Renal Capsule Of Nude Mice Research Articles

Anti-relapse effect of trametinib on a local minimal residual disease neuroblastoma mouse model

PurposeWe reported the in vitro and in vivo anti-tumor effects of trametinib, an MEK inhibitor, on neuroblastoma. However, long-term trametinib administration for bulky tumors failed to prevent local relapse. In this study, we established a local minimal residual disease (L-MRD) model to develop an optimal clinical protocol. MethodsWe prepared an l-MRD model by implanting neuroblastoma cells (SK-N-AS) into the renal capsule of nude mice with total tumorectomy or sham operation 14 days later. These mice received post-operative administration of trametinib or vehicle for eight weeks. Relapse was measured once weekly. Flow cytometry was performed with SK-N-AS cells treated by trametinib. ResultsTumorectomy+trametinib dramatically suppressed relapse, and all mice survived during trametinib administration, while other treatments failed to suppress relapse. The survival rates for other groups were 20% in sham+trametinib, 17% in tumorectomy+vehicle, and 0% in sham+vehicle. Relapse occurred in the tumorectomy+trametinib group after withdrawal of trametinib administration. Flow cytometry revealed G1 arrest in SK-N-AS cells treated with trametinib. ConclusionThese findings suggested that trametinib was able to suppress relapse from minimal residual tumor cells. Therefore, we propose that trametinib be administered as an option for maintenance therapy after surgical and chemotherapeutic treatments for neuroblastoma in future clinical protocols.

Effects of Inorganic Arsenic on Human Prostate Stem-Progenitor Cell Transformation, Autophagic Flux Blockade, and NRF2 Pathway Activation.

Background:Inorganic arsenic (iAs) is an environmental toxicant associated with an increased risk of prostate cancer in chronically exposed populations worldwide. However, the biological mechanisms underlying iAs-induced prostate carcinogenesis remain unclear.Objectives:We studied how iAs affects normal human prostate stem-progenitor cells (PrSPCs) and drives transformation and interrogated the molecular mechanisms involved.Methods:PrSPCs were enriched by spheroid culture from normal human primary or immortalized prostate epithelial cells, and their differentiation capability was evaluated by organoid culture. Microarray analysis was conducted to identify iAs-dysregulated genes, and lentiviral infection was used for stable manipulation of identified genes. Soft agar colony growth assays were applied to examine iAs-induced transformation. For in vivo study, PrSPCs mixed with rat urogenital sinus mesenchyme were grafted under the renal capsule of nude mice to generate prostatelike tissues, and mice were exposed to () iAs in drinking water for 3 months.Results:Low-dose iAs () disturbed PrSPC homeostasis in vitro, leading to increased self-renewal and suppressed differentiation. Transcriptomic analysis indicated that iAs activated oncogenic pathways in PrSPCs, including the KEAP1-NRF2 pathway. Further, iAs-exposed proliferative progenitor cells exhibited NRF2 pathway activation that was sustained in their progeny cells. Knockdown of NRF2 inhibited spheroid formation by driving PrSPC differentiation, whereas its activation enhanced spheroid growth. Importantly, iAs-induced transformation was suppressed by NRF2 knockdown. Mechanistically, iAs suppressed Vacuolar ATPase subunit VMA5 expression, impairing lysosome acidification and inhibiting autophagic protein degradation including p62, which further activated NRF2. In vivo, chronic iAs exposure activated NRF2 in both epithelial and stroma cells of chimeric human prostate grafts and induced premalignant events.Conclusions:Low-dose iAs increased self-renewal and decreased differentiation of human PrSPCs by activating the p62-NRF2 axis, resulting in epithelial cell transformation. NRF2 is activated by iAs through specific autophagic flux blockade in progenitor cells, which may have potential therapeutic implications. https://doi.org/10.1289/EHP6471

Generation of functional salivary gland tissue from human submandibular gland stem/progenitor cells

