Comparative serum metabolomics between SCID mice and BALB/c mice with or without Schistosoma japonicum infection: Clues to the abnormal growth and development of schistosome in SCID mice.

The small blood flukes of genus Schistosoma, which cause one of the most prevalent and serious parasitic zoonosis schistosomiasis, are dependent on immune-related factors of their mammalian host to facilitate their growth and development, and the formation of granulomatous pathology caused by eggs deposited in host's liver and intestinal wall. Schistosome development is hampered in the mice lacking just T cells, and is even more heavily retarded in the severe combined immunodeficient (SCID) mice lacking both T and B lymphocytes. Nevertheless, it's still not clear about the underlying regulatory molecular mechanisms of schistosome growth and development by host's immune system. This study, therefore, detected and compared the serum metabolic profiles between the immunodeficient mice and immunocompetent mice (SCID mice vs. BALB/c mice) before and after S. japonicum infection (on the thirty-fifth day post infection using liquid chromatography-mass spectrometry (LC-MS). Totally, 705 ion features in electrospray ionization in positive-ion mode (ESI+) and 242 ion features in ESI- mode were identified, respectively. First, distinct serum metabolic profiles were identified between SCID mice and BALB/c mice without S. japonicum worms infection. Second, uniquely perturbed serum metabolites and their enriched pathways were also obtained between SCID mice and BALB/c mice after S. japonicum infection, which included differential metabolites due to both species differences and differential responses to S. japonicum infection. The metabolic pathways analysis revealed that arachidonic acid metabolism, biosynthesis of unsaturated fatty acids, linoleic acid metabolism, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, alpha-linolenic acid metabolism, glycerophospholipid metabolism, sphingolipid metabolism and purine metabolism were enriched based on the differential serum metabolites between SCID mice and BALB/c mice after S. japonicum infection, which was addressed to be related to the retarded growth and development of S. japonicum in SCID mice. These findings provide new clues to the underlying molecular events of host's systemic metabolic changes on the growth and development of S. japonicum worms, and also provide quite promising candidates for exploitation of drugs or vaccines against schistosome and schistosomiasis.

Hematological and biochemical profile of BALB/c nude and C57BL/6 SCID female mice after ovarian xenograft.

Hematological and biochemical profile studies help to evaluate functional changes of animals used in experiments. The aim of this study was to determine the hematological and biochemical profile of immunosuppressed BALB/c nude and C57BL/6 SCID mice after bovine ovarian xenotransplantation. Therefore, a total of 74 female mice were divided into four groups: non-xenotransplanted animals, xenotransplanted animals, xenotransplanted animals treated with eCG and xenotransplanted animals treated with FSH + LH. After anesthesia, blood samples were collected and hematologic and biochemical values were evaluated. The results showed no significant differences (p ≤ 0.05) for hematological parameters between the control group and the treatment groups of both strains. However, considering the biochemical profile, it was observed an increase of AST concentrations (p ≤ 0.05) in both strains and a decrease of ALT concentrations (p ≤ 0.05) only in C57BL/6 SCID strain of the groups subjected to hormonal treatment compared with those non subjected. Additionally, the values of the renal enzymes, urea and creatinine, did not differ (p ≤ 0.05) between the groups. Our findings suggest that the xenotransplantation procedure as well as the hormonal dosages had no significant effect on the well-being of the animals considering the evaluated hematological and biochemical profile.

Semaphorin-3A inhibits multiple myeloma progression in a mouse model.

Previous studies revealed that progression of multiple myeloma (MM) is associated with downregulation of semaphorin-3A (sema3A) expression in bone marrow endothelial cells. We therefore determined if serum sema3A concentrations are correlated with MM progression and if sema3A can affect MM progression. We find that the concentration of sema3A in sera of MM patients is strongly reduced and that the decrease is correlated with disease progression. A similar depletion is found in patients having acute myeloid leukemia and acute lymphoblastic leukemia but not in cancer forms that do not involve the bone marrow such as in colon cancer. Expression of a modified sema3A [furin-resistant sema3A (FR-sema3A)] stabilized against cleavage by furin-like proprotein convertases in CAG MM cells did not affect their behavior in-vitro. CAG cells injected into the tail vein of severe combined immunodeficient (SCID) mice home to the bone marrow and proliferate, mimicking MM disease progression. Disease progression in mice injected with CAG cells expressing FR-sema3A was inhibited, resulting in prolonged survival and a lower incidence of bone lesions. Histological examination and fluorescence-activated cell sorting analysis revealed that FR-sema3A expression reduced the infiltration of the CAG cells into the bone marrow, reduced bone marrow necrosis and reduced angiogenesis induced by the MM cells in the bone marrow. Our results suggest that measurement of sema3A serum concentrations may be of use for the diagnosis and for the monitoring of malignancies of the bone marrow such as MM. Furthermore, our results suggest that FR-sema3A may perhaps find use as an inhibitor of MM disease progression.

