Bioengineering human microvascular networks in immunodeficient mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs); however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs (5-7). We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.     

Establishment of human tumor xenografts in immunodeficient mice

Heterotransplantation of human cancer cells or tumor biopsies into immunodeficient rodents (xenograft models) has, for the past two decades, constituted the major preclinical screen for the development of novel cancer therapeutics. Despite limitations, these models have identified clinically efficacious agents, and remain the 'workhorse' of the pharmaceutical industry. However, if therapeutic approaches to treating tumors according to their molecular characteristics are to be achieved, additional new models of human cancer will be required to represent the genetic diversity that exists within tumor histologies. This protocol details a method for establishing xenografts from primary solid-tumor isolates or cells grown in culture. The procedure relies on immunodeficient mice to provide a host for the establishment of human xenografts. The procedure can be completed in 1-2 h with results being obtained in 1-4 months.     

Fate and functions of human adult lymphoid cells in immunodeficient mice

Laboratory models enabling to study in vivo human leukocyte functions have been developed. Most of the models consist of human immunocytes transferred to mice homozygous for the scid mutation. Mice with additional immunodeficient-prone genetic background or with immunodeficiency-induced conditioning have also been used. Human grafts mainly consisted of human immune cells in suspension injected intraperitoneally, or in pieces of human organs containing immunocytes implanted subcutaneously. Cells in suspension could be easily manipulated in vitro before transfer to the animal, but disseminated within the mouse body. In opposition, human cells mostly remained within implantation areas of animals given human organ pieces. This favorizes cell interactions and helps for cell recovery after their in vivo passage. Moreover, the diversity of antibodies in animals transplanted with human lymphoid organ pieces appeared broader than that of mice transferred with lymphocytes in suspension. Spontaneous recall antibody and autoantibody productions have been generally observed in animals transferred with cells from donors with such antibodies. In vivo boosting of recall antibody by antigen has been most successful, but such a manipulation inconstantly boosted autoantibodies. Primary human T and B cell responses were difficult to obtain in xenochimeric animals, and success has been generally obtained by optimizing human immune response parameters, such as antigen presentation.     

Immunotherapy of metastases of human neoplastic cells grown in immunodeficient mice

Summary Hereditarily athymic (nude) and asplenic-athymic (lasat) mice were inoculated neonatally with 10⁷ K-562 pluripotential leukemia cells of human origin. Meningeal infiltration and/or multiple metastases were found in the lungs, kidneys, and lymph nodes in nearly 60% of mice. Twice as many lasat mice as nude mice had meningeal and lymph node infiltrations. This result indicates that the spleen of nude mice influences the infiltrations and/or the distribution of metastases. Metastases of K-562 cells were found as early as 10 days and as late as 115 days of age. Goat immune gamma ()-globulin, prepared from antiserum to K-562 cells and absorbed with peripheral leukocytes and bone marrow cells from normal individuals, markedly diminished the incidence of metastases of K-562 cells. About 16% of the mice treated with immune -globulin had metastases in the lungs only. All mice receiving the immune -globulin had peripheral monocytosis and lymphocytosis as well as a hyperplasia of the bone marrow monocytic series. Immune -globulin may lyse heterotransplanted leukemia cells by direct binding to leukemia cells in the presence of complement and/or may activate antibody-dependent effector cells, for example macrophages or killer cells, which would destroy the transplanted leukemia cells.     

Study on placental transmission of Pneumocystis carinii in mice using immunodeficient SCID mice as a new animal model.

Placental transmission of Pneumocystis carinii in mice was examined in 39 animals obtained by caesarean section from 17 pregnant SCID females experimentally infected with P. carinii. When examined with toluidine blue O, DAPI and immunofluorescent antibody stains, P. carinii was detected in the lungs of infected mothers but not in the lungs of caesarean section-derived neonates even after the neonates were treated with dexamethasone for 8 weeks. However, 13 neonates born to five infected females developed P. carinii pneumonia. These results indicate that P. carinii cannot be transmitted transplacentally in mice.     

