人造血干细胞人源化小鼠的疝气表型分析

目的研究成年雄性人源化小鼠个体出现疝气症状的原因和对人源化小鼠的影响。方法利用显微注射法构建人造血干细胞人源化小鼠,对疝气表型特征、小鼠行为、生理和病理变化进行了研究。结果 2月龄雄性人源化小鼠出现直接性疝气症状,腹股沟区致密结缔组织结构减少可能是疝气形成的主要原因。疝气小鼠同时伴有耐力和运动协调性下降,但疝气对小鼠的繁殖系统无显著影响。结论雄性人造血干细胞人源化小鼠具有显著的疝气症状,其发生机理有待进一步研究。     

Reduced Viability,Fertility and Fecundity in Mice Lacking the Cajal Body Marker Protein,Coilin

Background

Coilin is the signature protein of the Cajal body, a conserved nuclear organelle involved in multiple aspects of small ribonucleoprotein (RNP) biogenesis. Coilin is required for Cajal body homeostasis in both plants and animals. Mice lacking coilin are viable when the mutation is crossed to an outbred strain but only partially viable when crossed to inbred lines.

Methodology/Principal Findings

In order to clarify this issue, we backcrossed the coilin deletion onto the C57BL6/J background for ten generations and then investigated the consequences of coilin removal on overall viability and reproductive success. We conclude that semi-lethal phenotype observed in mixed-background crosses is due to loss of the Coilin gene (or a very tightly-linked locus). Interestingly, coilin knockout embryos die relatively late in gestation, between E13.5 and birth. We show that the maternal contribution of coilin is not important for organismal viability. Importantly, coilin knockout mice display significant fertility and fecundity defects. Mutant males that escape the embryonic lethality display reduced testis size, however, both males and females contribute to the observed reduction in reproductive fitness.

Conclusions/Significance

The evolutionary conservation of coilin from plants to animals suggests that the protein plays an important role, perhaps coordinating the activities of various RNA-processing machineries. Our observations are consistent with the idea that coilin functions to ensure robust organismal development, especially during periods of rapid growth.     

Short-Term PTEN Inhibition Improves In Vitro Activation of Primordial Follicles,Preserves Follicular Viability,and Restores AMH Levels in Cryopreserved Ovarian Tissue From Cancer Patients

IntroductionIn vitro activation and growth of primordial dormant follicles to produce fertilizable oocytes would provide a useful instrument for fertility preservation. The employment of Phosphatase and TENsin homolog (PTEN) inhibitors, in combination with Protein kinase B (Akt) stimulating molecules, has been previously employed to increase follicular activation through the stimulation of the PTEN-Akt pathway.MethodsWe aim to establish improved in vitro activation also for cancer patients whose ovarian tissue has already been cryopreserved. Fresh and previously cryopreserved human ovarian cortex were exposed to short-term, low-concentration and ovary-specific treatment with only a PTEN inhibitor.ResultsOur in vitro activation protocol enhances the activation mechanisms of primordial follicles in both fresh and cryopreserved samples, and enlarges growing populations without inducing apoptosis in either follicles or the surrounding stroma. Treatment augments estradiol secretion and restores the expression levels of the previously diminished Anti-Müllerian hormone by means of cryopreservation procedures. Genomic modulation of the relative expression of PTEN pathway genes was found in treated samples.ConclusionThe in vitro activation protocol offers new alternatives for patients with cryopreserved tissue as it increases the pool of viable activated follicles available for in vitro growth procedures. The combination of ovarian tissue cryopreservation and in vitro activation of primordial follicles, the main ovarian reserve component, will be a major advancement in fertility preservation.     

