Regulation of human cell engraftment and development of EBV-related lymphoproliferative disorders in Hu-PBL-scid mice

Human PBMC engraft in mice homozygous for the severe combined immunodeficiency (Prkdcscid) mutation (Hu-PBL-scid mice). Hu-PBL-NOD-scid mice generate 5- to 10-fold higher levels of human cells than do Hu-PBL-C.B-17-scid mice, and Hu-PBL-NOD-scid beta2-microglobulin-null (NOD-scid-B2mnull) mice support even higher levels of engraftment, particularly CD4+ T cells. The basis for increased engraftment of human PBMC and the functional capabilities of these cells in NOD-scid and NOD-scid-B2mnull mice are unknown. We now report that human cell proliferation in NOD-scid mice increased after in vivo depletion of NK cells. Human cell engraftment depended on CD4+ cells and required CD40-CD154 interaction, but engrafted CD4+ cells rapidly became nonresponsive to anti-CD3 Ab stimulation. Depletion of human CD8+ cells led to increased human CD4+ and CD20+ cell engraftment and increased levels of human Ig. We further document that Hu-PBL-NOD-scid mice are resistant to development of human EBV-related lymphoproliferative disorders. These disorders, however, develop rapidly following depletion of human CD8+ cells and are prevented by re-engraftment of CD8+ T cells. These data demonstrate that 1) murine NK cells regulate human cell engraftment in scid recipients; 2) human CD4+ cells are required for human CD8+ cell engraftment; and 3) once engrafted, human CD8+ cells regulate human CD4+ and CD20+ cell expansion, Ig levels, and outgrowth of EBV-related lymphoproliferative disorders. We propose that the Hu-PBL-NOD-scid model is suitable for the in vivo analysis of immunoregulatory interactions between human CD4+ and CD8+ cells.     

Human alpha-globin gene expression following chromosomal dependent gene transfer into mouse erythroleukemia cells.

We have used the genetic marker, adenine phosphoribosyl transferase (APRT), an enzyme known to be on human chromosome 16, to establish a method for the transfer of human α-globin genes into mouse erythroleukemia cells. Mouse erythroleukemia cells devoid of detectable levels of APRT were fused with fractions of human marrow enriched in human erythroid cells. The hybrid cells arising from this fusion were isolated in medium supplemented with aminopterin and thymidine, and used adenine as the sole purine source. This population of hybrid cells was dominated by cells (80%) in which human chromosome 16 was present. Human chromosomes 4, 5 and 6 were also found in these cells. The hybrid cells were then placed in medium supplemented with diaminopurine (DAP), which is lethal for cells containing APRT. Greater than 95% of the DAP-selected hybrid cells lacked human chromosome 16. Cytoplasmic RNA was extracted from the two hybrid cell populations and assayed by molecular hybridization for sequences coding for human α-globin. Carboxymethyl cellulose chromatography was used to study the level of synthesis of human a-globin in the hybrids. The original hybrid cell, which contained a high frequency of human chromosome 16, also contained high levels of human a-globin mRNA and human α-globin chains. Hybrid cells counter-selected in DAP and thus lacking human chromosome 16 were devoid of detectable levels of human APRT, human α-globin mRNA and human α-globin chains. This work shows that transfer of human chromosome 16 into the MEL cell is possible using a chromosomedependent, APRT-mediated method of gene transfer. Using this system in which expression of the human α-globin gene occurs, we were also able to confirm our earlier assignment of the human α-globin gene to human chromosome 16. This system may be of further use in identifying genetic elements governing expression of the human α-globin gene which can be carried with human chromosome 16 as it is donated to the mouse erythroleukemia cell by donor cells of different epigenotypes.     