BackgroundOrgan replacement regenerative therapy based on human adult stem cells may be effective for salivary gland hypofunction. However, the generated tissues are immature because the signaling factors that induce the differentiation of human salivary gland stem cells into salivary glands are unknown.MethodsIsolated human submandibular gland stem/progenitor cells (hSMGepiS/PCs) were characterized and three-dimensionally (3D) cultured to generate organoids and further induced by fibroblast growth factor 10 (FGF10) in vitro. The induced spheres alone or in combination with embryonic day 12.5 (E12.5) mouse salivary gland mesenchyme were transplanted into the renal capsules of nude mice to assess their development in vivo. Immunofluorescence, quantitative reverse transcriptase-polymerase chain reaction, calcium release analysis, western blotting, hematoxylin–eosin staining, Alcian blue–periodic acid-Schiff staining, and Masson’s trichrome staining were performed to assess the structure and function of generated tissues in vitro and in vivo.ResultsThe isolated hSMGepiS/PCs could be long-term cultured with a stable genome. The organoids treated with FGF10 [FGF10 (+) group] exhibited higher expression of salivary gland–specific markers; showed spatial arrangement of AQP5+, K19+, and SMA+ cells; and were more sensitive to the stimulation by neurotransmitters than untreated organoids [FGF10 (−) group]. After heterotopic transplantation, the induced cell spheres combined with mouse embryonic salivary gland mesenchyme showed characteristics of mature salivary glands, including a natural morphology and saliva secretion.ConclusionFGF10 promoted the development of the hSMGepiS/PC-derived salivary gland organoids by the expression of differentiation markers, structure formation, and response to neurotransmitters in vitro. Moreover, the hSMGepiS/PCs responded to the niche in mouse embryonic mesenchyme and further differentiated into salivary gland tissues with mature characteristics. Our study provides a foundation for the regenerative therapy of salivary gland diseases.

Sequential stimulation with different concentrations of BMP4 promotes the differentiation of human embryonic stem cells into dental epithelium with potential for tooth formation

BackgroundTooth loss caused by caries or injuries has a negative effect on human health; thus, it is important to develop a reliable method of tooth regeneration. Research on tooth regeneration has mainly focused on mouse pluripotent stem cells, mouse embryonic stem cells, and adult stem cells from various sources in mice, whereas little has examined the differentiation of human embryonic stem (hES) cells into dental epithelium (DE) and odontogenic potential in vivo.MethodsIn this study, we induced hES cells to differentiate into dental epithelium using different concentrations of bone morphogenetic protein 4 (BMP4). With 1 pM BMP4, the hES cells differentiated into oral ectoderm (OE). These cells were then stimulated with 30 pM BMP4. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunofluorescence showed the differentiation of OE and DE. The DE generated was mixed with embryonic day 14.5 mouse dental mesenchyme (DM) and transplanted into the renal capsules of nude mice. Thirty days later, the resulting tooth-like structures were examined by micro-computed tomography and hematoxylin and eosin staining.ResultsAfter 4 days of 1 pM BMP4 stimulation, Pitx1-positive OE formed. qRT-PCR and immunofluorescence revealed that induction with 30 pM BMP4 for 2 days caused the OE to differentiate into Pitx2/Dlx2/AMBN-positive DE-like cells. These cells also expressed β-catenin and p-Smad1/5/8, which are key proteins in the Wnt/β-catenin and Bmp signaling pathways, respectively. Thirty days after in vivo transplantation, six teeth with enamel and dentin had formed on the kidney.ConclusionsThese results show that hES cells differentiated into DE after sequential stimulation with different concentrations of BMP4, and the DE thus generated showed odontogenic potential.

Periodontal ligament-like tissue regeneration with drilled porous decalcified dentinmatrix sheet composite.