MHC Class I Expression by Donor Hematopoietic Stem Cells Is Required to Prevent NK Cell Attack in Allogeneic,but Not Syngeneic Recipient Mice

NK cells resist engraftment of syngeneic and allogeneic bone marrow (BM) cells lacking major histocompatibility (MHC) class I molecules, suggesting a critical role for donor MHC class I molecules in preventing NK cell attack against donor hematopoietic stem and progenitor cells (HSPCs), and their derivatives. However, using high-resolution in vivo imaging, we demonstrated here that syngeneic MHC class I knockout (KO) donor HSPCs persist with the same survival frequencies as wild-type donor HSPCs. In contrast, syngeneic MHC class I KO differentiated hematopoietic cells and allogeneic MHC class I KO HSPCs were rejected in a manner that was significantly inhibited by NK cell depletion. In vivo time-lapse imaging demonstrated that mice receiving allogeneic MHC class I KO HSPCs showed a significant increase in NK cell motility and proliferation as well as frequencies of NK cell contact with and killing of HSPCs as compared to mice receiving wild-type HSPCs. The data indicate that donor MHC class I molecules are required to prevent NK cell-mediated rejection of syngeneic differentiated cells and allogeneic HSPCs, but not of syngeneic HSPCs.     

Bradyzoite-Induced Murine Toxoplasmosis: Stage Conversion, Pathogenesis, and Tissue Cyst Formation in Mice Fed Bradvzoites of - Different Strains of Toxoplasma gondii

ABSTRACT. The development of Toxoplasma gondii was studied in mice fed bradyzoites. At one hour after oral inoculation (HAI), bradyzoites were found in cells of the surface epithelium and the lamina propria of the small intestine, primarily the ileum. Division into two tachyzoites was first observed at 18 HA1 in the intestine. At 24 HAI, organisms were also seen in mesenteric lymph nodes. Organisms were first detected in the brain at six days after oral inoculation with bradyzoites (DAI) but not consistently until 10 DAI. Immunohistochemical staining with bradyzoite specific (BAG-5 antigen) anti-serum showed that bradyzoites retained their BAG-5 reactivity even after the first division into two tachyzoites in the intestine at 18 HAL BAG-5 positive organisms were not seen 2–5 DAI. BAG-5 antigens reappeared in T. gondii at 6 DAI. Whole mice and individual tissues of mice fed bradyzoites were bioassayed in cats and mice for the presence of bradyzoites. Feces of cats fed murine tissues were examined for oocyst shedding for short prepatent periods. Bradyzoites were present in the intestines of mice up to 12 HA1 but not at 18 HAI, and tachyzoites and not bradyzoites disseminated to other tissues from the intestine. Bradyzoites were again detected 6 DAI. Using the mouse bioassay, T. gondii was first detected in peripheral blood at 24 HA1 and more consistently at 48 HAL Using a pepsin-digestion procedure and mouse bioassay, organisms were demonstrated in many tissues of mice 15 and 49 DAI.     

Changes in the Bacterial Microbiota in Gut,Blood, and Lungs following Acute LPS Instillation into Mice Lungs

Introduction

Previous reports have shown that the gastrointestinal (GI) bacterial microbiota can have profound effects on the lungs, which has been described as the “gut-lung axis”. However, whether a “lung-gut” axis exists wherein acute lung inflammation perturbs the gut and blood microbiota is unknown.

Methods

Adult C57/Bl6 mice were exposed to one dose of LPS or PBS instillation (n = 3 for each group) directly into lungs. Bacterial microbiota of the bronchoalveolar lavage fluid, blood, and cecum were determined using 454 pyrotag sequencing and quantitative polymerase chain reaction (qPCR) at 4 through 168 hours post-instillation. We then investigated the effects of oral neomycin and streptomycin (n = 8) on the microbiota at 4 and 24 hours post LPS instillation versus control treatment (n = 5 at baseline and 4 hours, n = 7 at 24 hours).

Results

At 24 hours post LPS instillation, the total bacterial count was significantly increased in the cecum (P<0.05); whereas the total bacterial count in blood was increased at 4, 48, and 72 hours (P<0.05). Antibiotic treatment reduced the total bacteria in blood but not in the cecum. The increase in total bacteria in the blood correlated with Phyllobacteriaceae OTU 40 and was significantly reduced in the blood for both antibiotic groups (P<0.05).

