Cells In SCID Mice Research Articles

BCR/ABL Kinase Elevates ROS-Mediated Oxidative DNA Damage in CML Stem/Progenitor Cells and Affects the Efficiency and Fidelity of DNA Repair To Induce Genetic Instability.

BCR/ABL kinase transforms hematopoietic stem cells (HSCs) to induce chronic myelogenous leukemia in chronic phase (CML-CP), which eventually evolves into fatal blast crisis (CML-BC). CML is a stem cell-derived but a progenitor-driven disease. In CML-CP leukemia stem (LSCs) and progenitor (LPCs) cells reside in CD34+CD38− and CD34+CD38+ populations, respectively, whereas in CML-BC LSCs are found also in CD34+CD38+ population. BCR/ABL kinase stimulates genomic instability causing imatinib-resistant point mutations and chromosomal aberrations associated with progression to CML-BC. Genomic instability may result from enhanced DNA damage and/or aberrant DNA repair mechanisms. We showed that CD34+ stem/progenitor CML cells contain higher levels of reactive oxygen species (ROS) than these from healthy donors (CML-BC>CML-CP>Normal). In addition, ROS were elevated in CD34+CD38− and CD34+CD38+ sub-populations isolated from CML-BC and CML-CP patients in comparison to cells from healthy donor. Higher ROS levels induced more oxidative DNA lesions such as oxidized bases (e.g., 8-oxoG) and DNA double-strand breaks (DSBs). ROS and oxidative DNA damage in CML stem/progenitor cells could be diminished by an antioxidant N-acetyl-cysteine. Moreover, inhibition of ROS by vitamin E reduced the frequency of imatinib-resistant BCR/ABL point mutants and chromosomal aberrations in leukemia cells in SCID mice. Cellular DNA repair systems act to remove DNA damage and ultimately preserve the informational integrity of the genome. Base excision repair (BER) and mismatch repair (MMR) are responsible for removal of oxidized bases. BER was assessed using single- and double-stranded DNA substrates containing 5-OH-U (a derivative of ROS-damaged hydroxy-deoxycytidine). MMR activity was measured by restoration of the expression of GFP from the construct containing T-G mismatch in the start codon. BCR/ABL kinase severely inhibited BER and MMR in cell lines and CD34+ CML cells, and promoted accumulation of point mutations in genes encoding BCR/ABL kinase and Na+/K+ ATPase. Inhibition of BCR/ABL kinase by imatinib restored BER and MMR activities. Oxidized bases, if not repaired, may lead to accumulation of DSBs observed in LSCs and LPCs. DSBs may be processed by homologous recombination (HR), non-homologous and-joininig (NHEJ), and single-strand annealing (SSA). HR represents faithful repair, NHEJ usually produces small deletions, and SSA causes very large deletions. Genome-integrated repair-specific reporter cassettes containing two disrupted fragments of the gene encoding GFP were used where a single DSB induced by I-SceI endonuclease in one of the fragments stimulated HR, NHEJ, or SSA. In general, BCR/ABL kinase enhanced DSBs repair activities, however at the expense of their fidelity. Numerous point mutations were introduced in HR repair products. NHEJ generated larger than usual deletions. SSA, rather rare but very unfaithful, was also induced in BCR/ABL-positive leukemia cells. In summary, BCR/ABL kinase enhanced ROS-mediated oxidative DNA damage in LSCs and LPCs. In addition, BCR/ABL inhibited BER and MMR of usually non-lethal oxidized DNA lesions leading to accumulation of point mutations. Moreover, BCR/ABL kinase stimulated HR, NHEJ and SSA of lethal DSBs, but compromised the fidelity of repair.