In this study, we constructed a composite by combining the human dental follicle cell sheet and a manual drilled porous decalcified dentin matrix that was used to construct ectopic tissue-engineered periodontal ligament-like tissues in renal capsules of nude mice. Human dental follicle cells were harvested from human lower third molars and then embedded into atemperature-sensitiveculture dish. These cells were thenplaced into frozen porousdecalcified dentin matrix sheets andinduced by 50g/mlascorbic acid. This established a "sandwichstructure" in vitro implant that was placed in nude mice under the renal capsule. The mice were sacrificed at 4 and 8weeks after implantation, andthe implants were assessed after haematoxylin-eosin staining,Massonstaining andimmunohistochemical staining. The experimental group showed a fibre structurebetweenthe dentin and HA-TCP after 4weeks. After 8weeks, the collagen fibresincreased, and the direction was perpendicular to thedentin. Immunohistochemistry showed positive staining in the osteopontin and periostin. The composite can induce ectopic bone and fibre formation, and the fibre had a certain directionality. Besides, the composite can maintain the stability of the periodontal ligamentwidth.

Generation of tooth\u2013periodontium complex structures using high-odontogenic potential dental epithelium derived from mouse embryonic stem cells

BackgroundA number of studies have shown that tooth-like structures can be regenerated using induced pluripotent stem cells and mouse embryonic stem (mES) cells. However, few studies have reported the regeneration of tooth–periodontium complex structures, which are more suitable for clinical tooth transplantation. We established an optimized approach to induce high-odontogenic potential dental epithelium derived from mES cells by temporally controlling bone morphogenic protein 4 (BMP4) function and regenerated tooth–periodontium complex structures in vivo.MethodsFirst, immunofluorescence and quantitative reverse transcription-polymerase chain reaction were used to identify the watershed of skin and the oral ectoderm. LDN193189 was then used to inhibit the BMP4 receptor around the watershed, followed by the addition of exogenous BMP4 to promote BMP4 function. The generated dental epithelium was confirmed by western blot analysis and immunofluorescence. The generated epithelium was ultimately combined with embryonic day 14.5 mouse mesenchyme and transplanted into the renal capsules of nude mice. After 4 weeks, the tooth–periodontium complex structure was examined by micro-computed tomography (CT) and hematoxylin and eosin (H&E) staining.ResultsOur study found that the turning point of oral ectoderm differentiation occurred around day 3 after the embryoid body was transferred to a common culture plate. Ameloblastin-positive dental epithelial cells were detected following the temporal regulation of BMP4. Tooth–periodontium complex structures, which included teeth, a periodontal membrane, and alveolar bone, were formed when this epithelium was combined with mouse dental mesenchyme and transplanted into the renal capsules of nude mice. Micro-CT and H&E staining revealed that the generated tooth–periodontium complex structures shared a similar histological structure with normal mouse teeth.ConclusionsAn optimized induction method was established to promote the differentiation of mES cells into dental epithelium by temporally controlling the function of BMP4. A novel tooth–periodontium complex structure was generated using the epithelium.

Induction of Salivary Gland–Like Cells from Dental Follicle Epithelial Cells

The dental follicle (DF), most often associated with unerupted teeth, is a condensation of ectomesenchymal cells that surrounds the tooth germ in early stages of tooth development. In the present study, we aim to isolate epithelial stem-like cells from the human DF and explore their potential differentiation into salivary gland (SG) cells. We demonstrated the expression of stem cell–related genes in the epithelial components of human DF tissues, and these epithelial progenitor cells could be isolated and ex vivo expanded in a reproducible manner. The human DF-derived epithelial cells possessed clonogenic and sphere-forming capabilities, as well as expressed a panel of epithelial stem cell–related genes, thus conferring stem cell properties (hDF-EpiSCs). When cultured under in vitro 3-dimensional induction conditions, hDF-EpiSCs were capable to differentiate into SG acinar and duct cells. Furthermore, transplantation of hDF-EpiSC–loaded native de-cellularized rat parotid gland scaffolds into the renal capsule of nude mice led to the differentiation of transplanted hDF-EpiSCs into salivary gland–like cells. These findings suggest that hDF-EpiSCs might be a promising source of epithelial stem cells for the development of stem cell–based therapy or bioengineering SG tissues to repair/regenerate SG dysfunction.