Human BLyS facilitates engraftment of human PBL derived B cells in immunodeficient mice

The production of fully immunologically competent humanized mice engrafted with peripheral lymphocyte populations provides a model for in vivo testing of new vaccines, the durability of immunological memory and cancer therapies. This approach is limited, however, by the failure to efficiently engraft human B lymphocytes in immunodeficient mice. We hypothesized that this deficiency was due to the failure of the murine microenvironment to support human B cell survival. We report that while the human B lymphocyte survival factor, B lymphocyte stimulator (BLyS/BAFF) enhances the survival of human B cells ex vivo, murine BLyS has no such protective effect. Although human B cells bound both human and murine BLyS, nuclear accumulation of NF-kappaB p52, an indication of the induction of a protective anti-apoptotic response, following stimulation with human BLyS was more robust than that induced with murine BLyS suggesting a fundamental disparity in BLyS receptor signaling. Efficient engraftment of both human B and T lymphocytes in NOD rag1(-/-) Prf1(-/-) immunodeficient mice treated with recombinant human BLyS is observed after adoptive transfer of human PBL relative to PBS treated controls. Human BLyS treated recipients had on average 40-fold higher levels of serum Ig than controls and mounted a de novo antibody response to the thymus-independent antigens in pneumovax vaccine. The data indicate that production of fully immunologically competent humanized mice from PBL can be markedly facilitated by providing human BLyS.     

Identification of Pneumocystis carinii in immunodeficient mice

Various procedures were utilized to determine the most sensitive, cost and labor effective techniques for detection of Pneumocystis carinii in immunologically compromised mice. Immunoperoxidase staining techniques that utilized polyclonal antibodies directed against purified rat or mouse P. carinii were more sensitive and specific than staining with Gomori's methenamine silver. Indirect immunofluorescence microscopy on frozen sections was comparable to immunoperoxidase staining, but lacked fine cytologic detail. Impression smears were of limited value when stained with Diff-Quik Stain, Harleco's Hemacolor, Wright-Giemsa or Wright-Leishman stains. However, cysts could be detected consistently in imprints stained with Gomori's methanamine silver. Transmission electron microscopy showed ultrastructural detail of P. carinii, but this technique was too costly and time consuming for routine use. Thus, because of its sensitivity and specificity, immunohistochemistry on paraffin sections was the most satisfactory method for screening and identifying P. carinii in lungs of immunocompromised mice.     

Morphological and functional evaluation of angiogenesis of a xenografted human sarcoma in nude mice

The implantation of tumour cells in normal tissues and the subsequent induction of angiogenesis by the growing xenograft were studied by means of immunohistochemistry and digital image analysis. Tumour growth was induced by injection of a human spindle cell sarcoma (ES3) into the subcutis of HsdCpb:NMRI-nu/nu mice. In vivo injection of Hoechst 33342 was used as a marker of perfusion. The vasculature was stained with specific antibodies and subsequently analysed by digital image analysis. Starting at day 3 up to day 6, angiogenesis could be detected and the relative amount of perfusion within the investigated area reached a peak at day 6. This method, which allows investigation of both functional and morphometric characteristics of human xenograft vasculature, serves as an excellent assay for evaluation of antiangiogenic therapies in translational research of experimental tumours.     

Quantification of cerebral oxygenation by full-spectrum near-infrared spectroscopy using a two-point method

The aim of this study was to quantify the relative concentrations of oxyhemoglobin and deoxyhemoglobin within the light path of the brain and to estimate cerebral hemoglobin (Hb) oxygen saturation using full-spectrum near-infrared spectroscopy (fsNIRS). For this purpose, we developed a novel exponential correction equation as well as a two-point spectroscopy method to estimate the relative concentrations of Hb and Hb oxygen saturation in biological tissues. The results of evaluation of measurements using an in vitro model indicated that our fsNIRS method enables accurate and non-invasive measurements of Hb content and saturation in a highly scattered medium such as the human brain. According to the results of analysis using a hypoxic piglet model, the mean cerebral Hb oxygen saturation (SbO(2)) of newborn piglets at an inspired oxygen gas concentration of 0.21 was estimated to be 63+/-4% (mean+/-S.D.). Umbilical arterial and left internal jugular venous Hb oxygen saturation were simultaneously estimated to be 96+/-2% and 52+/-11%, respectively. SbO(2) and arterial Hb oxygen saturation values had a linear relationship. The average oxygenation state of cerebral tissue is comparable with that of the cerebral vein. The results of this study showed that our method can be used to monitor Hb oxygen saturation in the neonatal brain at the bedside in an intensive care unit.     