Stevens-Johnson syndrome/toxic epidermal necrolysis mouse model generated by using PBMCs and the skin of patients.

BACKGROUND: Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are life-threatening cutaneous reactions caused by drugs or infections and exhibiting widespread epidermal necrosis. Currently, there is no animal model that reproduces SJS/TEN symptoms. OBJECTIVE: We sought to develop a novel mouse model of SJS/TEN by using PBMCs and skin from patients who had recovered from SJS/TEN. METHODS: For our mouse model, patients' PBMCs were injected intravenously into immunocompromised NOD/Shi-scid, IL-2Rγ(null) (NOG) mice, followed by oral administration of a causative drug. Subsequently, to replace human skin, unaffected skin specimens obtained from patients who had recovered from SJS/TEN were grafted onto NOG mice, after which patient-derived PBMCs and the causative drug were applied. RESULTS: Mice injected with PBMCs from patients with SJS/TEN and given the causative drug showed marked conjunctival congestion and numerous cell death of conjunctival epithelium, whereas there were no symptoms in mice injected with PBMCs from patients with ordinary drug skin reactions. CD8(+) T lymphocyte-depleted PBMCs from patients with SJS/TEN did not elicit these symptoms. In addition, skin-grafted mice showed darkening of the skin-grafted areas. Cleaved caspase-3 staining showed that dead keratinocytes were more numerous in the skin-grafted mice than in the healthy control animals. CONCLUSION: We have established a novel human-oriented SJS/TEN mouse model and proved the importance of CD8(+) T lymphocytes in SJS/TEN pathogenesis. The mouse model promises to promote diagnostic and therapeutic approaches.

Sulfasalazine inhibits the growth of primary brain tumors independent of nuclear factor-kappaB.

Nuclear factor-kappaB (NF-kappaB) is a pleiotropic transcription factor that generally enhances cellular resistance to apoptotic cell death. It has been shown to be constitutively active in some cancers and is being pursued as potential anticancer target. Sulfasalazine which is used clinically to treat Crohn's disease has emerged as a potential inhibitor of NF-kappaB and has shown promising results in two pre-clinical studies to target primary brain tumors, gliomas. Once digested, sulfasalazine is cleaved into sulfapyridine and 5-aminosalicylic acid (5-ASA; mesalamine) by colonic bacteria, and the latter, too, is reported to suppress NF-kappaB activity. We now show that glioma cells obtained from patient biopsies or glioma cell lines do not show significant constitutive NF-kappaB activation, unless exposed to inflammatory cytokines. This does not change when gliomas are implanted into the cerebrum of severe combined immun-deficient mice. Nevertheless, sulfasalazine but not its cleaved form 5-ASA caused a dose-dependent inhibition of glioma growth. This effect was entirely attributable to the inhibition of cystine uptake via the system x(c)(-) cystine-glutamate transporter. It could be mimicked by S-4-carboxy-phenylglycine (S-4-CPG) a more specific system x(c)(-) inhibitor, and lentiviral expression of a constitutively active form of IkappaB kinase b was unable to overcome the growth retarding effects of sulfasalazine or S-4-CPG. Both drugs inhibited cystine uptake causing a chronic depletion of intracellular GSH and consequently compromised cellular redox defense which stymied tumor growth. This data suggests that system x(c)(-) is a promising therapeutic target in gliomas and possibly other cancers and that it can be pharmacologically inhibited by Sulfasalazine, an FDA-approved drug.

Antitumor alkyl ether lipid edelfosine: tissue distribution and pharmacokinetic behavior in healthy and tumor-bearing immunosuppressed mice.

