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.     

嗜中性粒细胞是人抵抗素表达的主要细胞

抵抗素(resistin)是小鼠白色脂肪组织大量表达的富含半胱氨酸的 分泌型蛋白.近年研究发现,人与啮齿类动物的抵抗素组织表达分布存在很 大差异．小鼠抵抗素主要在白色脂肪组织表达,而人抵抗素主要在单核细 胞/巨噬细胞表达,且在骨髓组织中大量表达,但目前骨髓中的细胞定位还 不清楚．本研究的目的是明确成人骨髓及外周血白细胞中抵抗素表达细胞 的类型．免疫荧光法检测骨髓中抵抗素表达细胞,结果显示,抵抗素主要表 达在细胞核呈杆状和分叶核状的成熟粒细胞中,其中杆状核粒细胞表达较 高,分叶核粒细胞表达减弱．Anti-hresistin IgG-Biotin-PE单色荧光流 式细胞术分选外周血白细胞中抵抗素表达细胞后经瑞氏化学染色,结果显 示,抵抗素表达细胞主要为杆状和分叶核状的嗜中性粒细胞,还有少量嗜酸 性粒细胞,且抵抗素蛋白分布在细胞质中. RT-qPCR结果在RNA水平上证明, 人抵抗素在嗜中性粒细胞中大量表达．Anti-hresistin IgG-FITC和anti- HNL IgG-Biotin-PE 双色荧光流式细胞术进一步证明,抵抗素的主要表达细 胞为成熟的嗜中性粒细胞．嗜中性粒细胞在机体免疫防御中起重要作用, 人骨髓及外周血中抵抗素主要在成熟嗜中性粒细胞中表达,这一研究结论 为人抵抗素与炎症反应的关联性及其功能的进一步研究奠定了基础.     

Synergistic inhibition of human marrow granulocyte-macrophage progenitor cells by prostaglandin E and recombinant interferon-alpha, -beta, and -gamma and an effect mediated by tumor necrosis factor

The effects of prostaglandin E (PGE) and recombinant human interferon-alpha, -beta, and -gamma alone and in combination were tested for their effects on the proliferation of human bone marrow granulocyte-macrophage colony-forming units (GM-CFU). When tested alone, both classes of cytokines inhibited GM-CFU proliferation. In combination, PGE and all three types of recombinant interferons synergized in their ability to inhibit GM-CFU proliferation. Progressive enrichment for marrow GM-CFU indicated that the synergistic effects of PGE and interferon were dependent upon the presence of marrow-adherent cells. Studies using conditioned media from marrow-adherent cells prepared in the presence of interferon-alpha, -beta, and -gamma indicated that adherent cells produced a soluble factor in the presence of interferons that subsequently synergized with PGE in inhibiting GM-CFU proliferation. Neutralization of this conditioned media with a monoclonal antibody to tumor necrosis factor abrogated the synergistic inhibition of GM-CFU observed in the presence of PGE. The addition of recombinant tumor necrosis factor and PGE to accessory cell-depleted bone marrow resulted in synergystic inhibition of GM-CFU proliferation.     

Effects of age and gender on WNT gene expression in human bone marrow stromal cells

WNT signaling pathways play important roles in the behavior of human bone marrow stromal cells. Although WNT expression has been examined in human bone marrow stromal cells (hMSCs) with limited numbers of subjects or from commercial sources, there are conflicting results on WNT gene expression in hMSCs. Furthermore, the effects of age and gender on WNT expression in hMSCs are largely unknown. In this study, we evaluated RNA expression of all the WNT genes in hMSCs from 19 subjects, 12 women and 7 men, aged from 36 to 85 years. Analysis of WNT gene expression in young and old groups indicated that WNT7B and 14 were expressed significantly higher in the young group. WNT2 and WNT13 showed a trend of higher expression in young group. WNT7B, 13, and 14 were inversely correlated with age. Further analysis for gender‐specific difference indicated that WNT16 was expressed significantly higher in men than in women. WNT11 showed a trend of higher expression in hMSCs from women. For the hMSCs from women, WNT13 was inversely correlated with age and WNT4 was positively correlated with age. For the hMSCs from men, WNT7B and WNT14 were inversely correlated with age. These data indicated that most of the age‐related WNT genes belong to the canonical WNT signaling pathway. Further, there are gender‐specific differences in the expression of WNT4, 7B, 13, 14, and 16 in hMSCs. Age and gender account for many of the sample‐to‐sample variations in WNT gene expression in human marrow stromal cells. J. Cell. Biochem. 106: 337–343, 2009. © 2008 Wiley‐Liss, Inc.     