Transdifferentiation of human male germline stem cells to hepatocytes in vivo via the transplantation under renal capsules.

Here we proposed a new concept that human spermatogonial stem cells (SSCs) can transdifferentiate into hepatocytes in vivo. We first established liver injury model of mice by carbon tetrachloride to provide proper environment for human SSC transplantation. Liver mesenchymal cells were isolated from mice and identified phenotypically. Human SSC line was recombined with liver mesenchymal cells, and they were transplanted under renal capsules of nude mice with liver injury. The grafts expressed hepatocyte hallmarks, including ALB, AAT, CK18, and CYP1A2, whereas germ cell and SSC markers VASA and GPR125 were undetected in these cells, implicating that human SSCs were converted to hepatocytes. Furthermore, Western blots revealed high levels of PCNA, AFP, and ALB, indicating that human SSCs-derived hepatocytes had strong proliferation potential and features of hepatocytes. In addition, ALB–, CK8–, and CYP1A2– positive cells were detected in liver tissues of recipient mice. Significantly, no obvious lesion or teratomas was observed in several important organs and tissues of recipient mice, reflecting that transplantation of human SSCs was safe and feasible. Collectively, we have for the first time demonstrated that human SSCs can be transdifferentiated to hepatocyte in vivo. This study provides a novel approach for curing liver diseases using human SSC transplantation.

Heterogeneous Uptake of Nanoparticles in Mouse Models of Pediatric High-Risk Neuroblastoma.

Liposomal chemotherapeutics are exemplified by DOXIL® are commonly used in adult cancers. While these agents exhibit improved safety profile compared to their free drug counterparts, their treatment response rates have been ~ 20%, often attributed to the heterogeneous intratumoral uptake and distribution of liposomal nanoparticles. Non-invasive and quantitative monitoring of the uptake and distribution of liposomal nanoparticles in solid tumors could allow for patient stratification and personalized cancer nanomedicine. In this study, the variability of liposomal nanoparticle intratumoral distribution and uptake in orthotopic models of pediatric neuroblastoma was investigated using a liposomal nanoprobe visualized by high-resolution computed tomography (CT). Two human neuroblastoma cell lines (NGP: a MYCN-amplified line, and SH-SY5Y a MYCN non-amplified line) were implanted in the renal capsule of nude mice to establish the model. Intratumoral nanoparticle uptake was measured at tumor ages 1, 2, 3 and 4 weeks post implantation. The locations of uptake within the tumor were mapped in the 3-dimensional reconstructed images. Total uptake was measured by integration of the x-ray absorption signal over the intratumoral uptake locations. Both tumor models showed significant variation in nanoparticle uptake as the tumors aged. Observation of the uptake patterns suggested that the nanoparticle uptake was dominated by vascular leak at the surface/periphery of the tumor, and localized, heterogeneous vascular leak in the interior of the tumor. Slow growing SH-SY5Y tumors demonstrated uptake that correlated directly with the tumor volume. Faster growing NGP tumor uptake did not correlate with any tumor geometric parameters, including tumor volume, tumor surface area, and R30 and R50, measures of uptake localized to the interior of the tumor. However, uptake for both SH-SY5Y and NGP tumors correlated almost perfectly with the leak volume, as measured by CT. These results suggest that the uptake of nanoparticles is heterogeneous and not governed by tumor geometry. An imaging nanoprobe remains the best measure of nanoparticle uptake in these tumor models.