Effective Ablation of Pancreatic Cancer Cells in SCID Mice Using Systemic Adenoviral RIP-TK/GCV Gene Therapy

Studies have demonstrated that adenovirus subtype 5 mediated rat insulin promoter directed thymidine kinase (A-5-RIP-TK)/ganciclovir (GCV) gene therapy resulted in significant enhanced cytotoxicity to both PANC-1 and MIA PaCa2 pancreatic cancer cells in vitro. However, little is known about the effect in vivo. In this study we examine the in vivo safety and efficacy of intravenous A-5-RIP-TK/GCV gene therapy. 1 x 10(6) Mia PaCa2 cells were injected intraperitoneally (i.p.) into SCID mice to create a mouse model of human pancreatic cancer. A-5-RIP-TK gene construct was administered intravenously (i.v.), followed by i.p. GCV administration. Intravenous injection of A-5-RIP-lacZ reporter gene constructs was used for evaluation of Ad-RIP-gene expression in tumors and other tissues. Optimal adenoviral and GCV doses and treatment duration were determined. Tumor volume, serum insulin, and glucose levels were measured. Immunohistochemical staining of pancreata and tumors were performed to assess morphology and hormone expression and apoptotic rates were determined. All A-5-RIP-TK/GCV-treated mice had reduced tumor volume compared with controls, but maximal tumor volume reduction was observed with 10(8) vp followed by GCV treatment for 4 wk. A-5-RIP-TK/GCV gene therapy contributed to significant survival advantage in MIA PaCa2 bearing mice, and the greatest survival benefit was observed with 10(8) vp and was not affected by length of treatment of GCV. A-5-RIP-TK/GCV therapy increased PDX-1 expression and tumor cells apoptosis, and altered islet morphology. However, A-5-RIP-TK/GCV gene therapy caused diabetes associated with islet cell apoptosis, increased delta-cells and reduced pancreatic polypeptide (PP)-cell numbers. Systemically administered A-5-RIP-TK/GCV is an effective treatment of pancreatic cancer. A-5-RIP-TK/GCV cytotoxicity to malignant cells varies with adenoviral dose and length of GCV treatment. However, A-5-RIP-TK/GCV is associated with islet cell toxicity and diabetogenesis. The type of diabetes observed is distinct from Types 1 and 2 and is associated with islet cell apoptosis and reduced delta- and PP-cells.

IL-28 Elicits Antitumor Responses against Murine Fibrosarcoma

IL-28 is a recently described antiviral cytokine. In this study, we investigated the biological effects of IL-28 on tumor growth to evaluate its antitumor activity. IL-28 or retroviral transduction of the IL-28 gene into MCA205 cells did not affect in vitro growth, whereas in vivo growth of MCA205IL-28 was markedly suppressed along with survival advantages when compared with that of controls. When the metastatic ability of IL-28-secreting MCA205 cells was compared with that of controls, the expression of IL-28 resulted in a potent inhibition of metastases formation in the lungs. IL-28-mediated suppression of tumor growth was mostly abolished in irradiated mice, indicating that irradiation-sensitive cells, presumably immune cells, are primarily involved in the IL-28-induced suppression of tumor growth. In vivo cell depletion experiments displayed that polymorphonuclear neutrophils, NK cells, and CD8 T cells, but not CD4 T cells, play an equal role in the IL-28-mediated inhibition of in vivo tumor growth. Consistent with these findings, inoculation of MCA205IL-28 into mice evoked enhanced IFN-gamma production and cytotoxic T cell activity in spleen cells. Antitumor action of IL-28 is partially dependent on IFN-gamma and is independent of IL-12, IL-17, and IL-23. IL-28 increased the total number of splenic NK cells in SCID mice and enhanced IL-12-induced IFN-gamma production in vivo and expanded spleen cells in C57BL/6 mice. Moreover, IL-12 augmented IL-28-mediated antitumor activity in the presence or absence of IFN-gamma. These findings indicate that IL-28 has bioactivities that induce innate and adaptive immune responses against tumors.

32: Effective ablation of human pancreatic cancer cells in SCID mice using systemic adenoviral rat insulin promoter directed thymidine kinase gene therapy

32: Effective ablation of human pancreatic cancer cells in SCID mice using systemic adenoviral rat insulin promoter directed thymidine kinase gene therapy

Cotransplantation of Cord Blood Hematopoietic Stem Cells and Culture-Expanded and GM-CSF-/SCF-Transfected Mesenchymal Stem Cells in SCID Mice