Scanning spontaneous photon emission from transplanted ovarian tumor of mice using a photomultiplier tube

A scanning system for the detection of spontaneous ultraweak photon emission from nude mice with transplanted tumors is presented. A photomultiplier tube (PMT) with an effective area of 15 mm diameter was used for measuring photon emission in a wavelength range from 300 to 650 nm. Tumors were induced in nude mice by transplantation of an ovarian cancer cell line into the back of mice. The PMT was moved for scanning over the whole body of a mouse placed in a dark box. The profiles of the intensities of photon emissions from the tumor mice are presented and compared with those obtained from the control mice.     

Mycobacterium genavense infection in normal and immunodeficient mice

Mycobacterium genavense is a recently described microorganism causing disseminated infections in AIDS patients. In this study, we investigate its pathogenicity in mice and some mechanisms of the host response to this bacterium. Following an intravenous challenge of 10(6) organisms, M. genavense grew progressively in the spleens and livers of BALB/c and CBA mice over at least an 8-month period. Granulomas were present in the spleens, livers and lungs of the animals. The numbers of bacteria recovered from the spleens and livers were higher in BALB/c (Bcg(s)) than in CBA (Bcg(r)) mice from day 30. The role of the Bcg gene, in the early phase of infection, was supported by the fact that the bacterial load, on day 15, was higher in BALB/c than in the congenic C.D2 (Bcg(r)) mice. The role of T cells in the host response was suggested by the high susceptibility of nude mice to M. genavense infection. In vivo depletion experiments in CBA mice indicated that gamma interferon and both CD4(+) and CD8(+) T cells participate in the containment of the bacterial load.     

Quantification of the spatial strain distribution of scoliosis using a thin-plate spline method

The objective of this study was to quantify the three-dimensional spatial strain distribution of a scoliotic spine by nonhomogeneous transformation without using a statistically averaged reference spine. The shape of the scoliotic spine was determined from computed tomography images from a female patient with adolescent idiopathic scoliosis. The shape of the scoliotic spine was enclosed in a rectangular grid, and symmetrized using a thin-plate spline method according to the node positions of the grid. The node positions of the grid were determined by numerical optimization to satisfy symmetry. The obtained symmetric spinal shape was enclosed within a new rectangular grid and distorted back to the original scoliotic shape using a thin-plate spline method. The distorted grid was compared to the rectangular grid that surrounded the symmetrical spine. Cobb's angle was reduced from 35° in the scoliotic spine to 7° in the symmetrized spine, and the scoliotic shape was almost fully symmetrized. The scoliotic spine showed a complex Green–Lagrange strain distribution in three dimensions. The vertical and transverse compressive/tensile strains in the frontal plane were consistent with the major scoliotic deformation. The compressive, tensile and shear strains on the convex side of the apical vertebra were opposite to those on the concave side. These results indicate that the proposed method can be used to quantify the three-dimensional spatial strain distribution of a scoliotic spine, and may be useful in quantifying the deformity of scoliosis.     

Phenotypic characterisation of a Neospora caninum temperature-sensitive strain in normal and immunodeficient mice

The in vivo persistence, immunogenicity and pathogenicity of a recently described temperature-sensitive (ts) strain from Neospora caninum, NCts-8, was investigated in normal and immunodeficient mice. Groups of BALB/c and SCID/Bg mice were infected s.c. with 5 x 10(6) wild-type NC-1, control NCts-8 (pass 0) or NCts-8 tachyzoites prepared at four in vitro passage levels (pass 7, 13, 21 and 28). For persistence and immunogenicity studies, BALB/c mice were bled and sacrificed at 4, 6 or 8 weeks p.i. Sera were analysed by IFAT and brain tissues examined for lesions by histology and tested for parasite presence by PCR. For pathogenicity studies, SCID/Bg mice were monitored by clinical signs and survival time. Results from parasite persistence experiments demonstrated microscopic lesions and PCR positive brain tissues in NC-1 infected mice. In contrast, brain tissues from NCts8-infected groups were consistently negative by histology and PCR. Based on IFAT titres, all parasite strains were immunogenic, although parasite-specific IgG levels were lower in the NCts-8 infected groups. Results from pathogenicity studies in SCID/Bg mice demonstrated a significantly (P < 0.0001) longer mean survival time in NCts-8 vs NC-1 infected groups. In addition, there was no significant difference in mean survival time between control NCts-8 and experimental passage NCts-8 infected mice. Collectively, these studies demonstrate that the NCts-8 strain maintains a stable phenotype following multiple passages in vitro, and possesses an attenuated, shorter persistence phenotype in vivo compared with the parental wild-type NC-1.     