Growth and fertilization of porcine fetal oocytes grafted under the renal capsules of nude mice

The fetal ovary contains a larger pool of oocytes than the adult ovary, but utilization of the fetal oocytes of large animals has hardly been examined. In this study, we investigated the developmental competence of oocytes grown in host mice harboring ovarian grafts obtained from fetal pigs. Ovarian fragments from fetuses at 55, 70, and 90 days postartificial insemination (dpi) were grafted into ovariectomized nude mice (Crlj:CD1-Foxn1nu; 55-, 70- and 90-dpi groups, respectively). For comparison, ovarian fragments from 20-day postpartum (dpp) piglets were also grafted (20-dpp group). About 60 days after detection of vaginal opening, the mice were given 62.5 U/mL porcine FSH for 13 days by infusion to enhance their follicular development. In the fetal ovaries before grafting, the percentage of germ cells in primordial follicles (termed primordial oocytes) relative to the total number of germ cells was 0.06% at 55 dpi, 2.4% at 70 dpi, and 7.2% at 90 dpi, but the majority was contained within egg nests. At 20 dpp, primordial oocytes accounted for 91.7% of the total number of germ cells and the rest were mostly in primary follicles. After FSH stimulation of host mice, formation of antral follicles was promoted in the grafts of the 70- and 90-dpi groups as well as the 20-dpp group, but a very small number of antral follicles developed in the 55-dpi group consistent with the lowest (P < 0.05) levels of circulating inhibin in that group. The mean number of full-sized oocytes with meiotic competence recovered per mouse was 6.0 in the 70-dpi, 18.0 in the 90-dpi, and 21.2 in the 20-dpp groups, whereas virtually no oocytes were recovered from mice in the 55-dpi group. Moreover, the mature oocytes in the 70- and 90-dpi groups were fertilized in vitro, as shown by formation of male and female pronuclei, but the percentage of oocytes penetrated by sperm was low in the 70- (49%) and 90-dpi (29%) groups as compared with the 20-dpp group (88%). These results clearly indicate that porcine fetal ovaries during the late pregnancy period are able to grow and produce oocytes with fertilization ability after being grafted into nude mice. The presence of primordial follicles is likely to be associated with the ability of fetal ovaries to produce antral follicles after xenografting.

Estrogen-initiated transformation of prostate epithelium derived from normal human prostate stem-progenitor cells.

The present study sought to determine whether estrogens with testosterone support are sufficient to transform the normal human prostate epithelium and promote progression to invasive adenocarcinoma using a novel chimeric prostate model. Adult prostate stem/early progenitor cells were isolated from normal human prostates through prostasphere formation in three-dimensional culture. The stem/early progenitor cell status and clonality of prostasphere cells was confirmed by immunocytochemistry and Hoechst staining. Normal prostate progenitor cells were found to express estrogen receptor α, estrogen receptor β, and G protein-coupled receptor 30 mRNA and protein and were responsive to 1 nm estradiol-17β with increased numbers and prostasphere size, implicating them as direct estrogen targets. Recombinants of human prostate progenitor cells with rat urogenital sinus mesenchyme formed chimeric prostate tissue in vivo under the renal capsule of nude mice. Cytodifferentiation of human prostate progenitor cells in chimeric tissues was confirmed by immunohistochemistry using epithelial cell markers (p63, cytokeratin 8/18, and androgen receptor), whereas human origin and functional differentiation were confirmed by expression of human nuclear antigen and prostate-specific antigen, respectively. Once mature tissues formed, the hosts were exposed to elevated testosterone and estradiol-17β for 1-4 months, and prostate pathology was longitudinally monitored. Induction of prostate cancer in the human stem/progenitor cell-generated prostatic tissue was observed over time, progressing from normal histology to epithelial hyperplasia, prostate intraepithelial neoplasia, and prostate cancer with local renal invasion. These findings provide the first direct evidence that human prostate progenitor cells are estrogen targets and that estradiol in an androgen-supported milieu is a carcinogen for human prostate epithelium.

The effect of streptozotocin on the function of fetal porcine and rat pancreatic (pro-)islets.