Mesenchymal stem cells (MSC) are multipotent in nature and believed to facilitate the engraftment of hematopoietic stem cells (HSC) when transplanted simultaneously in animal studies and even in human trials. In this study, we transfected culture-expanded MSC with granulocyte macrophage-colony stimulating factor (GM-CSF) and stem cell factor (SCF) cytokine genes and then cotransplanted with mononuclear cells (MNC) to further promote HSC engraftment. MNC were harvested from cord blood and seeded in long-term culture for ex vivo MSC expansion. A total of 1×107 MNC plus MSC/µL were introduced to the tail vein of nonobese diabetic/severe combined immunodeficiency mice. After 6-8 weeks later, homing and engraftment of human cells were determined by flow cytometry and fluorescence in situ hybridization studies. The total nucleated cell count and the engraftment of CD45+/CD34+ cells and XX or XY positive human cells were significantly increased in cotransplanted mice and even higher with the cytokine gene-transfected MSC (GM-CSF>SCF, p<0.05) than in transplantation of MNC alone. These results suggest that MSC transfected with hematopoietic growth factor genes are capable of enhancing the hematopoietic engraftment. Delivering genes involved in homing and cell adhesions, CXCR4 or VLA, would further increase the efficiency of stem cell transplantation in the future.

Recombinant Bispecific Monoclonal Antibody (bsMAb) Against CD20 and CD22 Active In Vitro and In Vivo Against B-Cell Lymphomas.

Background: Preclinical and clinical results suggest improved anti-lymphoma activity of combining anti-CD20 and anti-CD22 MAbs. Our aim was to develop a recombinant bispecific MAb against CD20 and CD22 antigens, and to evaluate its anti-tumor potency compared to the parental MAbs.Methods: Tetravalent anti-CD20/CD22 bsMAb in the form of anti-CD20 IgG linked to two anti-CD22 scFv's was prepared recombinantly. The ability of the bsMAb to inhibit cell growth and mediate CDC and ADCC was evaluated by cell-based assays. Phosphorylation and distribution of CD22 in B-lymphoma cells treated with the bsMAb were studied by immunoblotting. The extension of survival of disseminated Daudi lymphoma cells in SCID mice also was evalauted.Results: In contrast to the parental anti-CD22 MAb, epratuzumab, the bsMAb did not internalize in Ramos cells. In CDC tests, the bsMAb showed no cytotoxic effects, similar to epratuzumab, although it did bind C1q complement protein, unlike epratuzumab. In ADCC, the bsMAb was as potent as the parental humanized anti-CD20 Mab, hA20, in inducing target-cell lysis. The anti-CD20/CD22 bsMAb has distinct effects on NHL B-cell lines compared to each parental monospecific MAb or in combination:<10 nM bsMAb causes cells to aggregate;in the soluble, non-crosslinked form, while none of the parental MAbs alone or mixed had significant anti-proliferative activity, the recombinant bsMAb was strongly anti-proliferative;the anti-proliferation activities induced by an anti-IgM Ab and the bsMAb were synergistic; andthe bsMAb resulted in rapid redistribution of CD22 into detergent-resistant membrane microdomains on the cell surface.Initial animal studies show that the bsMab significantly extended survival over control SCID mice.Conclusion: The recombinantly fused anti-CD20/CD22 bsMAb has new properties compared to the parental monospecific MAbs, and may represent a new class of potential therapeutic agents that could replace binary combinations of antibodies.

Restored Expression of the MYO18B Gene Suppresses Orthotopic Growth and the Production of Bloody Pleural Effusion by Human Malignant Pleural Mesothelioma Cells in SCID Mice

Malignant pleural mesothelioma (MPM) is closely related to exposure to asbestos, and a rapid increase in the number of MPM patients is therefore estimated to occur from 2010 to 2040 in Japan. Because MPM is refractory to conventional chemotherapy and radiotherapy, the prognosis of MPM patients is extremely poor. MYO18B, a novel member of the myosin family, is a tumor suppressor gene isolated from a homozygously deleted region at 22q12.1 in a lung cancer cell line. The inactivation of the MYO18B gene plays an important role in several malignant diseases. However, the role of MYO18B in the progression of MPM is still unknown. Six different human MPM cell lines were used in this study. Western blot revealed that none of the cell lines expressed a detectable level of MYO18B protein. One of the MPM cell lines, EHMES-10, was transfected with the MYO18B gene. We found that a restored expression of the MYO18B protein in EHMES-10 cells resulted in the inhibition of their anchorage-independent growth and motility in vitro. In addition, it also inhibited their ectopic (subcutaneous space) and orthotopic (thoracic cavity) growth in SCID mice, in association with an increased degree of cell apoptosis. Furthermore, it also suppressed the production of bloody pleural effusion after orthotopic injection. These findings suggest that the restored expression of MYO18B may be a useful therapeutic strategy for the treatment of locally advanced MPM in humans.