Dysregulation of gene expression in the artificial human trisomy cells of chromosome 8 associated with transformed cell phenotypes

A change in chromosome number, known as aneuploidy, is a common characteristic of cancer. Aneuploidy disrupts gene expression in human cancer cells and immortalized human epithelial cells, but not in normal human cells. However, the relationship between aneuploidy and cancer remains unclear. To study the effects of aneuploidy in normal human cells, we generated artificial cells of human primary fibroblast having three chromosome 8 (trisomy 8 cells) by using microcell-mediated chromosome transfer technique. In addition to decreased proliferation, the trisomy 8 cells lost contact inhibition and reproliferated after exhibiting senescence-like characteristics that are typical of transformed cells. Furthermore, the trisomy 8 cells exhibited chromosome instability, and the overall gene expression profile based on microarray analyses was significantly different from that of diploid human primary fibroblasts. Our data suggest that aneuploidy, even a single chromosome gain, can be introduced into normal human cells and causes, in some cases, a partial cancer phenotype due to a disruption in overall gene expression.     

蓝舌病毒HbC3对几株人和动物肿瘤细胞的感染特性研究

用BTV-HbC3感染人肺癌SPC—A-1细胞,人宫颈癌HeLa细胞,人星型胶质瘤U251细胞,小鼠星形胶质瘤C6细胞及人胚肺HEL细胞后,观察细胞病变效应(CPE);运用透射电镜技术及琼脂双扩散试验检测BTV-HbC3对各种不同肿瘤细胞及人胚肺HEL细胞的感染性;并用RT-PCR技术检测蓝舌病毒的增殖情况。结果显示,BTV-HbC3对正常HEL不感染,但能在不同来源的某些肿瘤细胞中选择性增殖,产生不同程度的细胞病变效应(CPE)及调亡现象,终致肿瘤细胞死亡。其中以人肺癌SPC-A-1细胞对其最为敏感。因此,初步认为BTV-HbC3株能靶向性杀死某些肿瘤细胞,从而为深入开展BTV-HbC3靶向性抗肿瘤的研究提供了第一手实验室依据。     

Effect of IL-3 and stem cell factor on the appearance of human basophils and mast cells from CD34+ pluripotent progenitor cells.

Hemopoietic stem cell factor (SCF), which is the ligand for the proto-oncogene c-kit receptor (allelic with W locus) and the product of Sl locus of the mouse, has recently been cloned. The human homologue has also been cloned, and recombinant protein (human rSCF) expressed and purified to homogeneity. To determine the effect of human rSCF in the presence or absence of human rIL-3 on human bone marrow-derived mast cells and basophils, human CD34+ pluripotent progenitor cells, highly enriched (greater than 99%) from bone marrow mononuclear cells, were cultured over agarose surfaces (interphase cultures) in the presence of human rIL-3, human rIL-3 and increasing concentrations of human rSCF, or human rSCF alone. Over 3 to 4 wk, human rSCF acted synergistically with human rIL-3 at all concentrations, producing a three- to fivefold increase in total, mast cell, and basophil numbers over human rIL-3 alone when used at 100 ng/ml. The percentage of cell types in the human rIL-3 and human rIL-3 plus human rSCF cultures, however, remained the same, with basophils constituting 18 to 35% of the final cultured cells, and mast cells 3% or less of the final cell number. In the presence of human rSCF alone, the combined total percentage of mast cells and basophils was 0 to 1.0%, the majority of cells being macrophages. Mast cells cultured in human rIL-3 plus human rSCF, but not human rIL-3 alone, were berberine sulfate positive, suggesting the presence of heparin proteoglycans within granules. Electron microscopic examination of cultures supplemented with human rIL-3 and rSCF, but not human rIL-3 alone, revealed that after 3 wk in culture, mast cell granules contained tryptase and exhibited scroll, reticular, and homogeneous patterns as seen previously in CD34+/3T3 fibroblast cocultures. Thus, CD34+ cells cultured in the presence of both human rIL-3 and rSCF give rise to cultures containing increased numbers of basophils and mast cells, with the mast cells by ultrastructural studies showing evidence of maturation although the percentages of basophils and mast cells arising in these cultures remained unchanged.     