Streptozotocin (STZ) is a broad spectrum antibiotic with anti-tumor and diabetogenic properties. Although STZ has been studied for many years, the exact mechanism of its diabetogenic action has not yet been fully elucidated. The present study investigated the effect of STZ on both fetal porcine proislets (FPP) and fetal rat islets (FRI) in an attempt to elucidate the diabetogenic effect of STZ on fetal pancreatic beta cells. This study demonstrates that after in vitro exposure of both FPP and FRI for 30 min to 2.2 mM and 4.4 mM STZ, respectively, FPP showed microscopically an intact structure, a spherical shape and a translucent color, while, in contrast, most FRI were disrupted and showed a slight white color with dark centers. Based on these data, we first transplanted FPP and FRI beneath the renal capsules of nude mice. Three to four weeks later, a single dosage of streptozotocin (180 mg/kg) was intravenously administered. Six of the seven nude mice pretransplanted with FPP became diabetic (blood glucose, BG, 308.08 +/- 33.62 mg/dl) within 2-5 days and then gradually achieved normoglycemia 51.56 +/- 7.71 days after STZ injection. After removal of the grafts, all of the six diabetic mice with normoglycemia returned to hyperglycemia (BG > 300 mg/dl). In contrast, all of the five nude mice pretransplanted with FRI persistently maintained hyperglycemia (BG > 300 mg/dl) and died 5 +/- 0.84 days after STZ injection.(ABSTRACT TRUNCATED AT 250 WORDS)

Formation of amyloid in human pancreatic islets transplanted to the liver and spleen of nude mice

In previous studies we have shown that apparently normal human islets, transplanted under the renal capsule of nude mice, frequently and rapidly develop amyloid deposits derived from the beta-cell hormone islet amyloid polypeptide (IAPP). In the present study, we show for the first time that human islets, transplanted into the liver or spleen of nude mice, also develop islet amyloid rapidly. Ultrastructural studies of such islets showed that the first aggregation of IAPP takes place within the beta-cells and that extracellular deposits show up later in the amyloid formation process. We also found that the amount of amyloid formed in human islet grafts placed under the kidney capsule increased with extended (26 weeks) observation time. Moreover, prolonged in vitro culture (14 days) prior to the implantation under the renal capsule seemed to enhance the formation of amyloid in the grafted islets. Since aggregated IAPP has been shown to be toxic to beta-cells, the finding of amyloid deposits in transplanted islets offers a possible explanation to the frequent loss of function of islets transplanted into diabetic patients.

Isolation, Culture, and Characterization of Endocrine Cells from 6-Month-Old Porcine Pancreas

Porcine endocrine cells were isolated from pancreas of 6-month-old pigs by two-step enzymatic digestion procedures. They were separated by the density gradient (isopycnic) centrifugation method using Histopaque-1077. Isolated cells were cultured and divided into two groups: suspension cells and adhesion cells. Suspension cells maintained their cell numbers on and after 7 days in culture. Approximately 1 x 10(7) cells were obtained from single pancreas of a 6-month-old pig. The cultured suspension cells took up dithizone (DTZ) staining 14 days after isolation in culture and indicated the presence of beta-cells. In in vitro study, the suspension cells were capable of secreting insulin into the culture medium. The suspension cells were tested for insulin and glucagon staining by Western blot analysis. These results indicated the maintenance of endocrine cell function after isolation. However, cultured adhesion cells failed to maintain their function during culture. In in vivo study, the suspension cells were transplanted into diabetes-induced nude mice. Reduction in blood glucose level was obtained after transplantation. Intraperitoneal glucose tolerance test (IPGTT) results showed a normal pattern of blood glucose clearance. After 1 week, the transplanted endocrine cells were detected with anti-insulin antibody by immunostaining and it showed the presence of viable beta-cells under the renal capsule of nude mice. Collectively, our results suggest that isolated and cultured suspension porcine endocrine cells maintained their endocrine function. These endocrine cells can be used as isolated islets for further study, including transplantation experiments.

Impairment of glucose-induced insulin secretion in human pancreatic islets transplanted to diabetic nude mice.