Establishment of a Waldenstrom's Macroglobulinemia Cell Line (BCWM.1) with Productive In Vivo Engraftment in SCID-hu Mice.

Waldenstrom's Macroglobulinemia (WM) is incurable B-cell malignancy characterized by the infiltration of the bone marrow infiltration with lymphoplasmacytic cells (LPC) and the presence of an IgM monoclonal gammopathy. A significant impairment in understanding the biology and advancing therapeutics for WM has been the lack of a representative cell line and animal model. We therefore report on work accomplished in our laboratories in establishing the BWMC.1 cell line, which was derived from long term culture of CD19+ selected lymphoplasmacytic cells isolated from the bone marrow (BM) aspirate of a previously untreated 55-year-old female patient with IgMk secreting WM. BCWM.1 cells morphologically resembled LPC and had prominent nucleoli and ample cytoplasm by Wright-Giemsa staining, and were propagated in RPMI 1640 medium supplemented with 10% FBS. Phenotypic characterization by flow cytometric analysis demonstrated typical WM LPC characteristics: CD5−, CD10−, CD19+, CD20+, CD23+, CD27−, CD38+, CD138+, CD40+, CD52+, CD70+, CD117+, cIgM+, cIgG−, cIgA−, ck+, cl−. BCWM.1 cells also expressed the survival proteins APRIL and B-Lymphocyte Stimulator (B-LYS), and their receptors TACI, BCMA and BAFF-R. ELISA studies demonstrated secretion of IgMk in culture. Karyotypic and M-FISH studies did not demonstrate cytogenetic abnormalities. Molecular analysis of BCWM.1 cells confirmed clonality by determination of IgH rearrangements through FR1c/JHc amplification of genomic DNA. BCWM.1 cells were positive for LMP1 expression by histochemical staining and PCR analysis. Importantly, inoculation of BCWM.1 cells directly into the human BM milieu of SCID-hu mice resulted in engraftment of tumor cells and serum detection of human IgMk in four of five SCID-hu mice within two weeks. In contrast, subcutaneous injection of BCWM.1 cells in SCID mice did not lead to engraftment, suggesting a role for the human BM microenvironment in supporting the expansion of BCWM.1 cells. These studies therefore support the use of BCWM.1 cells as an appropriate model for the study of WM, which in conjunction with the SCID-hu mouse model may be used as a convenient model for studies focused on both the pathogenesis and development of targeted therapies for WM.

The Vascular-Ablative Agent VEGF121/rGel Inhibits Pulmonary Metastases of MDA-MB-231 Breast Tumors

VEGF121/rGel, a fusion protein composed of the growth factor VEGF121 and the recombinant toxin gelonin (rGel), targets the tumor neovasculature and exerts impressive cytotoxic effects by inhibiting protein synthesis. We evaluated the effect of VEGF121/rGel on the growth of metastatic MDA-MB-231 tumor cells in SCID mice. VEGF121/rGel treatment reduced surface lung tumor foci by 58% compared to controls (means were 22.4 and 53.3, respectively; P < .05) and the mean area of lung colonies by 50% (210 ± 37 m2vs 415 ± 10 m2 for VEGF121/rGel and control, respectively; P < .01). In addition, the vascularity of metastatic foci was significantly reduced: (198 ± 37 vs 388 ± 21 vessels/mm2 for treated and control, respectively). Approximately 62% of metastatic colonies from the VEGF121/rGel-treated group had fewer than 10 vessels per colony compared to 23% in the control group. The VEGF receptor Flk-1 was intensely detected on the metastatic vessels in the control but not in the VEGF121/rGel-treated group. Metastatic foci present in lungs had a three-fold lower Ki-67 labeling index compared to control tumors. Thus, the antitumor vascular-ablative effect of VEGF121/rGel may be utilized not only for treating primary tumors but also for inhibiting metastatic spread and vascularization of metastases.

