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.

Platelet-rich plasma with keratinocytes and fibroblasts enhance healing of full-thickness wounds.

Advances in tissue engineering led to the development of various tissue-engineered skin substitutes (TESS) for the treatment of skin injuries. The majority of the autologous TESS required lengthy and costly cell expansion process to fabricate. In this study, we determine the possibility of using a low density of human skin cells suspended in platelet-rich plasma (PRP)-enriched medium to promote the healing of full-thickness skin wounds. To achieve this, full-thickness wounds of size 1.767 cm2 were created at the dorsum part of nude mice and treated with keratinocytes (2 × 104 cells/cm2) and fibroblasts (3 × 104 cells/cm2) suspended in 10% PRP-enriched medium. Wound examination was conducted weekly and the animals were euthanized after 2 weeks. Gross examination showed that re-epithelialization was fastest in the PRP+cells group at both day 7 and 14, followed by the PRP group and NT group receiving no treatment. Only the PRP+cells group achieved complete wound closure by 2 weeks. Epidermal layer was presence in the central region of the wound of the PRP+cells and PRP groups but absence in the NT group. Comparison between the PRP+cells and PRP groups showed that the PRP+cells-treated wound was more mature as indicated by the presence of thinner epidermis with single cell layer thick basal keratinocytes and less cellular dermis. In summary, the combination of low cell density and diluted PRP creates a synergistic effect which expedites the healing of full-thickness wounds. This combination has the potential to be developed as a rapid wound therapy via the direct application of freshly harvested skin cells in diluted PRP.

HepaCAM­PIK3CA axis regulates the reprogramming of glutamine metabolism to inhibit prostate cancer cell proliferation.

Energy metabolism reprogramming is becoming an increasingly important hallmark of cancer. Specifically, cancers tend to undergo metabolic reprogramming to upregulate a cell­dependent glutamine (Gln) metabolism. Notably, hepatocellular cell adhesion molecule (HepaCAM) has been previously reported to serve a key role as a tumour suppressor. However, the possible regulatory role of HepaCAM in Gln metabolism in prostate cancer (PCa) remains poorly understood. In the present study, bioinformatics analysis predicted a significant negative correlation among the expression of HepaCAM, phosphatidylinositol­4,5­bisphosphate 3­kinase catalytic subunit α (PIK3CA), glutaminase (GLS) and solute carrier family 1 member 5 (SLC1A5), components of Gln metabolism, in clinical and genomic datasets. Immunohistochemistry results verified a negative correlation between HepaCAM and PIK3CA expression in PCa tissues. Subsequently, liquid chromatography­tandem mass spectrometry (LC­MS/MS) and gas chromatography­mass spectrometry (GC­MS) assays were performed, and the results revealed markedly reduced levels of Gln and metabolic flux in the blood samples of patients with PCa and in PCa cells. Mechanistically, overexpression of HepaCAM inhibited Gln metabolism and proliferation by regulating PIK3CA in PCa cells. In addition, Gln metabolism was discovered to be stress­resistant in PCa cells, since the expression levels of GLS and SLC1A5 remained high for a period of time after Gln starvation. However, overexpression of HepaCAM reversed this resistance to some extent. Additionally, alpelisib, a specific inhibitor of PIK3CA, effectively potentiated the inhibitory effects of HepaCAM overexpression on Gln metabolism and cell proliferation through mass spectrometry and CCK­8 experiments. In addition, the inhibitory effect of PIK3CA on the growth of tumor tissue in nude mice was also confirmed by immunohistochemistry in vivo. To conclude, the results from the present study revealed an abnormal Gln metabolic profile in the blood samples of patients with PCa, suggesting that it can be applied as a clinical diagnostic tool for PCa. Additionally, a key role of the HepaCAM/PIK3CA axis in regulating Gln metabolism, cell proliferation and tumour growth was identified. The combination of alpelisib treatment with the upregulation of HepaCAM expression may serve as a novel method for treating patients with PCa.

Induction in vivo and in vitro of terminal deoxynucleotidyl transferase by thymosin in bone marrow cells from athymic mice.

