Combined Immune Deficiency Mice Research Articles

HSP90 antagonist, geldanamycin, inhibits proliferation, induces apoptosis and blocks migration of rhabdomyosarcoma cells in vitro and seeding into bone marrow in vivo

In this study, geldanamycin (GA) was found to have an antiproliferative effect on both embryonal and alveolar rhabdomyosarcoma (RMS) cell lines. The maximum level of inhibition reached 80% for both embryonal and alveolar RMS. After GA treatment, cells also became apoptotic as judged by Annexin V-positive staining, activation of caspase-3 pathway and poly(ADP ribose) polymerase cleavage. GA was responsible for the arrest of RMS cells in both G1 and G2/M phases of the cell cycle. G1 blockade, however, was transient and was seen only in the first 24 h of GA treatment. RMS often gives distant metastases to various organs including bone marrow. RMS cells express high levels of MET receptor and respond to hepatocyte growth factor with increased motility. In our study, we found that GA decreased the level of MET expression and inhibited the chemotaxis of RMS cells toward the hepatocyte growth factor gradient. GA also blocked the homing of RMS cells into bone marrow of severe combined immune deficient mice. In all our experiments embryonal RMS cell lines were significantly more sensitive, and lower concentrations of GA were sufficient to block embryonal RMS cell proliferation, induce apoptosis and inhibit motility. Our data show that the HSP90 inhibitor GA has the potential to become a new drug in RMS treatment. It blocks RMS proliferation, decreases cell survival and inhibits motility of RMS cells.

The apoptosis-inducing effect of gastrin on colorectal cancer cells relates to an increased IEX-1 expression mediating NF-κB inhibition

Addressing the puzzling role of amidated gastrin(17) (G17) and the gastrin/CCKB/CCK2 receptor in colorectal carcinogenesis, we analysed potential candidate genes involved in G17-dependent NF-kappaB inhibition and apoptosis. The colorectal carcinoma cell line Colo320 overexpressing the wild-type CCK2 receptor (Colo320wt) underwent G17-induced apoptosis along with suppressed NF-kappaB activation and decreased expression of the antiapoptotic NF-kappaB target genes cIAP1 and cIAP2, whereas G17 was without effect on Colo320 cells expressing a CCK2 receptor bearing a loss of function mutation (Colo320mut). Gene microarray analysis revealed an elevated expression of the stress response gene IEX-1 in G17-treated Colo320wt but not Colo320mut cells. Quantitative real-time PCR and conventional RT-PCR confirmed this G17-dependent increase of IEX-1 expression in Colo320wt cells. If these cells were subjected to IEX-1 knockdown by small interfering RNA transfection, the apoptosis-inducing effect of G17 was abolished. Moreover, tumor necrosis factor alpha (TNFalpha)- or 5-FU-induced apoptosis that is greatly enhanced by G17 treatment in Colo320wt cells was prevented if IEX-1 expression was repressed. Under these conditions of blocked IEX-1 expression, the NF-kappaB activity remained unaffected by G17, in particular in Colo320wt cells co-treated with TNFalpha and also the suppressive effect of G17 on cIAP1 and cIAP2 expression was not observed anymore if IEX-1 expression was blocked. Conversely, IEX-1 overexpression in Colo320mut cells caused an increase of basal and TNFalpha- or 5-FU-induced apoptosis, an effect not further triggered by G17 treatment. Using a xenograft tumor model in severe combined immune deficiency mice, we could show that experimental systemic hypergastrinemia induced by the administration of omeprazole led to enhanced apoptosis as well as to a marked increase of IEX-1 expression in Colo320wt tumors, but not in Colo320mut tumors. These observations indicate that the proapoptotic effect of G17 on human colon cancer cells expressing the wild-type CCK2 receptor is mediated by IEX-1, which modulates NF-kappaB-dependent antiapoptotic protection and thereby exerts tumor-suppressive potential.