452. The Enhancement of Anticancer Effect of Chemotherapy by the Delivery of Rad51 siRNA

Top of pageAbstract Chemotherapy is one of the effective therapies to cancer, however, cancers gradually develop machineries to resist to chemotherapy. To overcome the problem of drug resistance, we tested the combination of short interfering RNA (siRNA) against Rad51, related to the homologous recombination repair, with cis-diamminedichloroplatinum (II) (CDDP; cisplatin). Moreover, to deliver siRNA into cancer cells effectively, we have developed the HVJ (hemagglutinating virus of Japan, Sendai virus) envelope vector which has the high membrane fusion ability. When synthetic Rad51 siRNA was delivered to HeLa cells using HVJ envelope vector, no Rad51 transcripts were detected on day 2, and Rad51 protein completely disappeared for 4 days after siRNA transfer. When HeLa cells were incubated with 0.02 ug/ml CDDP for 3 hours after siRNA transfer, the number of colonies decreased to approximately 10% of that with scrambled siRNA. The sensitivity to CDDP was enhanced in various human cancer cells, but not in normal human fibroblasts. The synthetic Rad51 siRNA was also introduced into subcutaneous tumor mass of HeLa cells in SCID mice with or without intraperitoneal injection of CDDP, and tumor growth was monitored. When Rad51 siRNA was delivered into tumors using HVJ envelope vector, the delivery efficiency was approximately 50%, and the Rad51 transcript level was reduced to approximately 25%. Rad51 siRNA combined with CDDP significantly inhibited the tumor growth when compared to siRNA or CDDP alone. Rad51 siRNA could enhance the sensitivity to CDDP in cancer cells both in vitro and in vivo. Rad51 siRNA also enhanced the sensitivity of cancer cells to bleomycin. Our results suggest that the combination of CDDP and Rad51 siRNA will be an effective anticancer protocol.

Identification of a detrimental role for NK cells in pneumococcal pneumonia and sepsis in immunocompromised hosts

Gram-positive sepsis is a major disease problem. However, the contribution of various immune cell types to pathogenesis remains unclear. By infecting scid and wild type BALB/c mice with Streptococcus pneumoniae we have found a situation in which natural killer (NK) cells can play a detrimental role in the response to infection. scid mice were found to be significantly more susceptible to local and systemic pneumococcal infection than controls; they had significantly higher bacterial loads, elevated inflammatory responses and more widespread lung pathology. Interestingly, depletion of NK cells in scid mice resulted in significantly lower bacteraemia and inflammatory cytokine production. Infection with pneumococci deficient in pneumolysin revealed the toxin was involved in cytokine production. Overall results indicate that elevated NK cell activity during pneumococcal pneumonia amplifies pulmonary and systemic inflammation, increases bacteraemia and results in poor outcome.

Rad51 siRNA delivered by HVJ envelope vector enhances the anti‐cancer effect of cisplatin

Every cancer therapy appears to be transiently effective for cancer regression, but cancers gradually transform to be resistant to the therapy. Cancers also develop machineries to resist chemotherapy. Short interfering RNA (siRNA) has been evaluated as an attractive and effective tool for suppressing a target protein by specifically digesting its mRNA. Suppression of the machineries using siRNA may enhance the sensitivity to chemotherapy in cancers when combined with an effective delivery system. To enhance the anti-cancer effect of chemotherapy, we transferred siRNA against Rad51 into various human cancer cells using the HVJ (hemagglutinating virus of Japan, Sendai virus) envelope vector in the presence or absence of cis-diamminedichloroplatinum(II) (CDDP, cisplatin). The inhibition of cell growth was assessed by a modified MTT assay, counting cell number, or fluorescence-activated cell sorting (FACS) analysis after Annexin V labeling. The synthetic Rad51 siRNA was also introduced into subcutaneous tumor masses of HeLa cells in SCID mice with or without intraperitoneal injection of CDDP, and tumor growth was monitored. When synthetic Rad51 siRNA was delivered into HeLa cells using the HVJ envelope vector, no Rad51 transcripts were detected on day 2, and Rad51 protein completely disappeared for 4 days after siRNA transfer. When HeLa cells were incubated with 0.02 microg/ml CDDP for 3 h after siRNA transfer, the number of colonies decreased to approximately 10% of that with scrambled siRNA. The sensitivity to CDDP was enhanced in various human cancer cells, but not in normal human fibroblasts. When Rad51 siRNA was delivered into tumors using the HVJ envelope vector, the Rad51 transcript level was reduced to approximately 25%. Rad51 siRNA combined with CDDP significantly inhibited tumor growth when compared to siRNA or CDDP alone. Rad51 siRNA could enhance the sensitivity to CDDP in cancer cells both in vitro and in vivo. Our results suggest that the combination of CDDP and Rad51 siRNA will be an effective anti-cancer protocol.