Conclusion

LPS instillation in lungs leads to acute changes in the bacterial microbiota in the blood and cecum, which can be modulated with antibiotics.     

The Effect on Intracytoplasmic Sperm Injection Outcome of Genotype,Male Germ Cell Stage and Freeze-Thawing in Mice

Background

Intracytoplasmic sperm injection (ICSI) has been widely used to study the mechanisms of mammalian fertilization and to rescue male-factor infertility in humans and animals. However, very few systematic analyses have been conducted to define factors affecting the efficiency of ICSI. In this study, we undertook a large-scale series of ICSI experiments in mice to define the factors that might affect outcomes.

Methodology/Principal Findings

We used a 5×3×2 factorial design with the following factors: mouse genotype (ICR, C57BL/6, DBA/2, C3H/He, and 129/Sv strains), type of male germ cells (epididymal sperm, elongated or round spermatids), and their freeze–thawing treatment. The efficiencies (parameters) of each developmental step were analyzed by three-way ANOVA (significance level P<0.01). The type of male germ cells affected all the four parameters observed: oocyte survival after injection, cleavage of oocytes, implantation, and birth of offspring. Genotype affected the oocyte survival, cleavage and birth rates, whereas freeze–thawing had no effects on any of the parameters. There were significant genotype/cell type interactions for oocyte survival and cleavage, indicating that they were determined by a combination of strain and germ cell maturity. Multiple comparisons revealed that spermatozoa and elongated spermatids gave better implantation and birth rates than did round spermatids, while spermatozoa and elongated spermatozoa were indistinguishable in their ability to support embryonic development. The best overall efficiency (birth rate per oocytes injected) was obtained with frozen–thawed DBA/2 strain elongated spermatids (23.2±4.2%).

Conclusions/Significance

The present study provides the first comprehensive information on ICSI using the mouse as a model and will contribute to the efficient use of materials, time, and efforts in biomedical research and clinics involving ICSI.     

Encephalitogenicity of Murine,But Not Bovine,DM20 in SJL Mice Is Due to a Single Amino Acid Difference in the Immunodominant Encephalitogenic Epitope

Myelin proteolipid protein (PLP) contains 2 immunodominant encephalitogenic epitopes in SJL mice, namely PLP residues 139–151 and 178–191. DM20, a minor isoform of PLP, lacks residues 116–150 and consequently contains only the single major encephalitogenic epitope 178–191. However, it has been found previously that bovine DM20 is not encephalitogenic in SJL mice. Since residue 188 within peptide 178–191 is phenylalanine (F) in murine DM20 and alanine (A) in bovine DM20, we tested the effect of this difference on the immune responses and induction of EAE. SJL mice were immunized with either highly purified murine or bovine DM20. Residues 178–191 were found to be immunodominant for each, but only murine and not bovine DM20 was encephalitogenic. A synthetic peptide corresponding to the murine 178–191 sequence (F188) was also encephalitogenic, whereas the peptide corresponding to the bovine sequence (A 188) was not. Both F188 and A188 bind with high affinity to I-A^s and both are recognized by the SJL T cell repertoire. A188-specific T cell lines reacted to both A188 and F188, but F188-specific T cell lines were not stimulated by A188. F188-specific T cell lines produced mRNA for the Thl cytokines IL2 and IFN, and, in passive transfer experiments, were encephalitogenic upon stimulation with F188, but not A188. In contrast, A188-specific T cell lines produced mRNA for IL4, IL5 and IL10, in addition to IL2 and IFN, and were not encephalitogenic after stimulation with either F188 or A188. Cotransfer of A188-specific T cell lines with F188-specific T cell lines resulted in protection from EAE. Thus, A188 induces a functionally different phenotype of T cells from that induced by F188. Taken together these data suggest that the failure of bovine DM20 to induce EAE may be attributable to induction of protective rather than pathogenic T cells by the immunodominant epitope.     