Ultra-Rapid Cardiotoxicity of the Hepatitis C Virus Protease Inhibitor BILN 2061 in the Urokinase-Type Plasminogen Activator Mouse

Because current therapies for chronic hepatitis C virus (HCV) infections are suboptimal and associated with severe side effects, novel treatment options are needed. A small animal model has recently been developed to study HCV infections. To examine the usefulness of this human liver-urokinase-type plasminogen activator (uPA)(+/+) severe combined immune deficient (SCID) mouse for the development of HCV-targeted drugs, we evaluated the antiviral efficacy and safety of an HCV NS3-protease inhibitor, BILN 2061. BILN 2061 was orally administered at clinical range doses for 4 days to SCID mice that differed in the presence of HCV infection, human hepatocyte grafts, and uPA zygosity. Treatment outcome was evaluated clinically, virologically, and morphologically. Using standard high-performance liquid chromatography-ultraviolet (HPLC-UV) methods and mass spectrometry, single-dose pharmacokinetics and multiple-dose drug exposures were analyzed. The (13)C-aminopyrine breath test was applied to compare in vivo liver function. A 4-day treatment with BILN 2061 of HCV genotype-1b infected chimeric animals reduced the viral load by >100-fold, but concomitant clinical and ultrastructural signs of cardiotoxicity appeared. BILN 2061 administration to uPA-transgenic mice induced mitochondrial swelling with aberrant cristae in cardiomyocytes, but not in skeletal muscle. Because both drug accumulation and liver function were identical in affected uPA-transgenic and nontransgenic SCID mice without cardiac involvement, the urokinase plasminogen activator transgene itself appears to be implicated. The human liver-uPA(+/+)SCID mouse is an interesting small animal model to evaluate the preclinical safety and efficacy of new antiviral compounds against HCV. The uPA-transgene increases the susceptibility of mice to BILN 2061-induced cardiotoxicity.

Analysis of Integrin α7 Mutations in Prostate Cancer, Liver Cancer, Glioblastoma Multiforme, and Leiomyosarcoma

Integrins are the major adhesive molecules in mammalian cells. Each integrin subtype plays a unique role in cell differentiation and embryo development. However, integrin involvement in carcinogenesis has not been well defined. We identified mutations in integrin alpha7 by sequencing genomic DNAs and cDNAs from 122 specimens, including 62 primary human tumor samples, four cell lines, and 56 matched normal tissues. We evaluated the tumor suppressor activity of integrin alpha7 with colony formation, soft agar colony growth, and cell migration assays by forcing its expression in PC-3 and Du145 prostate cancer cells and SK-UT-1 leiomyosarcoma cells. PC-3 and Du145 xenograft tumors with increased levels of integrin alpha7 in severe combined immune deficient mice were used to assess the effect of integrin alpha7 on tumor growth and metastasis. Immunostaining was used to localize and to measure the level of integrin alpha7 in 701 and 141 specimens of prostate and smooth muscle, respectively. A meta-analysis of integrin alpha7 mRNA microarray data from four studies was performed. Kaplan-Meier analyses were used to assess survival. All statistical tests were two-sided. Integrin alpha7 mutations that generate truncations were found in specimens of 16 of 28 prostate cancers (57%, 95% confidence interval [CI] = 37% to 76%), five of 24 hepatocellular carcinomas (21%, 95% CI = 7% to 42%), five of six glioblastomas multiforme (83%, 95% CI = 36% to 99%), and one of four leiomyosarcomas (25%, 95% CI = 0.6% to 81%). Integrin alpha7 mutations were associated with increased recurrence of human prostate cancer (nine recurrences among 13 patients with integrin alpha7 mutations versus one among eight without such mutations; odds ratio [OR] = 14, 95% CI = 1.15 to 782, P = .024) and hepatocellular carcinoma (five recurrences among eight patients with integrin alpha7 mutations versus one among 16 without such mutations, OR = 21, 95% CI = 1.6 to 1245; P = .007). Forced expression of normal integrin alpha7 in prostate cancer and leiomyosarcoma cell lines suppressed tumor growth and metastasis both in vitro and in vivo. Focal or no integrin alpha7 expression in human prostate cancer and soft tissue leiomyosarcoma was associated with a reduction of metastasis-free survival (for example, for prostate cancer with focal or no expression, 5-year metastasis-free survival was 32%, 95% CI = 24.4% to 40.3%, and for prostate cancer with at least weak expression, it was 85%, 95% CI = 79% to 91%; P<.001). Microarray analysis indicated that cyclin D kinase inhibitor 3 and GTPase-activating protein may be possible targets for integrin alpha7-mediated tumor suppressor activity and inhibition of cell motility. Integrin alpha7 appears to be a tumor suppressor that operates by suppressing tumor growth and retarding migration.