Terminal deoxynucleotidyl transferase (TdT) expression in bovine serum albumin (BSA) gradient-fractionated bone marrow cells was examined in NIH Swiss nu/nu and thymectomized C57BL/6 mice. In nude mice, TdT levels were approximately 10% of those of thymus-bearing littermates. In C57BL/6 mice, thymectomy caused a time-dependent loss of TdT activity in bone marrow cells. To determine whether or not not the apparent thymic requirement for TdT expression in bone marrow was mediated by thymic hormones, we examined the effects of thymosin fraction 5. Treatment of either NIH Swiss nu/nu or thymectomized C57BL/6 mice with thymosin fraction 5 restored the levels of TdT activity in BSA gradient-fractionated bone marrow cells to normal. Moreover, treatment of BSA gradient-fractionated bone marrow cells from NIH Swiss nu/nu or thymectomized C57BL/6 mice in tissue culture with thymosin fraction 5 induced TdT expression. In tissue culture, TdT induction was optimal with 25 ng/ml of thymosin fraction 5, it occurred within 6 h, and it was completely inhibited by actinomycin D. The effect was specific in that neither control nor spleen fraction 5-treated cells were induced to express TdT. These data demonstrate that TdT expression in bone marrow cells is under the direct control of thymic polypeptide hormones.

WW domain-binding protein 2 acts as an oncogene by modulating the activity of the glycolytic enzyme ENO1 in glioma.

WW domain-binding protein 2 (WBP2) has been demonstrated as oncogenic in breast cancer. Many studies have revealed the WBP2 gene as a high-risk gene for leukoariaosis and cerebral white matter lesions is important in the pathologic stage of glioma development. This study aimed to illustrate the underlying mechanism by which WBP2 regulates the process of glioma development. The expression pattern of WBP2 in several tumor cells was determined, clarifying the carcinogenic action of WBP2 in glioma cells. Overexpression of WBP2 in glioma cells promoted cell proliferation and migration, and the number of S-phase cells, whereas the depletion of WBP2 by RNAi-mediated knockdown restrained cell growth and cell cycle progression. Upregulation of WBP2 significantly enhanced the tumorigenic ability of U251 cells in vivo. MS/GST pulldown assay identified α-enolase (ENO1) and Homer protein homolog 3 (Homer3) as novel potent interaction partners of WBP2. Knockdown of ENO1 or Homer3 allowed cell growth and migration to return to normal levels. Furthermore, in vitro and in vivo experiments indicated that the oncogenic role of WBP2 in glioma was through modulating ENO1 and glycolysis activity via the ENO1-PI3K/Akt signaling pathway. Collectively, these results reveal that WBP2 plays a vital role in the occurrence and development of glioma, indicating a target gene for glioblastoma treatment.

PUVA suppresses the proliferative stimulus produced by stripping on hairless mice.

Hairless mice were fed with 8-methoxypsoralen by gastric tube and exposed to UVA light to produce threshold phototoxic reactions. 3H-thymidine autoradiography revealed no significant difference of the epidermal labelling index as compared to that of nonirradiated animals (4.8 vs 6.2). Single PUVA exposures performed 2,8,14 and 24 hr after tape stripping lead to suppression of a wave of synchronized DNA synthesis present in nonirradiated control animals. These data demonstrate different reactions of stripped and unstripped epidermis to PUVA exposures and offer indirect evidence for the suppression of DNA synthesis in hyperproliferative skin disorders by PUVA in vivo.

Antibody-induced cAMP accumulation in splenocytes from athymic nude mice.

Products from the hydrolysis of phosphatidylinositol 4,5-bisphosphate (IP3) can increase and/or potentiate cAMP accumulation in a variety of cells. Antibody to surface immunoglobulins activates IP3 hydrolysis in B-lymphocytes. In this study we have examined whether anti-Ig also stimulated and/or potentiated increases in the cAMP levels of splenocytes from athymic nude mice. Furthermore, since TPA potentiates anti-Ig-induced DNA synthesis and cAMP modulates DNA synthesis, the effects of TPA on any anti-Ig-induced changes in cAMP were also studied. Antibody (25 micrograms/ml) stimulated a rapid ris in cAMP which increased from 250 fmol/10(6) cells to 400 fmol/10(6) cells within 1 min and then subsided to 310 fmol/10(6) cells by 10 min. TPA (96 nM) suppressed the anti-Ig-induced cAMP accumulation at 1 min by 60%, but potentiated the forskolin (114 microM)-induced rise by 151%. Two other activators of protein kinase C, dioctanoylglycerol (5 microM), and anti-Ig (25 micrograms/ml), also potentiated the forskolin response by 198% and 52%, respectively. These results suggest that modulation of the adenylate cyclase system by anti-Ig may act in concert with cytokines and/or prostaglandins secreted by other lymphoid cells to define the state of proliferation or differentiation in B-cells.