Viability of bovine blastocysts obtained after 7, 8 or 9 days of culture in vitro following vitrification and one-step rehydration

This study examined morphological appearance, viability and hatching rates in relation to the total cell number following vitrification of in vitro produced bovine blastocysts and expanded blastocysts. In Experiment 1, embryos obtained after 7, 8 or 9 d of culture were pooled and equilibrated in either 10% ethylene glycol (EG) or 10% EG plus 0.3M trehalose in Dulbecco's phosphate buffered saline (DPBS) supplemented with 10% calf serum and 0.6% BSA for 5 min each, at room temperature, and then vitrified together in precooled vitrification solutions consisting of 40% EG (Treatment 1), 40% EG plus 0.3M trehalose (Treatment 2), 40% EG plus 0.3M trehalose and 20% polyvinylpyrrolidone (PVP, Treatment 3) in DPBS. The embryo viability and hatching rates of Treatment 1 (19 and 3%) differed significantly (P < 0.05) from those of Treatment 2 (56 and 31%) and Treatment 3 (70 and 43%). There was a significant difference (P < 0.05) in embryo viability between Treatment 2 (31%) and Treatment 3 (43%). In Experiment 2, Day 7, 8 and 9 embryos were vitrified separately, with higher viability and hatching rates in Experiment 1 than in Experiment 2. The viabilities of Day 7 (87%), 8 (71%) and 9 (46%) embryos differed significantly (P < 0.05). Again, there were significant differences (P < 0.01) among the hatching rates of Day 7 (75%), 8 (38%) and 9 (9%) embryos. The total cell number of hatched blastocysts was then determined by differential fluorochrome staining. The total cell number of Day 7, 8 and 9 embryos differed significantly (P < 0.05).     

Levels of Murine,but Not Human,CXCL13 Are Greatly Elevated in NOD-SCID Mice Bearing the AIDS-Associated Burkitt Lymphoma Cell Line, 2F7

Currently, few rodent models of AIDS-associated non-Hodgkin’s lymphoma (AIDS-NHL) exist. In these studies, a novel mouse/human xenograft model of AIDS-associated Burkitt lymphoma (AIDS-BL) was created by injecting cells of the human AIDS-BL cell line, 2F7, intraperitoneally into NOD-SCID mice. Mice developed tumors in the peritoneal cavity, with metastases to the spleen, thymus, and mesenteric lymph nodes. Expression of the chemokine receptor, CXCR5, was greatly elevated in vivo on BL tumor cells in this model, as shown by flow cytometry. CXCL13 is the ligand for CXCR5, and serum and ascites levels of murine, but not human, CXCL13 showed a striking elevation in tumor-bearing mice, with levels as high as 200,000 pg/ml in ascites, as measured by ELISA. As shown by immunohistochemistry, murine CXCL13 was associated with macrophage-like tumor-infiltrating cells that appeared to be histiocytes. Blocking CXCR5 on 2F7 cells with neutralizing antibodies prior to injection into the mice substantially delayed tumor formation. The marked elevations in tumor cell CXCR5 expression and in murine CXCL13 levels seen in the model may potentially identify an important link between tumor-interacting histiocytes and tumor cells in AIDS-BL. These results also identify CXCL13 as a potential biomarker for this disease, which is consistent with previous studies showing that serum levels of CXCL13 were elevated in human subjects who developed AIDS-lymphoma. This mouse model may be useful for future studies on the interactions of the innate immune system and AIDS-BL tumor cells, as well as for the assessment of potential tumor biomarkers for this disease.     

Induction of B-Cell Lymphoma in BALB/c Nude Mice with an Ecotropic, B-Tropic Helper Virus Present in the Murine AIDS Virus Stock

The pathogenicities of the murine AIDS (MAIDS) virus complex (LP-BM5) and ecotropic helper virus (BM5eco) isolated from the complex to BALB/c nude mice were studied to elucidate the possible role of replication-competent helper virus in inducing the monoclonal outgrowth of lymphoid cells. Neither LP-BM5 nor BM5eco was pathogenic in adult BALB/c nude mice. However, B-cell lymphoma developed with a very high frequency when either virus was inoculated into newborn BALB/c nude (nu/nu) mice. The cells from the B-cell lymphoma were easily transplanted into nude mice. These results suggested that ecotropic helper virus in the MAIDS virus complex plays an important role in inducing the monoclonal outgrowth of lymphoid cells under immunodeficient conditions caused by defective virus.     

