrated by the authors that histological characteristics of colony-forming units (CFUs) in normal mice prove a certain shift in their differentiation in erythroid direction comparing to the bone marrow CFUs. Thymectomy of mature animals is accompanied with weakening growth of granular colonies at cloning of the bone marrow CFUs and with loss of stability in direction of splenic CFUs differentiation. Polypeptide preparation of the thymus--thymalin stimulates growth of the granulocytic colonies from the splenic CFUs in thymectomized mice both in in vivo and in vitro experiments. Differentiation of the bone marrow CFUs is normalized under the effect of thymalin in in vivo experiment only. The data obtained confirm the suggestion made by R. V. Petrov on existence of T-cell clone, enhancing CFUs differentiation in granulocytic direction. Activation of this clone in the spleen is revealed at thymectomy and stimulation of the cells with thymalin both in in vivo and in vitro experiments. Thus, affirmations are obtained on differences of clonic T-cell regulation of the CFUs differentiation in the bone marrow and in the spleen.     

A newly established human acute lymphoblastic leukemia cell line with characteristics of the earliest B-cell maturation

A new human acute lymphoblastic leukemia (ALL) cell line, designated HBL-3, was established from the bone marrow of a patient with non-T-ALL. The HBL-3 cell line expressed B4 (CD 19), BA-1 (CD 24) and HLA-DR antigens, but not surface immunoglobulin (SIg) or cytoplasmic immunoglobulin (CIg). The cell line lacked the common acute lymphoblastic leukemia antigen (CALLA) and antigenic markers characteristic of T-cell and myeloid cell lineages. The HBL-3 cells had structural rearrangements of both the homologous chromosome 9s, including a translocation with chromosome 1 which has been reported in a patient with common ALL. The cell line had rearranged immunoglobulin heavy chain genes but retained germ-line kappa light chain genes and germ-line T-cell receptor beta- and gamma-chain genes. The HBL-3 cell line was strongly positive for terminal deoxynucleotidyl transferase (TdT). These findings indicate that the HBL-3 cell line is derived from the earliest B-cell committed to B-cell lineage.     

Generation and characterization of IL-2-activated bone marrow cells as a potent graft vs tumor effector in transplantation

The potential of bone marrow transplantation as an immunotherapeutic modality, using biomodulation of the marrow cells has been ignored in autologous transplantation. Furthermore, many common cancers such as lung, colon, prostate, and pancreas are resistant to even transplant doses of conventional agents and hence require novel approaches such as biomodulation. This study shows that we can generate cytotoxic killer cells similar to lymphokine-activated killer cells capable of lysing NK-resistant tumor cells in vitro if we incubate human or murine bone marrow in IL-2. This was accomplished without affecting the ability of the bone marrow to fully reconstitute mice similar to that of fresh nonactivated bone marrow. Studies evaluating the IL-2 activated human bone marrow in vitro also indicated that these activated bone marrow have similar CFU to that of fresh human marrow. Furthermore, in murine in vivo studies, the activated bone marrow (ABM) caused significant tumor regression in tumor-bearing mice. Also, these ABM cells had similar or higher tumoricidal activity and longer kinetics than spleen lymphokine-activated killer cells in vitro. Also, the ABM had purging ability in vitro. Therefore this IL-2 ABM could be used as an active therapeutic tool and not just as a passive rescue element in the autologous bone marrow transplantation setting.     

Adoptive transfer of autologous, HER2-specific, cytotoxic T lymphocytes for the treatment of HER2-overexpressing breast cancer

The human epidermal growth factor receptor 2 (HER2) has been targeted as a breast cancer-associated antigen by immunotherapeutical approaches based on HER2-directed monoclonal antibodies and cancer vaccines. We describe the adoptive transfer of autologous HER2-specific T-lymphocyte clones to a patient with metastatic HER2-overexpressing breast cancer. The HLA/multimer-based monitoring of the transferred T lymphocytes revealed that the T cells rapidly disappeared from the peripheral blood. The imaging studies indicated that the T cells accumulated in the bone marrow (BM) and migrated to the liver, but were unable to penetrate into the solid metastases. The disseminated tumor cells in the BM disappeared after the completion of adoptive T-cell therapy. This study suggests the therapeutic potential for HER2-specific T cells for eliminating disseminated HER2-positive tumor cells and proposes the combination of T cell-based therapies with strategies targeting the tumor stroma to improve T-cell infiltration into solid tumors.     