PURPOSE: The present study investigates and compares the dose-dependent pharmacokinetics and oral bioavailability of edelfosine in healthy, immunodeficient, and tumor-bearing immunosuppressed mouse animal models, as well as edelfosine uptake and apoptotic activity in the Z-138 mantle cell lymphoma (MCL) cell line. EXPERIMENTAL DESIGN: Biodistribution study of edelfosine was done in both BALB/c and severe combined immune deficiency (SCID) mice, and then the in vivo behavior of the drug after i.v. and oral administration was monitored. RESULTS: We found that edelfosine is incorporated and induces apoptosis in the Z-138 human mantle cell lymphoma cell line, whereas normal resting peripheral blood human lymphocytes were not affected. In vivo biodistribution studies revealed that accumulation of edelfosine in the tumor of a MCL-bearing mouse animal model was considerably higher (P < 0.01) than in the other organs analyzed. Besides, no statistical differences were observed between the pharmacokinetic parameters of BALB/c and SCID mice. Edelfosine presented slow elimination and high distribution to tissues. Bioavailability for a single oral dose of edelfosine was <10%, but a multiple-dose oral administration increased this value up to 64%. CONCLUSION: Our results show that edelfosine is widely scattered across different organs, but it is preferentially internalized by the tumor both in vitro and in vivo. Our data, together with the apoptotic action of the drug on cancer cells, support a rather selective action of edelfosine in cancer treatment, and that multiple oral administration is required to increase oral bioavailability.

Efficient human immunodeficiency virus-based vector transduction of unstimulated human mobilized peripheral blood CD34+ cells in the SCID-hu Thy/Liv model of human T cell lymphopoiesis.

The methods available to efficiently transduce human CD34(+) hematopoietic stem cells (HSCs) derived from mobilized peripheral blood, such that they fully retain their engraftment potential and maintain high levels of transgene expression in vivo, have been unsatisfactory. The current murine retrovirus-based gene transfer systems require dividing cells for efficient transduction, and therefore the target HSCs must be activated ex vivo by cytokines to cycle, which may limit their engrafting ability. Lentivirus-based gene transfer systems do not require cell division and, thus, may allow for efficient gene transfer to human HSCs in the absence of any ex vivo cytokine stimulation. We constructed human immunodeficiency virus (HIV)-based vectors and compared them in vitro and in vivo with MuLV-based vectors in their ability to transduce unstimulated human CD34(+) HSCs isolated from mobilized peripheral blood. Both sets of vectors contained the marker gene that expresses the enhanced green fluorescent protein (EGFP) for evaluating transduction efficiency and were pseudotyped with either vesicular stomatitis virus glycoprotein (VSV-G) or the amphotropic murine leukemia virus envelope (A-MULV Env). The VSV-G-pseudotyped HIV-based vectors containing an internal mouse phosphoglycerate kinase promoter (PGK) were able to transduce up to 48% of the unstimulated CD34(+) cells as measured by EGFP expression. When these cells were injected into the human fetal thymus implants of irradiated SCID-hu Thy/Liv mice, up to 18% expressed EGFP after 8 weeks in vivo. In contrast, the MULV-based vectors were effective at transducing HSCs only in the presence of cytokines. Our results demonstrate that the improved HIV-based gene transfer system can effectively transduce unstimulated human CD34(+) HSCs, which can then differentiate into thymocytes and provide long-term transgene expression in vivo.

A soluble form of the murine common gamma chain is present at high concentrations in vivo and suppresses cytokine signaling.

The common gamma-chain (gammac) is a component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 and is essential for their signal transduction. Western blotting and a newly established enzyme-linked immunosorbent assay detected substantial constitutive levels (50-250 ng/mL) of soluble gammac (sgammac) in sera of murine inbred strains. It was demonstrated that purified immune cells, such as T, B, and natural killer cells, and macrophages released this protein after activation. Transfection experiments with cDNA encoding the full-length gammac showed that shedding of the transmembrane receptor led to the release of sgammac. The shedding enzymes, however, appeared to be distinct from those cleaving other cytokine receptors because inhibitors of metalloproteases (eg, TAPI) did not influence sgammac release. In vivo, superantigen-induced stimulation of T cells enhanced sgammac serum concentrations up to 10-fold within 6 hours. Because these findings demonstrated regulated expression of a yet unknown molecule in the immune response, further experiments were performed to assess the possible function(s) of sgammac. A physiological role of sgammac was indicated by its capacity to specifically inhibit cell growth induced by gammac-dependent cytokines. Mutational analysis revealed that the C-terminus and the WSKWS motif are essential for the cytokine inhibitory effect of the sgammac and for binding of the molecule to cytokine receptor-expressing cells. Thus, competitive displacement of the transmembrane gammac by excess sgammac is the most likely mechanism of cell growth inhibition. It was implied that naturally produced sgammac is a negative modulator of gammac-dependent cytokines.

The SCID mouse: an experimental model for endometriosis.