In vitro and in vivo induction of bone formation based on adeno-associated virus-mediated BMP-7 gene therapy using human adipose-derived mesenchymal stem cells

To determine whether adeno-associated virus (AAV)-2-mediated, bone morphogenetic protein (BMP)-7-expressing human adipose-derived mesenchymal stem cells (ADMS) cells would induce bone formation in vitro and in vivo. ADMS cells were harvested from patients undergoing selective suction-assisted lipectomy and transduced with AAV carrying the human BMP-7 gene. Non-transduced cells and cells transduced with AAV serotype 2 carrying the enhanced green fluorescence protein gene served as controls. ADMS cells were qualitatively assessed for the production of BMP-7 and osteocalcin, and subjected to alkaline phosphatase (ALP) and Chinalizarin staining. A total of 2.5 x 10(6) cells mixed with type I collagen were implanted into the hind limb of severe combined immune-deficient (SCID) mice and subjected to a histological analysis 3 weeks post implantation. Transfection of the ADMS cells achieved an efficiency of 99% at d 7. Transduction with AAV2-BMP-7 induced the expression of BMP-7 until d 56, which was markedly increased by d 7. The cells were positively stained for ALP. Osteocalcin production and matrix mineralization further confirmed that these cells differentiated into osteoblasts and induced bone formation in vitro. A histological examination demonstrated that implantation of BMP-7-expressing ADMS cells could induce new bone formation in vivo. The present in vitro and in vivo study demonstrated that human ADMS cells would be a promising source of autologous mesenchymal stem cells for BMP gene therapy and tissue engineering.

Existence of a threshold-like dose for γ-ray induction of thymic lymphomas and no susceptibility to radiation-induced solid tumors in SCID mice

Severe combined immune deficiency (SCID) mice exhibit limited repair of DNA double-strand breaks and are sensitive to ionizing radiation due to a mutation of the DNA-dependent protein kinase catalytic subunit gene. To elucidate the effects of deficient DNA double-strand break repair on radiation-induced carcinogenesis, the dose–response relationship for the induction of all tumor types was examined in wild-type and SCID mice. In wild-type mice, the incidence of thymic lymphomas at γ-ray doses up to 1 Gy was almost equal to the background level, increased gradually above 1 Gy, and reached a maximum of 12.5% at 5 Gy, which is indicative of a threshold dose of less than 1 Gy. SCID mice were extremely susceptible to the induction of spontaneous and radiation-induced thymic lymphomas. The incidence of thymic lymphomas in SCID mice irradiated with 0.1 Gy or less was similar to the background level; that is, it increased markedly from 31.7% at 0.1 Gy to 51.4% at 0.25 Gy, and reached a maximum of 80.6% at 2 Gy, suggesting the presence of a threshold-like dose at low γ-ray doses, even in radiosensitive SCID mice. As the average latency for the induction of thymic lymphomas at 0.1 Gy was significantly shortened, the effect of 0.1 Gy γ-rays on thymic lymphoma induction was marginal. The high susceptibility of SCID mice to develop thymic lymphomas indicates that thymic lymphomas are induced by a defect in DNA double-strand break repair or V(D)J recombination. Excessive development of tumors other than thymic and nonthymic lymphomas was not observed in SCID mice. Furthermore, our data suggest that the defective double-strand break repair in SCID mice is not a major determinant for the induction of nonlymphoid tumors.