Regulation of human B lymphopoiesis: effect of a urinary activity associated with cyclic neutropenia

Urine from a patient with cyclic neutropenia was found to contain a lymphopoietic activity that acts as a growth factor for human pre-B cells. This biologic activity was detectable during the week preceding, but not during, the period of neutropenia. This corresponded with a periodic excessive accumulation of pre-B cells in the marrow of this patient. Urine preparations were added to cultures of normal human bone marrow that had been depleted of B cells. Pre-B cells were generated in these cultures but not in cultures containing urine preparations from normal donors. Pre-B cells were also generated from bone marrow that had been depleted of 177.17+ cells and the majority of pre-B cells. This is the first report of a hemopoietic activity which affects human pre-B cells. This activity may represent a normal regulatory molecule that is periodically produced in excess in this patient.     

In vivo and in vivo/in vitro kinetics of cyclophosphamide-induced sister-chromatid exchanges in mouse bone marrow and spleen cells

In several acute and chronic exposures to various chemicals in vivo and in vitro, the average sister-chromatid exchange (SCE) frequencies in human, mouse, rat, and rabbit lymphocytes generally decrease with time following treatment. The rate of this decline varies, but little data have been published pertaining to the comparative kinetics of SCEs both in vivo and in vivo/in vitro (exposure of animals to the test compound and culturing of cells) simultaneously in the same tissues. In this study, a single dose of cyclophosphamide (40 mg/kg) was injected for varying periods (6-48 h) and its effects, as assessed by the induction of SCEs, were analyzed under both in vivo and in vivo/in vitro conditions in mouse bone marrow and spleen cells. In vivo, the cyclophosphamide-induced SCEs increased with increasing time up to 12 h, stayed at approximately the same level until 24 h, and then decreased with increase in post-exposure time. However, the SCE levels remained significantly higher than controls at 48 h post-exposure time in both bone marrow and spleen cells. Under in vivo/in vitro conditions, the SCEs in bone marrow decreased with increase in post-exposure time until reaching control values by 48 h post exposure. However, in spleen cells, the decrease in SCE level was gradual, and by 48 h post-exposure time, the cells still had approximately 6 times higher SCEs than the control values. These results suggest that there are pharmacokinetic differences for cyclophosphamide in mouse bone marrow and spleen. Also, there is a differential SCE response to cyclophosphamide under in vivo and in vivo/in vitro conditions.     

Hierarchy of T cell dependency in antibody response among different antigens.

T cell dependency of antibody response to polyvinylpyrrolidone (PVP), sheep red blood cells (SRBC), bovine gamma globulin (BGG), and bovine serum albumin (BSA) was examined. PVP and the other three are known as a T cell-independent antigen and T cell-dependent antigens respectively. Adult mice were thymectomized, X-irradiated, reconstituted with syngeneic bone marrow cells (TxXB mice), with bone marrow cells plus thymus cells (TxXBT mice), or with bone marrow cells treated with anti-Thy-1.2 serum and complement (TxXB-theta mice) and used as experimental animals. The anti-PVP response of TxXBT mice was significantly lower than that of TxXB mice, suggesting that T cells exerted a suppressive effect on the response to PVP. Both IgM and IgG responses to SRBC and BGG occurred even in TxXB-theta mice with the aid of bacterial lipopolysaccharide (LPS). However, a significant response to BSA was not observed in TxXB mice even in the presence of LPS or several other adjuvants. These results indicate that the T cell dependency of antigens is different among so called thymus-dependent antigens, that antibody response less dependent on the helper action of T cells can be supported by LPS in the absence of T cells, and that anti-BSA response seems to be extremely T cell dependent.     

Comparative study of bone marrow mesenchymal stromal cells at different stages of ontogeny