Short-Term Metabolome Dynamics and Carbon, Electron, and ATP Balances in Chemostat-Grown Saccharomyces cerevisiae CEN.PK 113-7D following a Glucose Pulse

The in vivo kinetics in Saccharomyces cerevisiae CEN.PK 113-7D was evaluated during a 300-second transient period after applying a glucose pulse to an aerobic, carbon-limited chemostat culture. We quantified the responses of extracellular metabolites, intracellular intermediates in primary metabolism, intracellular free amino acids, and in vivo rates of O₂ uptake and CO₂ evolution. With these measurements, dynamic carbon, electron, and ATP balances were set up to identify major carbon, electron, and energy sinks during the postpulse period. There were three distinct metabolic phases during this time. In phase I (0 to 50 seconds after the pulse), the carbon/electron balances closed up to 85%. The accumulation of glycolytic and storage compounds accounted for 60% of the consumed glucose, caused an energy depletion, and may have led to a temporary decrease in the anabolic flux. In phase II (50 to 150 seconds), the fermentative metabolism gradually became the most important carbon/electron sink. In phase III (150 to 300 seconds), 29% of the carbon uptake was not identified in the measurements, and the ATP balance had a large surplus. These results indicate an increase in the anabolic flux, which is consistent with macroscopic balances of extracellular fluxes and the observed increase in CO₂ evolution associated with nonfermentative metabolism. The identified metabolic processes involving major carbon, electron, and energy sinks must be taken into account in in vivo kinetic models based on short-term dynamic metabolome responses.     

Development of a Murine Model with Optimal Routes for Bacterial Infection and Treatment, as Determined with Bioluminescent Imaging in C57BL/6 Mice

Mice intragastrically infected with Listeria monocytogenes EGDe and Staphylococcus aureus Xen 36 showed no visible signs of infection over 48 h. However, high numbers (6.2 × 10⁵ cfu/mg feces) of S. aureus Xen 36 were detected 4 h, and 3.3 × 10⁵ cfu/mg feces of L. monocytogenes EGDe 8 h, after administration. Mice intraperitoneally infected with S. aureus Xen 36 (1 × 10⁷ cfu) developed infection immediately after administration and for at least the following 48 h. Injection with higher cell numbers of S. aureus Xen 36 (2 × 10⁸ cfu) resulted in more intense bioluminescence (infection) of the peritoneal cavity. Injection of S. aureus Xen 36 in the tail and penile veins resulted in localized tissue infection for the first 120 h. Injection of S. aureus Xen 36 into the thigh produced a faint bioluminescent signal for 15 min. Nisin F injected into the peritoneal cavity at the same area of infection led to an immediate statistically significant decrease in infection (from 2 × 10⁶ p/s/cm²/sr to 3 × 10⁵ p/s/cm²/sr) within 2 h. Similar results were recorded when nisin F was injected subcutaneously. Intraperitoneal administration is an optimal administration route for bacterial infection and treatment with antimicrobial peptides.     

Changes in Biomass, Aboveground Net Primary Production, and Peat Accumulation following Permafrost Thaw in the Boreal Peatlands of Manitoba, Canada