The purpose of this study was to validate the suitability of the severe combined immunodeficient (SCID) mouse as an experimental model for endometriosis, by defining the morphological and histological features of induced endometrial implants, and characterizing specific biochemical properties of these implants. Human secretory endometrial tissues were injected into the peritoneal cavity of SCID/SCID CB17 mature female mice. Successful peritoneal implantation was observed in 55 of 57 (96.5%) SCID mice and consisted of circumscribed elevated nodules. Haematoxylin-eosin staining of implanting lesions demonstrated the presence of endometrial glandular tissue in a mixed background of stromal and inflammatory cells. When progesterone was administered to mice, epithelial glands underwent well-defined secretory changes. Immunohistochemical analysis using polyclonal human pan-cytokeratin antibodies demonstrated selective positive staining in the glandular epithelium of the human implants with none in the surrounding stroma. In-situ hybridization analysis using complement component 3 cDNA radiolabelled riboprobes yielded significantly more intense signals in glands compared to stroma. As human endometrial implants in SCID mice were shown to retain specific histological, functional and biochemical properties, we conclude that the SCID mouse is an attractive animal model for the study of endometriosis.

Human acute myeloblastic leukemia-ascites model using the human GM-CSF- and IL-3-releasing transgenic SCID mice.

To generate an appropriate model for human acute myeloblastic leukemia (AML), we have successfully established a human hematopoietic growth factor-dependent AML cell line (TF-1 and UT-7/GM)-ascites model using human granulocyte-macrophage colony-stimulating factor (hGM-CSF)- and human interleukin 3 (hIL-3)-releasing transgenic (Tg)-SCID mice. When 1 x 10(7) cells of TF-1, a human erythroleukemia cell line, were transplanted into the peritoneum of irradiated Tg-SCID mice (TF-1 ip/Tg-SCID mice), TF-1 cells grew in both the single cell suspension form (asTF-1) and solid form in ascites and invaded various tissues: lungs, liver, pancreas, and genitals, 3-6 weeks following transplantation. Subsequently, 0.5-1 x 10(7) cells of UT-7/GM, a subline of the UT-7 human megakaryoblastic leukemia cell line, grown in the back of hGM-CSF Tg-SCID mice after subcutaneous inoculation, were transplanted into the peritoneum of other irradiated hGM-CSF Tg-SCID mice. After 4 weeks, UT-7/GM cells (asUT-7/GM) also grew in the same manner as TF-1 cells in hGM-CSF Tg-SCID mice. Analysis of the cells from the peritoneum and tissues by PCR amplifying ALU and human GM-CSF receptor beta sequences and by immunohistochemical staining using anti-human CD45 revealed that they possessed the original characteristics of the parental cells. To confirm the usefulness of this human AML-ascites model, experimental treatment of AML cells grown in these mice was carried out with a differentiation inducer, delta-aminolevulinic acid (deltaALA), which induces hemoglobin synthesis for TF-1 in vitro and is thus regarded as an anti-leukemia drug candidate. Unexpectedly, growth promotion of TF-1 cells was observed in the treated TF-1 ip/hIL-3 Tg-SCID mice without differentiation to erythroid cells after treatment with delta-ALA (5 mM) for 7 days. These results indicate that Tg-SCID mice can support the growth of human hematopoietic growth factor-dependent AML cell lines which are usually rejected by SCID mice, without modification of the parental cell characteristics. In addition, this Tg-SCID leukemia-ascites model may become a useful preclinical tool for estimation of drug efficacy in vivo, since the drug candidate which was promising in vitro did not act in the same manner in vivo.

Induction of human gamma delta T cells in myasthenia gravis thymus transplanted SCID mice.

BACKGROUND: TCR-gamma delta cells develop by extrathymic differentiation and might play an important role in thymectomy-resistant myasthenia gravis (MG) patients. In this study, we show development of human TCR-gamma delta cells in periphery of SCID mice by transplantation of human MG thymus or thymoma. METHODS: Three pieces of thymoma tissue and four of non-thymoma thymic tissues were obtained from MG patients by thymectomy. Each tissue was transplanted into 5-6 weeks old female SCID mice by intraperitoneal injection or surgical implantation. Rate of human TCR-positive cell development and its subtypes (TCR-alpha beta, TCR-gamma delta) were measured in the mice blood at one, three, and six weeks after the transplantation. RESULTS: Human TCR-positive cells were detected three weeks after transplantation. Rate of TCR-gamma delta T cell development got higher in thymoma transplanted group than in non-thymoma transplanted group. CONCLUSIONS: We could successfully develop human mature T cells in SCID mice by transplantation of human MG thymus or thymoma.