In Vitro and In Vivo Targeting and Therapy of an Antibody-Drug Conjugate (IMMU-110) in B-Cell Malignancies.

IMMU-110 is a drug immunoconjugate comprised of doxorubicin (DOX) conjugated to the humanized anti-CD74 monoclonal antibody (mAb), hLL1, at a DOX:mAb (mol/mol) ratio of 8:1. CD74 is a rapidly internalizing type-II transmembrane chaperone molecule associated with HLA-DR, and has high expression on human non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM) clinical specimens and cell lines. Here, we investigated the in vitro and in vivo efficacy of IMMU-110 in xenograft models of human NHL (Raji, Daudi) and MM (MC/CAR). In vitro cell binding of IMMU-110 with the CD74-positive cells was significantly higher than that of a non-specific isotype-matched mAb-DOX conjugate (DOX conjugated to a mAb against epithelial glycoprotein-1; DOX-hRS7), and was similar to that of naked hLL1. Both IMMU-110 and naked hLL1 bound CD74 with subnanomolar affinity. The in vitro cytotoxicity of IMMU-110 was significantly higher than non-specific antibody-DOX conjugate, DOX-hRS7, and was similar to free DOX in MC/CAR, Raji or Daudi human Burkitt's lymphoma cells. In CD74-negative cell lines, IMMU-110 was significantly less toxic than free DOX, having similar cytotoxicity to DOX-hRS7. In vivo, IMMU-110 displayed a pharmacokinetic and biodistribution profile almost identical to that of hLL1 mAb. Both hLL1 mAb and IMMU-110 had a biphasic clearance from the circulation; the α and β half-life (t1/2) of IMMU-110 were 4.6 h and 157.9 h, respectively, and those of hLL1 were 5.4 h and 151.5 h, respectively. In biodistribution studies, no significant difference was observed between IMMU-110 and naked hLL1 with regards to normal tissue uptake. Neither IMMU-110 nor naked hLL1 mAb had a significant association with any normal body tissue. In therapy experiments, a single i.v. protein dose of 350 μg IMMU-110, injected 5 days after implantation of MC/CAR cells in SCID mice, resulted in curing 70% of the animals. Similar cure rates were observed when treatment with IMMU-110 was given 10 days after transplantation of MC/CAR cells. In the Raji xenograft model, 100% of animals were cured with a single protein dose of 120 μg IMMU-110, injected 5 days after implantation of cells. In survival studies, the efficacy of IMMU-110 was significantly better than naked hLL1, the combination of naked hLL1 and free DOX, or of a non-specific antibody-DOX conjugate, DOX-hRS7. In a tolerability study in SCID mice, no toxic effect of IMMU-110 was observed even at the highest dose tested (2.5 mg /mouse). In conclusion, treatment of B-cell lymphoma and myeloma xenograft models with single injections of IMMU-110 resulted in high levels of response and long-term survivors. IMMU-110 is being further developed as a potential therapeutic for the treatment of CD74-positive tumors.

Co-Transplantation of Hematopoietic Stem Cells and GM-CSF/G-CSF/CSF-Transfected Mesenchymal Stem Cells in SCID Mice.