Hyperglycemia-induced beta-cell dysfunction may be an important component in the pathogenesis of non-insulin-dependent diabetes mellitus. However, most available data in this field were obtained from rodent islets. To investigate the relevance of this hypothesis for human beta-cells in vivo, human pancreatic islets were transplanted under the renal capsule of nude mice. Experimental groups were chosen so that grafted islets were exposed to either hyper- or normoglycemia or combinations of these for 4 or 6 wk. Grafts of normoglycemic recipients responded with an increased insulin release to a glucose stimulus during perfusion, whereas grafts of hyperglycemic recipients failed to respond to glucose. The insulin content of the grafts in the latter groups was only 10% of those observed in controls. Recipients initially hyperglycemic (4 wk), followed by 2 wk of normoglycemia regained a normal graft insulin content, but a decreased insulin response to glucose remained. No ultrastructural signs of beta-cell damage were observed, with the exception of increased glycogen deposits in animals hyperglycemic at the time of killing. It is concluded that prolonged exposure to a diabetic environment induces a long-term secretory defect in human beta-cells, which is not dependent on the size of the islet insulin stores.

Antitumor effects of antiprogesterones on human meningioma cells in vitro and in vivo

The presence of the progesterone receptor (PR) in meningioma tissue has been confirmed by previous investigations. Studies have shown that the antiprogesterone drug, mifepristone, is a potent agent that inhibits the growth of cultured meningioma cells and reduces the size of meningiomas in experimental animal models and humans. However, these studies have not fully examined the relationship between the antitumor effects of an antiprogesterone agent and the expression of the PR. The present study examined the antitumor effects of mifepristone and a new potent antiprogesterone agent, onapristone; a correlation between the antitumor effects of these antiprogesterones and the presence of PR's in meningiomas in vitro and in vivo was also investigated. Meningioma tissue surgically removed from 13 patients was used in this study. In the in vitro arm of the study, mifepristone and onapristone exhibited cytostatic and cytocidal effects against cultured meningioma cells, regardless of the presence or absence of PR's; however, three PR-negative meningiomas showed no response to any dose of mifeprestone and/or onapristone. In the in vivo arm, meningioma cells, embedded in a collagen gel, were implanted into the renal capsules of nude mice. Antiprogesterone treatment resulted in a marked reduction of the tumor volume regardless of the presence or absence of PR's. No histological changes in the meningioma cells suggestive of necrosis or apoptosis were detected in any of the mice treated with antiprogesterones. These findings suggest that mifepristone and onapristone have an antitumor effect against meningioma cells via the PR's and/or another receptor, such as the glucocorticoid receptor.

Successful Cryopreservation of Fetal Porcine Proislets

This study reports the cryopreservation of purified fetal pig proislets (FPP) which were isolated by a culture technique. The FPP were equilibrated with 2 M dimethyl sulfoxide (Me 2SO), cooled at a rate of 0.25°C/min to -40°C, transferred to liquid nitrogen ( - 196°C), stored for 2 h to 2 months at - 196°C, and thawed in a 37°C water bath. The morphology of frozen-thawed FPP was similar to that of noncryopreserved FPP. One hundred percent recovery of response capacity was achieved when tested with 16.7 m M glucose plus 10 m M theophylline. We have recently shown that streptozotocin is not toxic to FPP. Accordingly, we transplanted a standardized dose (10-15 mg/mouse) of either cultured or frozen-thawed FPP beneath the renal capsule of nude mice. Three to four weeks later the recipients of the FPP were rendered diabetic by iv streptozotocin. All of the mice transplanted with cultured FPP and seven of eight mice receiving cryopreserved FPP achieved normoglycemia, 74.8 ± 32.9 and 54.7 ± 8.1 days ( P > 0.05) after transplantation. An intraperitoneal glucose tolerance test in the mice of both groups showed a flatter response to glucose compared to those of normal controls. Grafts removed from the mice with normoglycemia for > 3 weeks, in both groups, had the capacity to secrete insulin in response to 16.7 m M glucose alone and 16.7 m M glucose plus 10 m M theophylline during in vitro perifusion. Histological examination revealed that the extent of differentiation and development, in vivo of cryopreserved FPP was comparable with that of cultured FPP. These data indicate that cryopreservation, with the protocol used here, is successful in maintaining functional viability of frozen-thawed FPP. This study is valuable for clinical islet transplantation research.