Fusion of Human Hematopoietic Progenitor Cells and Murine Cardiomyocytes Is Mediated by α4β1 Integrin/Vascular Cell Adhesion Molecule-1 Interaction

Fusion of transplanted stem cells and host cells has been proposed as a major mechanism for the generation of hepatocytes, Purkinje neurons, and cardiomyocytes. However, the mechanism of cell fusion has not been precisely defined. Furthermore, the consequence of cell fusion remains unclear. We have previously shown that adult peripheral blood CD34-positive cells injected into severe combined immune deficiency (SCID) mice can transform into cardiomyocytes, endothelial cells, and smooth muscle cells following experimentally induced myocardial infarction and that most of the newly formed cardiomyocytes result from cell fusion. We therefore undertook this study to define the mechanism and consequences of cell fusion. Here we show that hypoxia and cytokines increase fusion of human peripheral blood CD34-positive cells and murine cardiomyocytes in vitro by up to 7-fold, and this is blocked by anti-alpha4beta1 or anti-vascular cell adhesion molecule (VCAM)-1. In vivo, fusion of progenitor cells and cardiomyocytes can also be blocked by anti-alpha4beta1 or anti-VCAM-1, but not by anti-vascular endothelial growth factor. On the other hand, generation of human-derived endothelial cells is blocked by anti-vascular endothelial growth factor but not by anti-alpha4beta1 antibodies. Two months following transplant, a high percentage of fused cells expressed cyclin B1 and incorporated bromodeoxyuridine. Thus, hematopoietic progenitor cell and cardiomyocyte fusion is mediated by alpha4beta1/VCAM-1 interaction, leading to cell cycle reentry and cellular proliferation.

Characterization of a progenitor cell population in childhood T-cell acute lymphoblastic leukemia

AbstractA significant proportion of children with T-cell acute lymphoblastic leukemia (T-ALL) continue to fail therapy. Consequently, characterization of the cells that proliferate to maintain the disease should provide valuable information on the most relevant therapeutic targets. We have used in vitro suspension culture (SC) and nonobese diabetic–severe combined immune deficient (NOD/SCID) mouse assays to phenotypically characterize and purify T-ALL progenitor cells. Cells from 13 pediatric cases were maintained in vitro for at least 4 weeks and expanded in 8 cases. To characterize the progenitors, cells were sorted for expression of CD34 and CD4 or CD7 and the subfractions were evaluated in vitro and in vivo. The majority of cells capable of long-term proliferation in vitro were derived from the CD34+/CD4− and CD34+/CD7− subfractions. Moreover, the CD34+/CD4− or CD7− cells were the only subfractions capable of NOD/SCID engraftment. These T-ALL cells successfully repopulated secondary and tertiary recipients with equivalent levels of engraftment, demonstrating self-renewal ability. The immunophenotype and genotype of the original leukemia cells were preserved with serial passage in the NOD/SCID mice. These data demonstrate the long-term repopulating ability of the CD34+/CD4− and CD34+/CD7− subfractions in T-ALL and suggest that a cell with a more primitive phenotype was the target for leukemic transformation in these cases.

Molecular mechanisms of paclitaxel and NM-3 on human gastric cancer in a severe combined immune deficiency mice orthotopic implantation model

To explore the molecular mechanisms of action of paclitaxel and NM-3 on human gastric cancer in severe combined immune deficiency (SCID) mice. Human gastric cancer cells SGC-7901 were implanted into SCID mice and mice were treated with paclitaxel and NM-3. The effects of paclitaxel and NM-3 on apoptosis of human gastric cancer cells were analyzed using flow cytometry, TUNEL assays, and DNA fragment analyses. Apoptosis of SGC-7901 cells was successfully induced by paclitaxel, NM-3, and the combination of paclitaxel and NM-3 24 h after injection as shown by the presence of apoptotic hypodiploid peaks on the flow cytometer before G1-S and a characteristic apoptotic band pattern in the DNA electrophoresis. The apoptotic rate detected by TUNEL assay was found to be significantly higher in the paclitaxel/NM-3 compared to the control group (38.5% +/- 5.14% vs 13.2% +/- 1.75%, P < 0.01). Paclitaxel in combination with NM-3 is able to induce apoptosis of the human gastric cancer cells in SCID mice effectively and synergistically.