A comparison of bone turnover in athymic (nude) and euthymic mice: biochemical, histomorphometric, bone ash and in vitro studies.

Bone turnover in T-cell deficient mice was investigated by comparing parameters of bone physiology in athymic (nude) and euthymic mice. Static and dynamic bone histomorphometry, serum biochemical assays, body weight and tibia length measurements, and bone ash determination were completed in 6- and 12-wk-old athymic (nude) mice (NIH: Swiss nu/nu) and euthymic mice (nu/+) (10 mice/group). In vitro bone resorbing activity stimulated by parathyroid hormone (PTH) or prostaglandin E2 (PGE2) was measured in calvaria of neonatal athymic and euthymic mice. Athymic mice had smaller vertebral tissue area (p less than 0.01), tibia length (p less than 0.001), and less body weight (p less than 0.01) than euthymic mice. The percent double labeled surface (p less than 0.05) and mineralizing perimeter (p less than 0.01) were reduced in athymic as compared to age-matched euthymic mice. Osteoclast number was reduced in the 6-wk athymic mice as compared to 6-wk euthymic mice. Osteoclastic perimeter was reduced in the 12-wk-old mice (athymic and euthymic) as compared to the 6-wk-old mice. Serum calcium was lower at both ages in athymic mice (p less than 0.01) as compared to euthymic mice. Serum alkaline phosphatase levels were reduced (p less than 0.01) in 12-wk-old athymic mice as compared to age-matched euthymic mice, and were greater in 6-wk-old mice than 12-wk-old mice. Athymic mice had greater femur density than euthymic mice (p less than 0.01), and lower (p less than 0.001) percent ash weight of dry bone compared to euthymic mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative biodistributions of yttrium- and indium-labeled monoclonal antibody B72.3 in athymic mice bearing human colon carcinoma xenografts.

The biodistribution of yttrium- and indium-labeled monoclonal antibody (MAb) B72.3 IgG using three different chelate conjugates (SCN-Bz-EDTA, CA-DTPA, and SCN-Bz-DTPA) was compared in athymic mice bearing LS-174T tumors. The 88Y-SCN-Bz-DTPA-B72.3 yielded 40% ID/g at 5-7 days in the tumors, while the 88Y-SCN-Bz-EDTA-B72.3 and 88Y-CA-DTPA-B72.3 showed only 6-8% ID/g. The yttrium uptake in the bone with the SCN-Bz-EDTA and CA-DTPA conjugated IgG was over 14 and 11% ID/g, respectively, while 88Y-SCN-Bz-DTPA-B72.3 showed only 3% ID/g. In contrast, the 111In-labeled B72.3 uptake in the bone with all three chelate conjugates was 2-3% ID/g. The differences in yttrium- versus indium-labeled MAb biodistributions demonstrate the difficulty in using 111In-labeled MAbs to predict the biodistribution and dosimetry of 90Y-labeled MAbs unless chelate conjugates such as SCN-Bz-DTPA are used.

Fetal human neural progenitors can be the target for tumor transformation.

The hypothesis that stem cells may seed cancer has emerged from the cancer stem cells concept. However, the experimental systems necessary to provide more direct evidence to support the hypothesis have been lacking. We have used fetal neural progenitor cells (hNPC) transduced with the telomerase hTERT gene to investigate the neoplastic potential of hNPCs. The hTERT-transduced line, hNPCs-G3 lost normal diploid karyotype, showed loss of contact inhibition, anchorage independence, and formed neuroblastoma-like tumours in all of 10 mice. These data suggest that hNPCs have the potential for neoplastic transformation. These data have implications for providing a novel tool to test the feasibility of new anticancer treatment strategies and raise the possibility of a risk for the use of hNPCs in cell transplantation.

Murine model for skeletal metastases of Ewing's sarcoma.