Levamisole plus 5-fluorouracil inhibits the growth of human colorectal xenografts in nude mice

Fragments of human colorectal adenocarcinomas were inserted under the renal capsule of nude mice. The growth of these tumour grafts was significantly inhibited by the combination of 5-fluorouracil (5-FU) and levamisole. An alternating regimen of levamisole 2.5 mg/kg and 5-FU 20 mg/kg decreased the size of tumour implants by 33–59% and/or increased the number of macroscopically disappeared fragments in the combined group compared with ineffective monotherapy with saline, levamisole or 5-FU. This model could be valuable for investigating the mechanism of action of levamisole and to evaluate the effects of this adjuvant therapy in other oncological settings.

Infectious cycle of human papillomavirus type 11 in human foreskin xenografts in nude mice

We have performed the first molecular analysis of a time course of infection by a papillomavirus. The Hershey isolate of the human papillomavirus type 11 was used to infect human foreskin tissues, which were then implanted under the renal capsules of nude mice. The xenografts were recovered every 2 weeks for 14 weeks, fixed in formalin, and embedded in paraffin. Four-micrometer serial sections were examined by light microscopy for morphological changes, by immunocytochemistry for virion antigen production, and by in situ hybridization with 3H-labeled RNA probes for viral DNA replication and expression of the major mRNA species. After a lag period, probes spanning the E4 and E5 open reading frames, which are present in all E region viral mRNAs, generated the first detectable signals at week 4. Signals of other E region probes were minimally detected at week 6. Between weeks 6 and 8, there was an abrupt change in the implant such that cellular proliferation, viral DNA replication, and E and L region mRNA transcription were robust and reached a plateau. By weeks 10 to 12, the experimental condylomata were morphologically and histologically indistinguishable from naturally occurring condylomata acuminata. These findings suggest that cellular hyperproliferation and the morphologic features of condylomata are direct results of viral genetic activities. Unlike other DNA viruses, the E region transcripts increased with cell age and cellular differentiation and persisted throughout the entire experiment. In particular, the mRNA encoding the E1iE4 and perhaps E5 proteins remained overwhelmingly abundant. In contrast, viral DNA replication, L region mRNA synthesis, and virion antigen production were restricted to the most differentiated, superficial cells.

Streptozotocin is not toxic to the human fetal B cell

It has been generally assumed that because streptozotocin is toxic to the adult B cell of most species, it should also damage B cells obtained at earlier stages of development. This paper examines whether this is true for human fetal pancreata obtained from the therapeutic termination of pregnancies during the first half of the second trimester. Experiments were carried out both in vivo and in vitro. For the former experiments diced explants of the human fetal pancreas were grafted beneath the renal capsule of nude mice 3 weeks before streptozotocin was administered to make the animals diabetic. The grafts were removed 1 week, 2-4 weeks or 3 months later, and were found to be of similar weight and insulin content to the control grafts. In 2 of the animals with grafts remaining for 3 months the diabetes had even been reversed by the implant, hyperglycaemia recurring when the graft was removed. In contrast, rat fetal pancreata grafted beneath the renal capsule of nude mice, subsequently rendered diabetic, were adversely affected by streptozotocin, the insulin content of the implants being 12% of levels in control grafts. Adequate uptake of streptozotocin by the implanted human fetal pancreas was established by measuring tissue levels of the drug 30 min after its injection. Histological examination of the grafted human fetal pancreas showed no deleterious effect of streptozotocin on the number of granulated B cells one day after injection, although by this time the host was diabetic.(ABSTRACT TRUNCATED AT 250 WORDS)