Transfer and expression of the human multiple drug resistance gene as potential human gene therapy

The human multiple drug resistance (MDR) gene has been used as a model for human gene transfer which could lead to human gene therapy. MDR is a transmembrane protein which pumps a number of toxic substances out of cells including several drugs used in cancer chemotherapy. Normal bone marrow cells express low levels of MDR and are particularly sensitive to the toxic effects of these drugs. There are two general applications of MDR gene therapy: (1) to provide drug-resistance to the marrow of cancer patients receiving chemotherapy, and (2) as a selectable marker which when co-transferred with a non-selectable gene such as the human beta globin gene can be used to enrich the marrow for cells containing both genes. We demonstrate efficient transfer and expression of the human MDR gene in a retroviral vector into live mice and human marrow cells including CD34⁺ cells isolated from marrow and containing the bulk of human hematopoietic progenitors. MDR gene transduction corrects the sensitivity of CD34⁺ cells to taxol, an MDR drug substrate, and enriches the marrow for MDR-transduced cells. The MDR gene-containing retroviral supernatant used has been shown to be safe and free of replication-competent retrovirus. Because of the safety of the MDR retroviral supernatant, and efficient gene transfer into mouse and human marrow cells, a phase 1 clinical protocol for MDR gene transfer into cancer patients has been approved to evaluate MDR gene transfer and expression in human marrow.     

Studies of sub-human primate (marmoset) pluripotent hemopoietic stem cells (CFU-GEMM) in vitro

Pluripotent hemopoietic progenitor cells (CFU-GEMM) grow in vitro from marmoset bone marrow using a modified human CFU-GEMM assay. Characteristics of growth are similar to those reported in the human CFU-GEMM assay. The number of CFU-GEMM/10(5) marrow cells from marmoset bone marrow is approximately four times that grown in human marrow in our laboratory. Data concerning EPO and other requirements for growth of CFU-GEMM demonstrate an assay for the pluripotent hemopoietic progenitor in the marmoset. This assay may be useful in designing preclinical primate bone marrow transplant experiments.     

Gene expression profile of mouse bone marrow stromal cells determined by cDNA microarray analysis

Bone marrow stromal cells (BMSC) have gained increased attention because of their multipotency and adult stem cell character. They have been shown to differentiate into other cell types of the mesenchymal lineage and also into non-mesenchymal cells. The exact identity of the original cells, which are isolated from bone marrow by their selective adherence to plastic, remains unknown to date. We have established and characterized mouse BMSC cultures and analyzed three independent samples by cDNA microarrays. The expression profile was compared with two previous expression studies of human BMSC and revealed a high degree of concordance between different techniques and species. To gain clues about the positional context and biology of the isolated cells within the bone marrow stroma, we searched our data for genes that encode proteins of the extracellular matrix, cell adhesion proteins, cytoskeletal proteins and cytokines/cytokine receptors. This analysis revealed a close association of BMSC with vascular cells and indicated that BMSC resemble pericytes.     

Demonstration of altered signaling responses in bone marrow extracellular fluid during increased hematopoiesis in rats using a centrifugation method

The composition and characteristics of the bone marrow extracellular fluid supposedly modify the transport of cytokines, drugs, and other signaling molecules involved in the regulation of bone marrow function. Direct access to the bone marrow extracellular fluid surrounding hematopoietic cells is complicated by the virtually noncompliant surrounding bone tissue. We examined the applicability of a centrifugation method to obtain representative samples of bone marrow extracellular fluid from rats and humans. Perforated rat bones or human bone marrow biopsies were wrapped in nylon mesh baskets before being centrifuged at 180-239 g. In the rats, we found an only minor contribution of fluid from other sources than the bone marrow extracellular fluid as indicated by the average ratio of centrifugate-to-plasma activity of the extracellular tracer fluid 51Cr-labeled EDTA of 0.85. The colloid osmotic pressure in the centrifugate was consistently lower than that in the corresponding plasma in both species. In rats and humans, high-performance liquid chromatography showed a protein elution pattern from the bone marrow fluid similar to that of plasma, except for a peak eluting in the approximately 40-kDa molecular mass range. Western blotting of the cytokines erythropoietin and granulocyte colony-stimulating factor revealed generally higher amounts in the centrifugate than in the plasma. This difference was augmented during increased hematopoietic activity induced by inflammation or bleeding in rats. We conclude that the centrifugation method provides representative samples of bone marrow extracellular fluid and that extracellular signaling responses to altered hematopoiesis are more clearly reflected locally in the bone marrow interstitium than in plasma.     