Ewing's sarcoma shows a strong tendency to metastasize to the lungs or the skeleton, or both. A peculiar feature of the secondary involvement of bone with this tumor is that it may also appear in the absence of clinically evident lung metastases, both at clinical presentation and during the course of the disease. Although osseous metastases are critically relevant for prognosis, the pathogenesis of this peculiar feature of Ewing's sarcoma is poorly understood, partly due to the lack of appropriate experimental in vivo models. We show that the intravenous injection of TC-71 Ewing's sarcoma cells into athymic 4-5-week-old Crl/nu/nu (CD1) BR mice reproducibly colonizes specific sites of the skeleton in addition to the lungs and lymph nodes. The distribution and the morphologic appearance of these experimental bone metastases mimic the pattern of skeletal involvement observed in humans. This experimental model of bone metastasis of Ewing's sarcoma may be the basis for future studies aimed at understanding the pathophysiology and treatment of Ewing's sarcoma.

Distribution of Theiler's virus in the CNS of athymic nude mice: effect of varying the route of inoculation.

Immunohistochemical and ultrastructural techniques were used to map the distribution of Theiler's virus in the central nervous system (CNS) of the nude mouse, after intracerebral (i.c.), intravenous (i.v.) or intraocular (i.o.) inoculation. Expression of viral antigen was largely restricted to the subthalamus, substantia nigra, ventral tegmental tract and the grey and white matter of the spinal cord. Electron microscopy showed paracrystalline arrays of viral particles within neurons in the substantia nigra and spinal cord. Inoculation of virus i.v. or i.o. rather than i.c. delayed the onset of neurological signs but did not affect the distribution of virus within the CNS. In particular, there was no evidence of spread along the optic pathways after i.o. inoculation. The localization of Theiler's virus within certain regions of the CNS seems to depend on differential susceptibility to infection or differential restriction of replication rather than on the route of inoculation.

Physical model evaluation of topical prodrug delivery--simultaneous transport and bioconversion of vidarabine-5'-valerate V: Mechanistic analysis of influence of nonhomogeneous enzyme distributions in hairless mouse skin.

The mathematical problem of simultaneous transport and metabolism in the case of nonuniform enzyme distributions in the skin was solved, and the solutions were used for analyzing experimental data. Experimental data were obtained from permeation experiments with 3H-vidarabine and its 5'-valerate using cellophane tape-stripped hairless mouse skin. Results of the analyses revealed that the esterase activity was four to nine times higher in the epidermis than in the dermis, whereas the deaminase activity was about the same in the two strata. These results were in good agreement with independent experiments using tissue homogenates. The enzyme distributions and the previously reported diffusivities were employed in generating concentration profiles for the prodrug and the drug in the skin. These results may be used in predicting the possible therapeutic effect of the prodrug when it is topically applied.

Physical model evaluation of topical prodrug delivery--simultaneous transport and bioconversion of vidarabine-5'-valerate III: Permeability differences of vidarabine and n-pentanol in components of hairless mouse skin.

The permeation behavior of 3H-vidarabine (3H-9-beta-D-ara-binofuranosyladenine) and 14C-n-pentanol through different strata of hairless mouse skin was studied using a diffusion cell at 37 degrees under steady-state conditions. Partition coefficients for the skin components verus 0.9% aqueous NaCl solution also were obtained. Various skin preparations including full-thickness skin, cellophane-stripped skin, and dermis membranes of different thicknesses were employed. The dermis membranes were considered to be diffusionally homogenous, and the product of the permeability coefficient and the thickness was taken as the apparent diffusivity. The apparent diffusivities for both compounds investigated were independent of thickness. Therefore, it was concluded that the molecular diffusivity is constant throughout the dermis. Comparisons of permeability coefficients in various strata of the skin revealed that, while the stratum corneum is the major diffusional barrier, the epidermis appears to be significantly less permeable than the dermis.

Physical model evaluation of topical prodrug delivery--simultaneous transport and bioconversion of vidarabine-5'-valerate IV: Distribution of esterase and deaminase enzymes in hairless mouse skin.

A semiquantitative assessment of estrase and deaminase distributions in hairless mouse skin was performed in vitro. The enzyme activities were quantified using 3H-vidarabine and tis 5'-valerate as the substrates. Full-thickness skin of the hairless mouse was cut into two halves, and each half was homogenized in pH 7.4 buffer. BQOTH THE SUPERNATE AND THE RESIDUE OF THE HOMOGENATE were assayed for esterase and deaminase activities. Results show that the outer half-thickness of the skin contained more esterase but slightly less deaminase than the other half. The characteristics of the esterase and the deaminase reactions also were studied employing the crude enzyme extract; these reactions were essentially irreversible. The deaminase reaction was in the linear region of Michaelis-Menten kinetics for substrate concentrations up to 4.5 x 10(-5) M.