Proteomic analysis of human epithelial ovarian cancer xenografts in immunodeficient mice exposed to chronic psychological stress

This work aims at comparing alterations in the proteomes of human epithelial ovarian cancer xenografts between stressed and non-stressed immunodeficient mice as well as exploring the molecular mechanisms linking chronic psychological stress to ovarian cancer oncogenesis and progression. SK-OV-3 cells were injected subcutaneously into nude mice. The stress group was subjected to a chronic physical restraint protocol for 6 h on 35 consecutive days, while the control group was unrestrained. All mice were sacrificed on day 36 after SK-OV-3 cell injection, and tumors were excised. Tumor tissues were processed for 2D gel electrophoresis, mass spectrometry (nanoUPLC-ESI-MS/MS) and Western blotting. The expression of 20 proteins was found to be significantly altered between the stress and control groups, of which 14 were up-regulated, five were down-regulated, and one protein was found only in the stress group. All proteins were identified by UPLC-ESI-MS/MS, and Western blotting results were consistent with those of proteomic methods. The present results provide new evidence relating to the molecular mechanism underlying the relationship between psychological stress and tumor progression.     

Quantification of angiogenesis by a computerized image analysis system in renal cell carcinoma

OBJECTIVE: To ascertain whether tumor angiogenesis quantitated by a computerized image analysis system correlates with clinical outcome in renal cell carcinoma. STUDY DESIGN: Microvessels were immunohistochemically labeled with antibodies to CD34 in sections from 62 cases of renal cell carcinoma. Computerized image analysis was used to evaluate the mean microvessel count (MMC) and mean percentage microvessel area (MPMA). RESULTS: MMC ranged from 19.3 to 315.0, while MPMA was 0.6-17.9%. There was a highly significant correlation between MMC and MPMA (r = .867, P < .01). Although MMC and MPMA decreased with increasing nuclear grade and TNM stage, this difference failed to achieve statistical significance. No statistically significant differences in survival were found for MMC or MPMA. CONCLUSION: Our results indicate that computerized image analysis can evaluate accurately tumor angiogenesis, but tumor angiogenesis in renal cell carcinoma does not provide significant prognostic information in renal cell carcinoma.     

Quantification of lung fibrosis and emphysema in mice using automated micro-computed tomography

Background

In vivo high-resolution micro-computed tomography allows for longitudinal image-based measurements in animal models of lung disease. The combination of repetitive high resolution imaging with fully automated quantitative image analysis in mouse models of lung fibrosis lung benefits preclinical research. This study aimed to develop and validate such an automated micro-computed tomography analysis algorithm for quantification of aerated lung volume in mice; an indicator of pulmonary fibrosis and emphysema severity.

Methodology

Mice received an intratracheal instillation of bleomycin (n = 8), elastase (0.25U elastase n = 9, 0.5U elastase n = 8) or saline control (n = 6 for fibrosis, n = 5 for emphysema). A subset of mice was scanned without intervention, to evaluate potential radiation-induced toxicity (n = 4). Some bleomycin-instilled mice were treated with imatinib for proof of concept (n = 8). Mice were scanned weekly, until four weeks after induction, when they underwent pulmonary function testing, lung histology and collagen quantification. Aerated lung volumes were calculated with our automated algorithm.

Principal Findings

Our automated image-based aerated lung volume quantification method is reproducible with low intra-subject variability. Bleomycin-treated mice had significantly lower scan-derived aerated lung volumes, compared to controls. Aerated lung volume correlated with the histopathological fibrosis score and total lung collagen content. Inversely, a dose-dependent increase in lung volume was observed in elastase-treated mice. Serial scanning of individual mice is feasible and visualized dynamic disease progression. No radiation-induced toxicity was observed. Three-dimensional images provided critical topographical information.

Conclusions

We report on a high resolution in vivo micro-computed tomography image analysis algorithm that runs fully automated and allows quantification of aerated lung volume in mice. This method is reproducible with low inherent measurement variability. We show that it is a reliable quantitative tool to investigate experimental lung fibrosis and emphysema in mice. Its non-invasive nature has the unique benefit to allow dynamic 4D evaluation of disease processes and therapeutic interventions.