Transcriptional Profiling of Adult Neural Stem-Like Cells from the Human Brain

There is a great potential for the development of new cell replacement strategies based on adult human neural stem-like cells. However, little is known about the hierarchy of cells and the unique molecular properties of stem- and progenitor cells of the nervous system. Stem cells from the adult human brain can be propagated and expanded in vitro as free floating neurospheres that are capable of self-renewal and differentiation into all three cell types of the central nervous system. Here we report the first global gene expression study of adult human neural stem-like cells originating from five human subventricular zone biopsies (mean age 42, range 33–60). Compared to adult human brain tissue, we identified 1,189 genes that were significantly up- and down-regulated in adult human neural stem-like cells (1% false discovery rate). We found that adult human neural stem-like cells express stem cell markers and have reduced levels of markers that are typical of the mature cells in the nervous system. We report that the genes being highly expressed in adult human neural stem-like cells are associated with developmental processes and the extracellular region of the cell. The calcium signaling pathway and neuroactive ligand-receptor interactions are enriched among the most differentially regulated genes between adult human neural stem-like cells and adult human brain tissue. We confirmed the expression of 10 of the most up-regulated genes in adult human neural stem-like cells in an additional sample set that included adult human neural stem-like cells (n = 6), foetal human neural stem cells (n = 1) and human brain tissues (n = 12). The NGFR, SLITRK6 and KCNS3 receptors were further investigated by immunofluorescence and shown to be heterogeneously expressed in spheres. These receptors could potentially serve as new markers for the identification and characterisation of neural stem- and progenitor cells or as targets for manipulation of cellular fate.     

Binding of human interferon alpha to cells of different sensitivities: studies with internally radiolabeled interferon retaining full biological activity.

The characteristics of interferon binding to various cells with different interferon sensitivity were studied by using [3H]leucine-labeled, pure human interferon alpha from Namalwa cells. Scatchard analysis of the binding data on cells sensitive to interferon alpha (human FL and fibroblasts and bovine MDBK) indicated the presence of two kinds of binding sites with high and low affinities. The binding constants of the high-affinity sites in these cells were similar (4 X 10(10) to 11 X 10(10) M-1). Cells insensitive to human interferon alpha (human HEC-1 and mouse L cells) were shown to have only low-affinity sites, suggesting that high-affinity binding sites are indispensable for interferon sensitivity and represent interferon receptors. However, the number of sites in three human diploid fibroblast strains and one strain trisomic for chromosome 21 were not proportionally correlated to the interferon sensitivity of the cells. The high-affinity binding to human cells was completely inhibited by both nonradioactive human interferons alpha and beta in a similar manner, but binding to bovine MDBK cells, on which human interferon beta is practically inactive, was inhibited effectively only by interferon alpha and not by beta. These results suggest that the receptor for human interferon alpha is common to human interferon beta in human cells, whereas the receptor on bovine cells binds only human interferon alpha.     

Humanized mice mount specific adaptive and innate immune responses to EBV and TSST-1

Here we show that transplantation of autologous human hematopoietic fetal liver CD34+ cells into NOD/SCID mice previously implanted with human fetal thymic and liver tissues results in long-term, systemic human T-cell homeostasis. In addition, these mice show systemic repopulation with human B cells, monocytes and macrophages, and dendritic cells (DCs). T cells in these mice generate human major histocompatibility complex class I- and class II-restricted adaptive immune responses to Epstein-Barr virus (EBV) infection and are activated by human DCs to mount a potent T-cell immune response to superantigens. Administration of the superantigen toxic shock syndrome toxin 1 (TSST-1) results in the specific systemic expansion of human Vbeta2+ T cells, release of human proinflammatory cytokines and localized, specific activation and maturation of human CD11c+ dendritic cells. This represents the first demonstration of long-term systemic human T-cell reconstitution in vivo allowing for the manifestation of the differential response by human DCs to TSST-1.     

Adenovirus type 2 expresses fiber in monkey-human hybrids and reconstructed cells.