The aim of this study was to analyze the changes that occur in the population of bone marrow mesenchymal stromal cells (MSCs) during the individual development of an organism. For this purpose, the basic characteristics of MSCs (the content of clonogenic cells, immunophenotype, and potencies to differentiate in vitro and in vivo) in the prenatal, early postnatal, and late postnatal ontogeny of the rat were compared. It is shown that the cloning efficiency of bone marrow MSCs in 10-day-old and adult rats is comparable and hundreds of times smaller than that of bone cells of 20-day-old fetuses with a bone marrow rudiment. The activity of alkaline phosphatase, a marker of osteogenic cells, was found in the majority of colonies formed by MSCs of postnatal bone marrow but not by the fetal bone. By the CD90 expression and potencies for in vitro adipogenesis, the stromal cells from the fetal bone and bone marrow of 9- to 10-day-old rats were comparable with those of the mature bone marrow MSCs but differed from them by the small number of CD73-bearing cells and a weaker ability to osteogenesis in an inductive environment. The analysis of the fate of MSCs from the studied sources after their transplantation to adult rats showed that their ectopic transplantation as part of tissue fragments into the kidney results in the formation of bone tissues and hematopoietic stroma. In diffusion chambers with MSCs that were precultured in vitro, transplantation into the peritoneal cavity led to osteogenesis and chondrogenesis. However, no significant differences in the potencies of bone marrow MSCs for differentiation in vivo depending on the developmental stage have been found. Thus, during ontogeny, bone marrow MSCs enhance the expression of CD73 and the ability to osteogenesis in vitro, whereas the expression of CD90 and the potencies for adipogenesis in induction medium and differentiation in different directions in vivo do not change significantly.     

Immunoregulatory activity of human bone marrow. Identification of suppressor cells possessing OKM1, SSEA-1, and HNK-1 antigens

Human bone marrow contains natural regulatory cells capable of suppressing the in vitro primary IgM response of normal tonsillar cells. The suppression is mediated by non-T cells possessing Fc receptors, OKM1, SSEA-1, and HNK-1 antigens on their surface. The suppression was abrogated by treatment of bone marrow cells (BMC) with anti-HNK-1 or anti-SSEA-1 antisera and complement. Furthermore, BMC depleted of HNK-1+ cells could respond in a primary in vitro antibody response when provided with accessory T cells and macrophages from tonsillar cells. Our findings support the idea that HNK-1+ and HNK-1- BMC populations act antagonistically in the regulation of antibody synthesis. Further, the finding of HNK-1+, SSEA-1+, and OKM1+ suppressor cells in human bone marrow may represent a precursor phenotype of mature natural killer cells with potent immunoregulatory activity.     

人红细胞生成素的分离、纯化及鉴定

 用自制的苯基-琼脂糖CL-4B和羟基邻灰石等层析材料,从再生障碍性贫血病人尿中分离、纯化制得了红细胞生成素(EPO)。用多血小鼠红细胞~(56)Fe参入法测定该制品在体内的生物活力。用小鼠与人骨髓红系祖细胞培养法测其在体外的生物活力。实验结果说明,我们自制的EPO制品,不仅能用于动物,也能用于人骨贿红系祖细胞的培养。用Azocoll法测该制品中蛋白水解酶活力为阴性。     

Increased Bone Marrow (BM) Plasma Level of Soluble CD30 and Correlations with BM Plasma Level of Interferon (IFN)-γ, CD4/CD8 T-Cell Ratio and Disease Severity in Aplastic Anemia

Idiopathic aplastic anemia (AA) is an immune-mediated bone marrow failure syndrome. Immune abnormalities such as decreased lymphocyte counts, inverted CD4/CD8 T-cell ratio and increased IFN-γ-producing T cells have been found in AA. CD30, a surface protein belonging to the tumor necrosis factor receptor family and releasing from cell surface as a soluble form (sCD30) after activation, marks a subset of activated T cells secreting IFN-γ when exposed to allogeneic antigens. Our study found elevated BM plasma levels of sCD30 in patients with SAA, which were closely correlated with disease severity, including absolute lymphocyte count (ALC) and absolute netrophil count (ANC). We also noted that sCD30 levels were positively correlated with plasma IFN-γ levels and CD4/CD8 T-cell ratio in patients with SAA. In order to explain these phenomena, we stimulated T cells with alloantigen in vitro and found that CD30⁺ T cells were the major source of IFN-γ, and induced CD30⁺ T cells from patients with SAA produced significantly more IFN-γ than that from healthy individuals. In addition, increased proportion of CD8⁺ T cells in AA showed enhanced allogeneic response by the fact that they expressed more CD30 during allogeneic stimulation. sCD30 levels decreased in patients responded to immunosuppressive therapy. In conclusion, elevated BM plasma levels of sCD30 reflected the enhanced CD30⁺ T cell-mediated immune response in SAA. CD30 as a molecular marker that transiently expresses on IFN-γ-producing T cells, may participate in mediating bone marrow failure in AA, which also can facilitate our understanding of AA pathogenesis to identify new therapeutic targets.