Effects of 2-methoxyestradiol on endometrial carcinoma xenografts

We have previously demonstrated that 2-methoxyestradiol (2-ME) inhibits the growth of human endometrial cancer HEC-1-A and RL-95-2 cells in vitro. In this study, we examined the effects of 2-ME on human endometrial carcinoma in severe combined immune deficient (SCID) mice. The potential side effects of 2-ME on SCID mice were also investigated. Severe combined immune deficient mice were injected with HEC-1-A cells (1 x 10(6)/mouse) and a 18 day administration of 2-ME was followed after 1 week cell implantation. Tumor volume, weight, body weight and blood chemistry were determined. Tumor tissues were examined with an antibody against the proliferative cell nuclear antigen (PCNA) and Ki-67. Liver, spleen, kidney, heart, lung and uterus were screened by pathological examinations. 2-ME (100 mg/kg p.o.) did not inhibit the growth of human endometrial carcinoma as compared to control. Necrotic areas were similar in both 2-ME-treated and -untreated tumor tissues. The expressions of PCNA and Ki-67 were similar in 2-ME-treated and untreated tumor sections. The wet weight of uterus was increased to more than threefold. The epithelial cells and glands in endometrium were increased. No significant difference was detected in blood AST, ALT and BUN. 2-ME has no antitumor effects on human endometrial carcinoma in our animal model. Its proliferative effects on endometrium and uterus might limit its use in gynecological cancers.

Dissociation between Inhibition and Killing by Levofloxacin in Human Granulocytic Anaplasmosis

Human granulocytic anaplasmosis (HGA) is a potentially fatal tick-borne infection caused by Anaplasma phagocytophilum. Treatment options are limited for this entity, with doxycycline being the drug of choice. Certain fluoroquinolones such as levofloxacin are active against A. phagocytophilum in vitro. We report a hospitalized patient with HGA who improved coincident with a 13-day course of levofloxacin therapy, but clinically and microbiologically relapsed 15 days after completion of treatment. Relapse of infection after levofloxacin therapy was reproduced in a severe combined immune-deficient (SCID) mouse infection model. Quinolone therapy should not be considered curative of HGA.

The Retinoid X Receptor Regulates Human Hematopoietic Stem Cell Fate.

Our laboratory has recently demonstrated that competitive inhibition of the enzyme, aldehyde dehydrogenase-1 (ALDH-1), in human hematopoietic stem cells (HSCs) facilitates the 3.4-fold expansion of primitive cells capable of repopulating non-obese diabetic/severe combined immune deficient (NOD/SCID) mice. Since ALDH-1 activity is required for the intracellular production of retinoic acids, we hypothesized that retinoid signaling could be fundamentally involved in human HSC fate determinations. In order to demonstrate a link between ALDH-1 activity and retinoid signaling in human HSCs, we cultured human CD34+CD38−lin- HSC-enriched cells with retinaldehyde, which is a specific substrate for ALDH-1, and found that this induced a 10-fold increase in the expression of cEBP-epsilon, which is a Retinoic Acid Receptor (RAR)-dependent transcription factor. Moreover, when the competitive inhibitor of ALDH-1, diethylaminobenzaldehyde (DEAB), was added to HSC cultures supplemented with retinaldehyde, the expression of cEBP-epsilon was completely blocked, confirming a causal relationship between ALDH-1 activity and retinoid signaling in HSCs. We then sought to determine whether direct agonism or antagonism of the RAR or Retinoid X Receptor (RXR) could alter human HSC fate in short-term culture. We cultured primary human bone marrow (BM) and cord blood (CB) CD34+CD38−lin- HSC-enriched cells with hematopoietic cytokines, thrombopoietin (T), stem cell factor (S) and flt-3 ligand (F) in the presence and absence of several agonists and antagonists of RAR and RXR. Seven day culture of BM or CB CD34+CD38−lin- cells with TSF alone resulted in a loss of CD34+CD38− cells in culture, a pronounced increase in colony forming cell (CFC) activity and a complete loss of primitive SCID-repopulating cells (SRCs) compared to input, reflecting HSC differentiation during culture. Interestingly, the addition of either all-trans retinoic acid (ATRA), an agonist of RAR and RXR, or TTNPB, a selective RAR agonist, induced an accelerated differentiation of HSCs in culture compared to cytokines alone. Surprisingly, the addition of LGD815 (courtesy of Ligand Pharmaceuticals, San Diego, CA), a selective RAR antagonist, had no effect on the differentiation of HSCs that otherwise occurred in response to cytokines alone. However, the addition of LGD101506, a partial antagonist of RXR, preferentially maintained phenotypic CD34+CD38− cells in culture and inhibited CFC production in response to cytokines. Moreover, RXR antagonism maintained or induced the expansion of SRC content in culture, whereas SRC content was completely lost following cytokine culture alone. Taken together, these data demonstrate for the first time that RXR signaling contributes to the intrinsic regulation of human HSC fate. Pharmacologic inhibition of RXR signaling impedes the normal differentiation of HSCs that otherwise occurs in response to proliferation-inducing cytokines. These data provide further evidence for the role of retinoid signaling in the differentiation program of human HSCs and indicate that RXR and its heterodimeric partners are novel targets for the purpose of facilitating human HSC expansion.