Adenovirus type 2 protein expression was measured by indirect immunofluorescence in monkey-human hybrids and in cells reconstructed from monkey and human cell karyoplasts and cytoplasts. Monkey-human hybrid clones infected with adenovirus type 2 expressed fiber protein, whereas infected monkey cells alone did not. Hybrids constructed after the parental monkey cells were infected with adenovirus type 2 demonstrated that fiber synthesis in these cells could be rescued by fusion to uninfected human cells. Thus, human cells contain a dominant factor that acts in trans and overcomes the inability of monkey cells to synthesize fiber. Cells reconstructed from infected human karyoplasts and monkey cytoplasts expressed fiber, whereas cells reconstructed from infected monkey karyoplasts and human cytoplasts did not. These results are consistent with the hypothesis that the block to adenovirus replication in monkey cells involves a nuclear event that prevents the formation of functional mRNA for some late viral proteins including fiber polypeptide. Furthermore, they suggest that the translational apparatus of monkey cells is competent to translate functional fiber mRNA synthesized in human cells.     

IL-3-dependent growth of basophil-like cells and mastlike cells from human bone marrow

Human bone marrow cultured in the presence of human rIL-3 has been reported to give rise to basophils. In contrast, mouse bone marrow, cultured in the presence of mouse IL-3, leads to the growth of mast cells. To determine if human rIL-3 might also stimulate the growth of human mast cells, we cultured human bone marrow in the presence of human rIL-3 in suspension cultures, methylcellulose, and in "interphase" cultures where cells are layered over agar. The presence of mast cells was determined using a variety of histochemical techniques. In agreement with previous reports, basophil-like cells were identified in all culture systems. Mastlike cells were identified only in interphase cultures. By 3 wk, such cultures consisted of basophil-like cells (20 to 50%) and mastlike cells (1 to 5%). Cultures supplemented with rIL-4 showed no additional increase in basophil-like and mastlike cells. Both basophil-like and mastlike cells fluoresced with o-phthaldialdehyde and exhibited IgE receptors. Unlike basophil-like cells, mastlike cells were chloroacetate esterase, amidase, and human mast cell tryptase positive. We conclude that human rIL-3 can support the growth of human mastlike cells under selected culture conditions.     

Unrepaired DNA damage facilitates elimination of uniparental chromosomes in interspecific hybrid cells

Elimination of uniparental chromosomes occurs frequently in interspecific hybrid cells. For example, human chromosomes are always eliminated during clone formation when human cells are fused with mouse cells. However, the underlying mechanisms are still elusive. Here, we show that the elimination of human chromosomes in human–mouse hybrid cells is accompanied by continued cell division at the presence of DNA damage on human chromosomes. Deficiency in DNA damage repair on human chromosomes occurs after cell fusion. Furthermore, increasing the level of DNA damage on human chromosomes by irradiation accelerates human chromosome loss in hybrid cells. Our results indicate that the elimination of human chromosomes in human–mouse hybrid cells results from unrepaired DNA damage on human chromosomes. We therefore provide a novel mechanism underlying chromosome instability which may facilitate the understanding of carcinogenesis.     

Role of integrin switch and transforming growth factor Beta 3 in hypoxia-induced invasion inhibition of human extravillous trophoblast cells

The physiological hypoxic condition favors the angiogenesis in the placenta. However, it remains unclear how hypoxia regulates the invasion of human extravillous trophoblast cells. In the present study, we first showed that alpha5 integrin expression increased and alpha1 integrin expression decreased in human extravillous trophoblast cells cultured in 1% oxygen as compared with control cells cultured in 8% oxygen. Further data showed that the neutralizing antibody against alpha5 integrin increased the invasion of human extravillous trophoblast cells and the neutralizing antibody against alpha1 integrin inhibited the invasion of human extravillous trophoblast cells. Human extravillous trophoblast cells cultured in 1% oxygen showed reduced invasive capacity, which can be effectively blocked by alpha5 integrin neutralizing antibody. Moreover, human extravillous trophoblast cells exposed to 1% oxygen demonstrated increased expression of transforming growth factor-beta3 (TGFB3), and recombinant human TGFB3 inhibited the invasion of human extravillous trophoblast cells in a dose-dependent manner. The neutralizing antibodies against alpha5 integrin and TGFB3 markedly abrogated hypoxia-induced invasion inhibition in human extravillous trophoblast cells. These data indicate that hypoxia may inhibit the invasion of human extravillous trophoblast cells through inducing the integrin switch from alpha1 integrin to alpha5 integrin and promoting TGFB3 expression.     