Late-onset chronic inflammatory encephalopathy in immune-competent and severe combined immune-deficient (SCID) mice with astrocyte-targeted expression of tumor necrosis factor.

To examine the role of tumor necrosis factor (TNF)-alpha in the pathogenesis of degenerative disorders of the central nervous system (CNS), transgenic mice were developed in which expression of murine TNF-alpha was targeted to astrocytes using a glial fibrillary acidic protein (GFAP)-TNF-alpha fusion gene. In two independent GFAP-TNFalpha transgenic lines (termed GT-8 or GT-2) adult (>4 months of age) animals developed a progressive ataxia (GT-8) or total paralysis affecting the lower body (GT-2). Symptomatic mice had prominent meningoencephalitis (GT-8) or encephalomyelitis (GT-2) in which large numbers of B cells and CD4+ and CD8+ T cells accumulated at predominantly perivascular sites. The majority of these lymphocytes displayed a memory cell phenotype (CD44high, CD62Llow, CD25-) and expressed an early activation marker (CD69). Parenchymal lesions contained mostly CD45+ high, MHC class II+, and Mac-1+ cells of the macrophage microglial lineage with lower numbers of neutrophils and few CD4+ and CD8+ T cells. Cerebral expression of the cellular adhesion molecules ICAM-1, VCAM-1, and MAdCAM as well as a number of alpha- and beta-chemokines was induced or upregulated and preceded the development of inflammation, suggesting an important signaling role for these molecules in the CNS leukocyte migration. Degenerative changes in the CNS of the GFAP-TNFalpha mice paralleled the development of the inflammatory lesions and included primary and secondary demyelination and neurodegeneration. Disease exacerbation with more extensive inflammatory lesions that contained activated cells of the macrophage/microglial lineage occurred in GFAP-TNFalpha mice with severe combined immune deficiency. Thus, persistent astrocyte expression of murine TNF-alpha in the CNS induces a late-onset chronic inflammatory encephalopathy in which macrophage/microglial cells but not lymphocytes play a central role in mediating injury.

Establishment of human acute myelogenous leukemia lines secreting interleukin-1 beta in SCID mice.

A reproducible in vivo model of human acute myelogenous leukemia (AML) was established in severe combined immunodeficient (SCID) mice. The AML-CL and AML-PS lines were originated from leukemic blasts purified respectively from the peripheral blood of a 27-year-old woman with previously untreated hyperleukocytotic AML and from the bone marrow of a 61-year-old man during the third leukemic relapse. The 2 lines were maintained and serially transplanted i.p. in SCID mice. AML-PS and AML-CL produced ascitogenous gross tumors after approximately 4 and 6 weeks, respectively, and all mice died within 6-8 weeks. Microscopic evaluation of different organs at autopsy showed massive involvement of bone marrow, liver and spleen, though with differences in the tumor burdens for the 2 lines (AML-CL > AML-PS). Flow cytometric analysis documented the spread of leukemic cells to bone marrow, peripheral blood and spleen. AML-PS and AML-CL cells show an immunophenotypic profile (CD13+, CD33+) and cytogenetic findings similar to freshly isolated blasts. Interleukin-1 beta (IL-1 beta) gene expression was observed by Northern blot analysis in leukemic cells from AML-CL and AML-PS SCID mice. After 24 hr of culture both lines released IL-1 beta in culture supernatants. High levels of circulating IL-1 beta were secreted in plasma of tumor-bearing mice. This AML-SCID murine model could contribute to an understanding of the mechanisms of AML growth in vivo and the possible role of the autocrine production of IL-1 beta in promoting cell growth.

Cellular and molecular composition of human skin in long-term xenografts on SCID mice.

We report on the immunophenotypical characterization of adult human skin transplanted onto severe combined immunodeficient (SCID) mice. Thirty animals were followed for up to 12 months after receiving split-thickness xenografts, of which 28 were tolerated for the whole test period. Antigen mapping revealed an almost complete preservation of human cellular and extracellular tissue components in long-term transplants including skin immune cells (Langerhans-cells, macrophages, lymphocytes) and also parts of the engrafted endothelium. Hence, xenografts on SCID mice offer a versatile experimental tool for the in vivo study of both human skin immune cell function and endothelial cell-mediated interactions in an environment completely devoid of interferences by adoptive host immune response.