Targeting of CD44 eradicates human acute myeloid leukemic stem cells

The long-term survival of patients with acute myeloid leukemia (AML) is dismally poor. A permanent cure of AML requires elimination of leukemic stem cells (LSCs), the only cell type capable of initiating and maintaining the leukemic clonal hierarchy. We report a therapeutic approach using an activating monoclonal antibody directed to the adhesion molecule CD44. In vivo administration of this antibody to nonobese diabetic-severe combined immune-deficient mice transplanted with human AML markedly reduced leukemic repopulation. Absence of leukemia in serially transplanted mice demonstrated that AML LSCs are directly targeted. Mechanisms underlying this eradication included interference with transport to stem cell-supportive microenvironmental niches and alteration of AML-LSC fate, identifying CD44 as a key regulator of AML LSCs. The finding that AML LSCs require interaction with a niche to maintain their stem cell properties provides a therapeutic strategy to eliminate quiescent AML LSCs and may be applicable to other types of cancer stem cells.

Lymphocyte-mediated regulation of β-endorphin in the myenteric plexus

Lymphocytes are antinociceptive and can modulate visceral pain perception in mice. Previously, we have shown that adoptive transfer of CD4+ T cells to severe combined immune-deficient (SCID) mice normalized immunodeficiency-related visceral hyperalgesia. Pain attenuation was associated with an increase in beta-endorphin release by T cells and an upregulation of beta-endorphin in the enteric nervous system. In this study, we investigated the relationship between T cells and opioid expression in the myenteric plexus. We examined opioid peptide and receptor expression in the myenteric plexus in the presence and absence of mucosal T cells. We found a positive association between T cells and beta-endorphin expression; this was accompanied by a downregulation of the micro-opioid receptor (MOR). In vitro, T helper (Th) type 1 and type 2 cytokine stimulation of CD4+ T cells or isolation of T cells from in vivo Th-polarized mice did not increase T cell release of beta-endorphin or the induction of beta-endorphin expression in the myenteric plexus. However, exogenous beta-endorphin did upregulate beta-endorphin expression, and both cycloheximide and naloxone methiodide inhibited peptide upregulation. Therefore, our results suggest that nonpolarized CD4+ T cells release beta-endorphin, which, through an interaction with MOR, stimulates an upregulation of beta-endorphin expression in the myenteric plexus. Thus, we propose that the mechanism underlying lymphocyte modulation of visceral pain involves T cell modulation of opioid expression in the enteric nervous system.

Plasticity of Hepatic Cell Differentiation: Bipotential Adult Mouse Liver Clonal Cell Lines Competent to Differentiate In Vitro and In Vivo

In fetal liver, bipotential hepatoblasts differentiate into hepatocytes and bile duct cells (cholangiocytes). The persistence of such progenitor cells in adult mouse liver is still debated. In damaged liver of adult murine animals, when hepatocyte proliferation is compromised, bipotential oval cells emerge, probably from bile ducts, proliferate, and differentiate to regenerate the liver. However, treatment to elicit oval cell proliferation is not necessary to obtain bipotential stem cells from adult mouse liver. Here, we have isolated bipotential clonal cell lines from healthy liver of 8-10-week-old C57BL/6 mice. Primary cultures established from hepatocyte-enriched suspensions were characterized by time-lapse image acquisition, immunocytology, and RNA transcript analysis. Although hepatocytes dedifferentiated with loss of apical polarity and other hepatocyte markers, they rapidly activated expression of bile duct/oval cell markers. Reversibility of these processes was achieved in part by culture under dilute Matrigel or by aging of confluent cultures. Cell lines were obtained at high frequency from mass cultures, from isolated colonies, and by primary cloning of the hepatocyte-enriched suspension. Cells of the clonal cell lines do not grow in soft agar and are nontumorigenic, and they express cytokeratin 19, A6 antigen, and alpha6 integrin, as well as a large panel of hepatocyte functions. Furthermore, they can participate in liver regeneration in albumin-urokinase-type plasminogen activator/severe combined immune-deficient mice, where they differentiate in clusters of hepatocytes and occasionally bile ducts. These results demonstrate the existence, in normal adult mouse liver, of a significant pool of clonogenic cells that are (or can become) bipotential.