Human Canalicular Multispecific Organic Anion Transporter (cMOAT) Is Expressed in Human Lung,Gastric, and Colorectal Cancer Cells

Human canalicular multispecific organic anion transporter (cMOAT), a glutathione conjugate membrane transporter, has been isolated from cisplatin-resistant cancer cells and is distributed mainly in normal liver. We analyzed the expression of human cMOAT in 14 lung, 11 gastric, and 9 colorectal non-drug-selected human cancer cells, two multidrug-resistant cells, and one cisplatin-resistant cells, using quantitative RT-PCR and newly developed anti-human cMOAT antibody. All cell lines analyzed here expressed human cMOAT at the level of mRNA and protein, and some of them expressed higher levels of human cMOAT than the cisplatin-resistant cells. The two multidrug-resistant cell lines co-expressed human cMOAT gene and both or either of MRP and MDR1 genes. Immunostaining showed that human cMOAT was predominantly localized to the cytoplasm of these single cells. Our results indicate that human cMOAT is expressed in various human cancer cells including drug-resistant cells.     

A peripheral mechanism preserves self-tolerance to a secreted protein in transgenic mice

We have examined mechanisms of tolerance to circulating self-proteins in mice that are transgenic for human insulin. Normal, nontransgenic mice develop serum antibody responses when injected with human insulin in CFA; syngeneic transgenic mice do not. B cell responsiveness was assessed by immunizing with human insulin coupled to a T-independent Ag, Brucella abortus. No differences were found in the numbers of insulin-specific splenic plaque-forming cells between transgenic and nontransgenic mice suggesting that insulin-specific B cells are not tolerant in transgenic mice. Similarly, APC from transgenic and nontransgenic mice display no differences in their ability to process and present human insulin to human insulin-specific T cells in vitro. However, marked differences were detected between transgenic and nontransgenic T cells. Lymph node T cells from transgenic mice primed with human insulin provided no detectable helper activity for secondary antibody responses to human insulin whereas, lymph node T cells from nontransgenic mice did. Nevertheless, lymph node T cells from transgenic mice developed significant proliferative responses to human insulin. Lymph node T cells obtained from transgenic and nontransgenic mice were fused to BW5147 and human insulin-specific T cell hybridomas were generated. The fact that human insulin-specific T cell hybridomas were obtained from the transgenic mice suggests that these T cells were not clonally deleted. In addition, APC from transgenic mice did not stimulate human insulin-specific hybridomas from normal mice in the absence of exogenous insulin. We suggest that T cells specific for human insulin are not deleted in the thymus of transgenic mice because APC in the thymus do not bear the requisite levels of endogenous human insulin/Ia complexes. Therefore, we conclude that tolerance in the transgenic mice is preserved by peripheral mechanisms.     

Species-specific posttranscriptional regulation of interferon synthesis

Human fibroblast and Syrian hamster embryo cells were induced to synthesize interferon (IF) with rIn . rCn and rIn . rCn + DEAE-dextran, respectively. Following induction, these cells synthesized IF for only a short time before entering into a repressed state and shutting off the synthesis of IF. Homologous and heterologous whole cell translational systems were developed to investigate the molecular basis for the shut-off of IF synthesis. These systems allowedd for the introduction of exogenous hamster and human IF-mRNAs into intact normal and repressed hamster and human cells via an improved CaCl2 precipitation technique. Human IF-mRNA was translated in normal human and hamster cells and in repressed hamster cells but not in repressed human cells. In contrast, the hamster IF-mRNA was translated in normal human, normal hamster, and repressed human cells but not in repressed hamster cells. These results indicate that a species-specific mechanism inhibiting translation of IF-mRNA is directly responsible for the shut-off of IF synthesis in human fibroblasts and Syrian hamster embryo cells.     

Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells

Ethical considerations constrain the in vivo study of human hemopoietic stem cells (HSC). To overcome this limitation, small animal models of human HSC engraftment have been used. We report the development and characterization of a new genetic stock of IL-2R common gamma-chain deficient NOD/LtSz-scid (NOD-scid IL2Rgamma(null)) mice and document their ability to support human mobilized blood HSC engraftment and multilineage differentiation. NOD-scid IL2Rgamma(null) mice are deficient in mature lymphocytes and NK cells, survive beyond 16 mo of age, and even after sublethal irradiation resist lymphoma development. Engraftment of NOD-scid IL2Rgamma(null) mice with human HSC generate 6-fold higher percentages of human CD45(+) cells in host bone marrow than with similarly treated NOD-scid mice. These human cells include B cells, NK cells, myeloid cells, plasmacytoid dendritic cells, and HSC. Spleens from engrafted NOD-scid IL2Rgamma(null) mice contain human Ig(+) B cells and lower numbers of human CD3(+) T cells. Coadministration of human Fc-IL7 fusion protein results in high percentages of human CD4(+)CD8(+) thymocytes as well human CD4(+)CD8(-) and CD4(-)CD8(+) peripheral blood and splenic T cells. De novo human T cell development in NOD-scid IL2Rgamma(null) mice was validated by 1) high levels of TCR excision circles, 2) complex TCRbeta repertoire diversity, and 3) proliferative responses to PHA and streptococcal superantigen, streptococcal pyrogenic exotoxin. Thus, NOD-scid IL2Rgamma(null) mice engrafted with human mobilized blood stem cells provide a new in vivo long-lived model of robust multilineage human HSC engraftment.     

Tracking of human cells in mice

Tracking and tracing of transplanted cells in mice is required in many fields of research. Examples are transplantation of stem cells into organs of mice to study their differentiation capacity and injection of tumor cells to examine metastatic behavior. In the present study we tested the lipid dye CM-DiI and red fluorescent nanoparticles Qdot655 for their applicability in tagging and tracing of human cells in mice. Labeling of different cell types, including MCF-7 human breast cancer cells, human cord blood derived cells, human NeoHep cells and human hepatopancreatic precursor cells, is technically easy and did not compromise further cell culture. After transplantation of CM-DiI or Qdot655 marked cells, red fluorescent structures could be detected already in unprocessed paraffin slices of the studied organs, namely liver, lung, pancreas, kidney, spleen and bone marrow. Next, we examined whether the red fluorescent structures represent the transplanted human cells. For this purpose, we established an in situ hybridization (ISH) technique that allows clear-cut differentiation between human and murine nuclei, based on simultaneous hybridization with human alu and mouse major satellite (mms) probes. We observed a high degree of coincidence between CM-DiI-marked cells and alu positive nuclei. However, also some mms positive cells contained CM-DiI, suggesting phagocytosis of the transplanted CM-DiI-marked cells. The degree of such CM-DiI-positive mouse cells depended on the cell type and route of administration. From a technical point of view it was important that CM-DiI-positive structures in paraffin slices remained fluorescent also after ISH. In contrast, Qdot655 positive structures faded during further staining procedures. In conclusion, marking of cells with CM-DiI or Qdot655 prior to transplantation facilitates recovery of human cells, since a high fraction of positive structures in the host's tissue originate from the transplanted cells. However, CM-DiI or Qdot655 positive staining of individual cells in transplanted tissues is not sufficient to prove their human origin. Additional procedures, such as ISH with alu-probes, are essential, when characterizing individual cells.