Immunohistochemical detection of cathepsin D, K, and L in the process of endochondral ossification in the human

Cathepsins D, K, and L were immunolocalized in tissue undergoing endochondral ossification in the human. Cathepsins D, K, and L were localized in osteoclasts and chondroclasts attached to bone matrix and cartilage matrix, respectively. Cathepsins D and L were immunostained in chondrocytes. Immunolocalization of cathepsin D was limited to hypertrophic chondrocytes adjacent to the osteochondral junction. In contrast, cathepsin L was immunolocalized in both proliferating and hypertrophic chondrocytes. In the bone marrow space, cathepsins D, K, and L were localized in multinucleated cells. Cathepsin D was diffusely detected in mononuclear bone marrow cells which were negative for cathepsins K and L. The present findings indicated that cathepsins K, D, and L were associated with the process of endochondral ossification in the human, and suggested that these cathepsins share roles in bone and cartilage turnover in the human.     

SGP28, a novel matrix glycoprotein in specific granules of human neutrophils with similarity to a human testis-specific gene product and to a rodent sperm-coating glycoprotein

A novel 28 kDa glycoprotein was purified from exocytosed material from human neutrophils and its primary structure partially determined. Degenerate oligonucleotide primers were used to amplify cDNA clones from a human bone marrow cDNA library. The deduced 245 amino acid sequence of the 2124 bp full-length cDNA showed high degrees of similarity to the deduced sequences of the human gene TPX-1 and of sperm coating glycoprotein from rat and mouse. Subcellular fractionation of human neutrophils indicated that the protein is localized in specific granules. The protein was named SGP28 (specific granule protein of 28 kDa).     

Establishment of a variety of human bone marrow stromal cell lines by the recombinant SV40-adenovirus vector.

Various human bone marrow stromal cell lines were established from the adherent cell populations by introduction of the recombinant SV40-adenovirus vector with an infection or electric poration procedure. As compared with DNA transfection, the vector introduction was able to immortalize the cells with more than 100 times higher efficiency. Morphological and cytochemical analyses indicated that various cloned cell lines with different properties were isolated by the vector introduction. All the established cell lines expressed SV40 large T antigen. These results provided the evidence indicating that the recombinant SV40-adenovirus vector was a useful tool to establish a variety of cell lines with different biological activities from human bone marrow stroma.     

Cryopreservation of human cadaveric bone marrow cells

The viability of cryopreserved human cadaveric bone marrow cells was studied using methylcellulose clonal cell culture assays. This is the first study done to reveal the persistence of hemopoietic proliferative and differentiative functions using the methylcellulose clonal cell assay in cryopreserved human cadaveric bone marrow cells which are several hours postmortem. Studies of cryopreserved human cadaveric bone marrows corresponded with those of murine bone marrows. These results strongly suggest that several hours postmortem human cadaveric bone marrow cells can be cryopreserved and may be useful for transplantation.     

Human marrow stromal precursors are alpha 1 integrin subunit-positive

In this work we studied the expression of adhesion molecules on primate human and non-human marrow stromal cells (primary cultures and lines) and on human CD34(+) hematopoietic normal and leukemic precursors. Differential expression of alpha1 integrin subunit was observed, since this molecule was intensely expressed by marrow stroma but not detected on CD34(+) cells. We used this difference to select, in fresh bone marrow samples, alpha 1-positive cells. We found that all stromal precursors giving rise to colony-forming units-fibroblasts (CFU-F) were present in the alpha 1-positive fraction. No colonies were detected in the alpha 1-negative fraction even after 2 weeks of culture. Phenotypic studies of stromal cells derived from alpha1-positive cells and grown in long-term marrow culture indicated that these cells were similar to stromal cells from primary cultures. We also observed early upregulation of alpha 4 and alpha 2 integrin subunits in cultures derived from alpha1-positive cells with maximal expression by day 10 (26 and 51%, respectively) preceding a gradual decline to low to nil values at day 30 (4.5 and 12%). These data indicate that alpha 1 integrin subunit is a marker for both mature stromal cells and stromal precursors, while alpha 2 and alpha 4 integrin subunits are expressed primarily by immature cells.     

A new protein expressed in bone marrow cells and osteoblasts with implication in osteoblast recruitment

To study osteoblast recruitment from bone marrow cells, a rat femur cDNA library was screened by in situ hybridization for novel mRNA sequences that are frequently expressed in both marrow cells and osteoblasts. One isolated clone, called RP59, is described here. Northern blots indicated two bands of 2.6 and 2.8 kb in femur and spleen, tissues containing high amounts of immature mesenchymal cells, and no or little expression in other tissues. The cDNA sequence revealed a reading frame for a repetitive protein composed of arrays of 14-mers and phased phosphorylation sites. Antisera versus RP59 detected a single band of 90 kDa by Western blotting of femur extract. Immunohistochemistry indicated strong RP59 presence in the cytoplasm of bone marrow cells and weaker presence in nuclei of osteoblasts. Intermediate stages were found between strongly labeled, round, free bone marrow cells and weaker labeled, fibroblast-like young osteoblasts associated with bone matrix. These data indicated that marrow cells with high RP59 content were recruited into growing bone tissue. RP59 may help to study the transition of bone marrow cell to osteoblast in more detail.     