In Vivo Selection of Phage for the Optical Imaging of PC-3 Human Prostate Carcinoma in Mice

There is an increasing medical need to detect and spatially localize early and aggressive forms of prostate cancer. Affinity ligands derived from bacteriophage (phage) library screens can be developed to molecularly target prostate cancer with fluorochromes for optical imaging. Toward this goal, we used in vivo phage display and a newly described micropanning assay to select for phage that extravasate and bind human PC-3 prostate carcinoma xenografts in severe combined immune deficiency mice. One resulting phage clone (G1) displaying the peptide sequence IAGLATPGWSHWLAL was fluorescently labeled with the near-infrared fluorophore AlexaFluor 680 and was evaluated both in vitro and in vivo for its ability to bind and target PC-3 prostate carcinomas. The fluorescently labeled phage clone (G1) had a tumor-to-muscle ratio of ~30 in experiments. In addition, prostate tumors (PC-3) were readily detectable by optical-imaging methods. These results show proof of principle that diseasespecific library-derived fluorescent probes can be rapidly developed for use in the early detection of cancers by optical means.

Autologous Human Fat Grafting: Effect of Harvesting and Preparation Techniques on Adipocyte Graft Survival

Autogenous fat transfer with lipoinjection for soft-tissue augmentation is a commonly used technique without a universally accepted approach. The high percentage and variable amount of fat resorption reduce the clinical efficacy of this procedure and often result in the need for further grafting. The purposes of this study were to evaluate the effect of different harvesting and preparation techniques on human fat tissue viability and to determine fat tissue viability rates among the different fat preparations transplanted into a severe combined immune deficiency mouse model at 3 months. Using standard liposuction and syringe aspiration, fat was removed from patients (n = 3) undergoing elective body contouring. Tissue was prepared by six different combinations of centrifugation and/or washing the cells with lactated Ringer's solution or normal saline. Metabolic activities of fat cell viability were monitored to assess overall cell viability. To analyze viability over 3 months, freshly harvested tissue specimens (minimum n = 5) were prepared by a combination of various procedures (wash, centrifugation, and different solutions) and subsequently injected under the dorsal flank skin of severe combined immune deficiency mice in two experiments. Mice were monitored for 12 weeks and the fat xenografts were removed for mass and histological evaluations. Metabolic analyses showed improved cell viability in tissue specimens undergoing minimal manipulation. No significant differences in fat cell viability, as assessed by graft weight maintenance or histologic evaluations, were observed with regard to harvesting or preparation techniques. Improved viability of freshly harvested but untreated fat specimens may be expected as compared with grafts that have undergone additional manipulations. No unique combination of preparation or harvesting techniques appeared to be more advantageous on transplanted fat grafts at 3 months. This study also demonstrated a reliable animal model for future investigation into examining novel applications for augmenting fat graft survival.