Mesenchymal stem cells (MSC) are multipotent and believed to facilitate the engraftment of hematopoietic stem cells (HSC) when transplanted simultaneously in animal studies and recently even in human trials. In this study, we transfected culture-expanded MSC with GM-CSF, G-CSF, and CSF cytokine genes and then co-transplanted with HSC to further promote HSC engraftment. Mononuclear cells were harvested from the various sources and seeded in long-term culture for ex vivo MSC expansion. The phenotype and purity of MSC were assessed by flow cytometry. We transferred the above three cytokine genes into ex vivo expanded MSC, confirmed transfection by fluorescent microscope of GFP, and thereafter did co-transplantation with HSC. A total of 1x107 HSC plus MSC/uL were introduced to tail vein of SCID mice. After 3–7 weeks later, with venous blood from the eyeballs, homing and engraftment of human cells were determined by flow cytometry and fluorescence in situ hybridization (FISH) studies. The total nucleated cell count and the engraftment of CD45+/CD34+ cells and XX/XY-positive human cells significantly increased in co-transplanted mice and even higher with the cytokine gene-transfected MSC in the order of GM-CSF, SCF, and G-CSF transfections (P<0.05). These results suggest that MSC transfected with hematopoietic growth factor genes are capable of enhancing the hematopoietic engraftment. Now we are planning to deliver genes involved in homing and cell adhesions, e.g., CXCR4, VLA, or TPO into ex vivo expanded MSC and do co-transplantation with HSC to further increase the efficiency of stem cell transplantation.

Anti-Her-2/neu-IL-2 or heregulin-IL-2 fusions proteins redirect non-tumor specific CTL to the tumor site for tumor eradication.

IL-2 is a T-cell growth factor that has pleiotropic functions on T cells, assisting in activating and expanding immune responses. Interestingly, we have previously found that in the presence of IL-2, CD4(+) or CD8(+) T cells kill tumor cells in an antigen-independent and non-MHC-restricted manner. In this study we took advantage of IL-2 capabilities to induce in this way the killing of tumor cells for immunotherpeutic purposes. We describe a novel mechanism where it is possible to bypass the usage of tumor-specific T cells for adoptive transfer by using non-tumor-specific T cells in combination with an anti-Her-2/neu-IL-2 (neu-Ab-IL-2) or heregulin-IL-2 fusion protein for tumor eradication. T cells in the presence of fusion proteins are capable of inducing the lysis of tumor cells in a non-MHC-restricted manner. This lysis is only observed in T cells that have been stimulated through the TCR, and it is mediated through the Fas-FasL pathway. Moreover, transfer of non-tumor-specific T cells in combination with the fusion proteins, induced the eradication of established Her-2, Her-3, and Her-4 expressing tumor cells in SCID mice. In contrast, the combination of non-tumor-specific T cells plus rIL-2 or irrelevant Ab-IL-2 (anti-hemagglutinin-IL-2) does not induce the elimination of tumors, indicating that the antitumor activity is dictated by the specificity of the fusion protein. These data demonstrate that neu-Ab-IL-2 or heregulin-IL-2 fusion proteins can direct the non-tumor-specific T cells to the tumor site and induce the elimination of tumors. Therefore, combination of non-tumor-specific T cells and antibody/ligand-IL-2 fusion proteins provides an alternative therapeutic strategy to control tumor growth in vivo.

Adrenomedullin antagonist suppresses in vivo growth of human pancreatic cancer cells in SCID mice by suppressing angiogenesis.

Since it is reported that adrenomedullin (AM) upregulated by hypoxia inhibits hypoxic cell death, we examined the effects of AM antagonist (AM C-terminal fragment; AM(22-52)) on the growth of pancreatic cancer cells. We, for the first time, demonstrated that AM antagonist significantly reduced the in vivo growth of the pancreatic cancer cell line. Immunohistochemical analysis demonstrated that the mean diameter of blood vessels was significantly smaller in the tumor tissues treated with AM antagonist than in those treated with AM N-terminal fragment (AM(1-25)), and that the PCNA-labeling index was lower in the former than in the latter. Then we demonstrated that AM antagonist showed no effect on the in vitro growth of the pancreatic cancer cell line. These results showed that AM played an important role in the growth of pancreatic cancer cells in vivo, suggesting that AM antagonist might be a useful tool for treating pancreatic cancers.

CD4+CD25+ regulatory T cells suppress differentiation and functions of Th1 and Th2 cells, Leishmania major infection, and colitis in mice.