胎肝造血干细胞的移植特性

胚胎发育中,肝脏是一个重要的造血器官。近年来胎肝移植的临床应用重新引起了人们的关注。本文应用染色体的 C-带染色法研究了小鼠骨髓和胎肝造血干细胞在照射受体小鼠中的增殖能力与相互间的竞争作用。实验结果表明胎肝造血干细胞在成年骨髓中的植入率比较同样条件下的成年骨髓造血干细胞低,但胎肝造血干细胞比较成年骨髓造血干细胞具有更强的自我更新或增殖能力。在同种胎肝造血干细胞移植中,为了降低同种移植抗力,提高移植的胎肝造血干细胞在受体中的耐受性,移植前对受体作适当的免疫抑制处理是必要的。因此,克服个体发育屏障和移植免疫屏障是提高同种胎肝造血干细胞移植效果中两个重要的研究课题。     

Requirement of SLD5 for Early Embryogenesis

SLD5 forms a GINS complex with PSF1, PSF2 and PSF3, which is essential for the initiation of DNA replication in lower eukaryotes. Although these components are conserved in mammals, their biological function is unclear. We show here that targeted disruption of SLD5 in mice causes a defect in cell proliferation in the inner cell mass, resulting in embryonic lethality at the peri-implantation stage, indicating that SLD5 is essential for embryogenesis. Moreover, this phenotype of SLD5 mutant mice is quite similar compared with that of PSF1 mutant mice. We have previously reported that haploinsufficiency of PSF1 resulted in failure of acute proliferation of bone marrow hematopoietic stem cells (HSCs) during reconstitution of bone marrow ablated by 5-FU treatment. Since SLD5 was highly expressed in bone marrow, we investigated its involvement in bone marrow reconstitution after bone marrow ablation as observed in PSF1 heterozygous mutant mice. However, heterozygous deletion of the SLD5 gene was found not to significantly affect bone marrow reconstitution. On the other hand, abundant SLD5 expression was observed in human cancer cell lines and heterozygous deletion of the gene attenuated tumor progression in a murine model of spontaneous gastric cancer. These indicated that requirement and dependency of SLD5 for cell proliferation is different in different cell types.     

Cytometry and Time-Dependent Variations in Peripheral Blood and Bone Marrow Cells: A Literature Review and Relevance to the Chronotherapy of Cancer

By flow cytometry of individual cells, multiple cell properties can be analyzed. Such parameters may be important in relation to cytotoxic treatment of cancer. For example, DNA measurements will answer questions regarding cell kinetics. Myelosuppression is the major dose-limiting toxicity during cancer treatment. Therefore, the study of cell cycle parameters in bone marrow cells is highly relevant. However, inattention to the existence and potential importance of biological rhythms may introduce artifacts and misleading results. The literature of rhythms in hematology is reviewed. Time-dependent variations in hematological variables have been extensively studied and rhythms have been described for all kinds of blood cells. Also the numbers of hemopoietic stem cells in the bone marrow undergo circadian variations. Our group has shown how such variations change with aging in mice. The relevance of time sequence studies in aging research of hemopoiesis was clearly demonstrated. In animal studies using cytometry, our group has demonstrated extensive circadian variations in cell cycle distribution of bone marrow cells, especially the DNA synthesis (S-phase). In humans a few and rather small time sequence studies of the bone marrow have been performed, so far. In this overview the clinical implications of circadian rhythms of S-phase variations measured by flow cytometry of human bone marrow cells are discussed. Male volunteers were examined every 4 h around-the-clock. The data indicated a lower proliferative activity during night, suggesting the possibility of reducing the bone marrow toxicity to cancer treatment when taking these time-dependent variations into consideration.     

Cloning and characterization of human CAGLP gene encoding a novel EF-hand protein.

The EF-hand proteins, containing conserved Ca2+ binding motifs, play important roles in many biological processes. Through data mining, a novel human gene, CAGLP (calglandulin-like protein) was predicted and subsequently isolated from human skeleton muscle. The open reading frame of CAGLP is 543 bp in length, coding a putative Ca2+ binding protein with four EF-hand motifs. The deduced amino acid sequence of CAGLP displays high similarity with Bothrops insularis snake protein calglandulin (80%). The results of PCR amplification using cDNA from 17 human tissues indicated that human CAGLP is expressed in prostate, thymus, heart, skeleton muscle, bone marrow and ovary. Functional CAGLP::EGFP (enhanced green fluorescent protein) fusion protein revealed that CAGLP accumulated through-out Hela cells. Western blot using anti-EGFP antibodies indicated that the CAGLP protein has a molecular weight of about 19 kD. A phylogenetic tree showed that CAGLP and calglandulin may be orthologous proteins representing a distinct group in the EF-hand proteins.