Prolonged dormancy of human liposarcoma is associated with impaired tumor angiogenesis

The disease state of cancer appears late in tumor development. Before being diagnosed, a tumor can remain for prolonged periods of time in a dormant state. Dormant human cancer is commonly defined as a microscopic tumor that does not expand in size and remains asymptomatic. Dormant tumors represent an early stage in tumor development and may therefore be a potential target for nontoxic, antiangiogenic therapy that could prevent tumor recurrence. Here, we characterize an experimental model that recapitulates the clinical dormancy of human tumors in mice. We demonstrate that these microscopic dormant cancers switch to the angiogenic phenotype at a predictable time. We further show that while angiogenic liposarcomas expand rapidly after inoculation of tumor cells in mice, nonangiogenic dormant liposarcomas remain microscopic up to one-third of the normal severe combined immune deficiency (SCID) mouse life span, although they contain proliferating tumor cells. Nonangiogenic dormant tumors follow a similar growth pattern in subcutaneous (s.c.) and orthotopic environments. Throughout the dormancy period, development of intratumoral vessels is impaired. In nonangogenic dormant tumors, small clusters of endothelial cells without lumens are observed early after tumor cell inoculation, but the nonangiogenic tumor cannot sustain these vessels, and they disappear within weeks. There is a concomitant decrease in microvessel density, and the nonangiogenic dormant tumor remains harmless to the host. In contrast, microvessel density in tumors increases rapidly after the angiogenic switch and correlates with rapid expansion of tumor mass. Both tumor types cultured in vitro contain fully transformed cells, but only cells from the nonangiogenic human liposarcoma secrete relatively high levels of the angiogenesis inhibitors thrombospondin-1 and TIMP-1. This model suggests that as improved blood or urine molecular biomarkers are developed, the microscopic, nonangiogenic, dormant phase of human cancer may be vulnerable to antiangiogenic therapy years before symptoms, or before anatomical location of a tumor can be detected, by conventional methods.

Characterization of the Babesia gibsoni P18 as a homologue of thrombospondin related adhesive protein

Thrombospondin related adhesive proteins (TRAPs) are well conserved among several apicomplexans. In this study, we reported the identification of the Babesia gibsoni P18, designated by our group previously, as a homologue of TRAP and renamed the P18 as the B. gibsoni TRAP (BgTRAP). The amino acid sequence of BgTRAP consists of several typical regions, including a signal peptide, a vonWillebrand factor A domain, a thrombospondin type 1 domain, a transmembrane region, and a cytoplasmic C-terminus. The B. gibsoni infected dog serum recognized recombinant BgTRAP expressed in E. coli by Western blotting. The antiserum against recombinant BgTRAP recognized an 80 kDa protein in the lysate of infected erythrocytes (RBCs), which was detectable in the micronemal area of the parasites by confocal microscopic observation. The BgTRAP showed a bivalent cation-independent binding to canine RBC, and the specific antiserum was found to inhibit the growth of B. gibsoni in the infected severe combined immune deficiency mice given canine RBC. These results suggest that the BgTRAP is a new member of TRAP family identified from the merozoites of B. gibsoni and functionally important in merozoite invasion; this protein may be useful as a vaccine candidate against canine B. gibsoni infection.

Immune suppression uncovers endogenous cytopathic effects of the hepatitis B virus.

It is generally accepted that the host's immune response rather than the virus itself is causing the hepatocellular damage seen in acute and chronic hepatitis B virus (HBV) infections. However, in situations of severe immune suppression, chronic HBV patients may develop a considerable degree of liver disease. To examine whether HBV has direct cytopathic effects in severely immune compromised hosts, we have infected severe combined immune deficient mice (uPA-SCID), harboring human liver cells, with HBV. Serologic analysis of the plasma of HBV-infected animals revealed the presence of extremely high amounts of viral genomes and proteins. Histological analysis of the livers of uPA-SCID chimeras infected with HBV for more than 2 months showed that the majority of human hepatocytes had a ground-glass appearance, stained intensely for viral proteins, and showed signs of considerable damage and cell death. This histopathologic pattern closely resembles the picture observed in the livers of immunosuppressed HBV patients. These lesions were not observed in animals infected with HBV for less than 1 month. Ultrastructural analysis of long-term-infected hepatocytes showed a highly increased presence of cylindrical HBsAg structures, core particles, and Dane particles compared to short-term-infected hepatocytes. These long-term-infected hepatocytes also contained elevated amounts of HBV cccDNA. In conclusion, HBV causes dramatic intracellular changes and hepatocellular damage in the human hepatocytes that reside in a severely immune deficient mouse. These lesions show much resemblance to the ones encountered in immunosuppressed chronic HBV patients. Our observations indicate that HBV may be directly cytopathic in conditions of severe immune suppression.