Regulatory T cells play a major role in modulating the immune response. However, most information on these cells centers on autoimmunity, and there is also considerable controversy on the functional characteristics of these cells. Here we provide direct in vitro and in vivo evidence that CD4+CD25+ regulatory T cells inhibit the differentiation and functions of both Th1 and Th2 cells. Importantly, CD4+CD25+ T cells suppressed the disease development of Leishmania major infection in SCID mice reconstituted with naive CD4+CD25- T cells. Furthermore, CD4+CD25+ T cells inhibited the development of colitis induced by both Th1 and Th2 cells in SCID mice. Our results therefore document that CD4+CD25+ regulatory T cells suppress both Th1 and Th2 cells and that these regulatory T cells have a profound therapeutic potential against diseases induced by both Th1 and Th2 cells in vivo.

The matrix metalloproteinase inhibitor batimastat inhibits the lung colonisation of orthotopically implanted malignant pancreatic tumor cells in SCID mice.

In this study, we investigated the effect of the matrix metalloproteinase inhibitor batimastat on the lung colonisation of orthotopically implanted malignant pancreatic tumor cells in SCID mice. Following intraperitoneal anaesthesia, 10(6) Panc-TU-1 cells were orthotopically implanted in the head of the pancreas in 20 SCID mice. Seven days later, treatment of 10 of these mice with an intraperitoneal injection of batimastat (30 mg/kg body weight) was begun and continued for 14 days. Of the mice in the untreated control group, 3 were sacrificed and examined after 7 days, a further 3 after 14 days and the remainder together with the group that had been treated after 21 days. Tumor growth was clearly visible between the 14th. and the 21st. postoperative day. The orthotopically implanted tumor cells metastasized between the 2nd. and 3rd. postoperative week in the lung. In the control group, a diffuse metastasis of the lung was observed, but in the group of treated mice no lung metastases were found. In this mouse model, a clear reduction and inhibition of lung metastases from orthotopically implanted pancreatic tumor cells was achieved by treatment with the matrix metalloproteinase inhibitor batimastat.

Lack of MHC expression and retention of ultrastructural characteristics by xenograft transmissible venereal tumor cells in SCID mice

Canine transmissible venereal tumor (CTVT) is primarily a tumor of adult dogs with a high incidence of spontaneous regression. We recently reported a xenograft model of CTVT (XTVT) in NOD/SCID mice. XTVT cells retain cytological and histological features of CTVT as well as characteristic rearranged LINE/c-MYC junction [Am. J. Vet. Res. 62 (2001) 907]. In this paper, we demonstrate that XTVT cells maintain ultrastructural characteristics of CTVT and do not express MHC classes I and II molecules.

In vitro and in vivo (SCID mice) effects of phytosterols on the growth and dissemination of human prostate cancer PC-3 cells.

The dietary effect of phytosterols (PS) versus cholesterol on the growth and metastasis of the PC-3 human prostate cancer cells in SCID mice was studied. Also, their direct effect on the growth and migration of these cells in vitro was analysed. In the in vivo experiment, SCID mice were fed a diet containing 2% of either PS mixture or cholesterol plus 0.2% cholic acid and implanted with 2 x 10(6) tumour cells per mouse. Tumour growth was monitored for 8 weeks post inoculation. Animals fed the PS diet had tumours 40-43% smaller than those fed the cholesterol diet. Furthermore, the number of mice with lymph node and lung metastasis was almost one-half that of the cholesterol-fed group. In the in vitro studies, both beta-sitosterol and campesterol inhibited the growth of PC-3 cells by 70% and 14%, respectively, while cholesterol supplementation increased the growth by 18% when compared with controls. PS inhibited the invasion of PC-3 cells into Matrigel-coated membranes by 78% while cholesterol increased it by 43% as compared with the cells in the control media. Migration of tumour cells through 8 microm pore membranes was reduced by 60-93% when the PC-3 cells were in PS media, as compared with a 67% increase after cholesterol supplementation. PS supplementation reduced the binding of PC-3 cells to laminin by 15-38% and fibronectin by 23% while cholesterol increased binding to type IV collagen by 36%. It was concluded that PS indirectly (in vivo as a dietary supplement) and directly (in tissue culture media) inhibited the growth and metastasis of PC-3 cells. beta-Sitosterol was more effective than campesterol in offering this protection in most of the parameters studied.