Permeation of hairless mouse skin I: Experimental methods and comparison with human epidermal permeation by alkanols.

The permeation of intact hairless mouse skin by alkanols was studied. The method is described, and data for the quasi-steady-state and nonstationary-state aspects of mass transfer are given. Partitioning data also are presented. The permeability coefficients increased exponentially up to a carbon chain length of about eight (octanol). There was a marked temperature dependency (Ea congruent to 19 kcal for the series), and the partitioning was biphasic, increasing exponentially for alkanols larger than butanol. These data are compared with literature data on human skin tissues, and great similarity in all facets of behavior is noted.

Methionine-enkephalin immunoreactivity in Merkel cell dense-core granules of nude mice sinus hair. A post-embedding immunogold electron-microscopic study.

The ultra-immunocytochemical technique applied in the present study revealed the occurrence of methionine-enkephalin (met-enkephalin)-like substance in the dense-core granules of Merkel cells of nude mice sinus hair. Incubation of ultra-thin sections of sinus hair with met-enkephalin antisera conjugated with gold particles showed specific association of gold particles on the dense-core granules of the Merkel cells. Gold particles were heavily and specifically located on the dense-core granules as well as in the adjacent cytoplasm. Dense-core granules of degenerating Merkel cells also exhibit met-enkephalin immunoreactivity. The nerve terminals associated with the Merkel cell did not show met-enkephalin immunoreactivity. Therefore, it is concluded that a met-enkephalin-like substance is present and stored in nude mice Merkel cell dense-core granules and it might act as a neurotransmitter or neuromodulator which could be involved in the functioning of the cell. Non-osmicated tissue should be used to locate this substance because of the possibility of cross-linkage of the amino acid sequence with osmium tetroxide.

A search for the "multiple sclerosis virus"--lack of effect of brain extracts on myelin development in chickens, mice and rats.

Attempts were made to transmit possible infectious agents from tissue of MS patients into three animal species, under conditions designed to enhance the development of such an agent. Myelination was monitored by measuring CNPase activity and myelin basic protein. Although no significant effects were observed, the usefulness of a new assay for CNPase was demonstrated. Nude mice were found to have a lower level of CNPase than their heterozygous littermates.

Nude mouse in studies on chemical carcinogenesis.

Enzyme of the monooxygenase complex were found in the skin of a nude mouse strain, and the activities increased after an intraperitoneal administration of 3-methylcholantrene. No comparable change was observed in UDPglucuronosyltransferase, an enzyme of the conjugation system. Fibroblast cultures prepared from nude mouse skin proved sensitive for transformation by carcinogenic chemicals. Nude mouse accepts transplants e.g. transformed cells more easily than conventional laboratory animals. The nude mouse model may therefore offer a useful alternative to other transformation assays using mammalian cells as targets.

Skin absorption of organic solvent vapors in nude mice in vivo.

Nude mice each attached to a respirator to avoid pulmonary uptake were exposed in a glass exposure chamber to 200, 1000 or 3000 ppm of benzene, toluene or tetrachloroethylene (perclene) for 2, 4 or 6 h. The animals were killed at the end of the study and the amount of each solvent retained in the whole body was determined by gas chromatography. Skin absorption rates were calculated from the amount retained in the whole body using the single compartment model (elimination rate constant) obtained in a previous experiment. There was a linear relationship between the amount of skin absorption and exposure time, and also a linear relationship between the skin adsorption rate and concentration of exposed vapors. Skin absorption of solvent vapors occurs by passive diffusion as defined by Fick's law. The skin absorption coefficient (cm/h) of each solvent vapor was calculated by dividing the skin absorption rate by exposure concentration; the values were 1.24 for toluene, 1.00 for perclene and 0.619 for benzene. The coefficient may be useful for evaluating the amount of skin absorption (ng) was calculated by multiplying the skin absorption coefficient (cm/h), concentration of solvent vapor (ng/cm3), exposure time (h) and exposed skin area (cm2).