Permafrost thaw resulting from climate warming may dramatically change the succession and carbon dynamics of northern ecosystems. To examine the joint effects of regional temperature and local species changes on peat accumulation following thaw, we studied peat accumulation across a regional gradient of mean annual temperature (MAT). We measured aboveground net primary production (AGNPP) and decomposition over 2 years for major functional groups and used these data to calculate a simple index of net annual aboveground peat accumulation. In addition, we collected cores from six adjacent frozen and thawed bog sites to document peat accumulation changes following thaw over the past 200 years. Aboveground biomass and decomposition were more strongly controlled by local succession than regional climate. AGNPP for some species differed between collapse scars and associated permafrost plateaus and was influenced by regional MAT. A few species, such as Picea mariana trees on frozen bogs and Sphagnum mosses in thawed bogs, sequestered a disproportionate amount of peat; in addition, changes in their abundance following thaw changed peat accumulation. ²¹⁰Pb-dated cores indicated that peat accumulation doubles following thaw and that the accumulation rate is affected by historical changes in species during succession. Peat accumulation in boreal peatlands following thaw was controlled by a complex mix of local vegetation changes, regional climate, and history. These results suggest that northern ecosystems may show responses more complex than large releases of carbon during transient warming. Received 8 August 2000; accepted 12 January 2001.     

Suppression of Graft Regeneration,Not Ischemia/Reperfusion Injury,Is the Primary Cause of Small-for-Size Syndrome after Partial Liver Transplantation in Mice

Background

Ischemia/reperfusion injury (IRI) is commonly considered to play a crucial role in the pathogenesis of small-for-size syndrome (SFSS) after liver transplantation. Rapid regeneration is also considered essential for the survival of SFS grafts.

Methods

Mouse models of full-size orthotopic liver transplantation, 50% partial liver transplantation and 30% partial liver transplantation were established. Survival rate and serum alanine aminotransferase were observed. IRI was assessed by hepatic pathologic alterations, apoptosis and necrosis. Regeneration response was detected by mitotic index, BrdU incorporation and PCNA, Cyclin D1 and Cyclin E expression. The expression of mTOR, AKT, ERK, JNK2 and p70S6K, also involved in regeneration signaling pathways, were analyzed as well.

Results

30% partial liver graft resulted in a significantly low 7-day survival rate (P = 0.002) with no marked difference in tissue injury compared with the 50% partial graft group. Serum alanine aminotransferase levels were not significantly different between partial transplantation and full-size transplantation. Western blot analysis of caspase-3 and TUNEL staining also indicated no significant difference in apoptosis response between 30% partial transplantation and half-size or full-size transplantation (P = 0.436, P = 0.113, respectively). However, liver regeneration response indicators, mitotic index (P<0.0001) and BrdU (P = 0.0022), were markedly lower in 30% LTx compared with 50% LTx. Suppressed expression of PCNA, cyclin D1, cyclin E, mTOR, JNK2, AKT, ERK and p70S6K was also detected by western blot.

Conclusions

Liver regeneration is markedly suppressed in SFSS, and is more likely the primary cause of SFSS, rather than ischemia/reperfusion injury. Therapy for recovering graft regeneration could be a potentially important strategy to reduce the incidence of SFSS.     

Viability of dried filaments, survivability and reproduction under water stress, and survivability following heat and UV exposure in Lyngbya martensiana, Oscillatoria agardhii, Nostoc calcicola, Hormidium fluitans, Spirogyra sp. and Vaucheria geminata

Dried vegetative filaments ofSpirogyra sp.,Vaucheria geminata andNostoc calcicola died within 1/2, 1 and 4 h, respectively; those ofHormidium fluitans, Oscillatoria agardhii andLyngbya martensiana retained under similar storage conditions viability for 3, 5 and 10 d, respectively. The viability of dried vegetative filaments ofL. martensiana, O. agardhii andH. fluitans decreased on storage at 20°C in the dark.L. martensiana andO. agardhii tolerated 0.8 mol/L NaCl. The resistance to desiccation inL. martensiana andO. agardhii exhibited similar dependence as that to frost, to heat and UV light.O. agardhii filaments became slightly broader and their cells developed large number of gas vacuoles when grown in 0.8 mol/L NaCl-containing medium. The water stress imposed on growing algae either on high-agar solid media or in NaCl-containing liquid media reduced hormogonium formation inL. martensiana andO. agardhii, heterocyst and akinete formation inN. calcicola and fragmentation inH. fluitans; it did not induce conjugation inSpirogyra sp. and formation of reproductive organs inV. geminata. In all studied algae the stress reduced at various levels the survival of vegetative parts. Generally, algal body form and composition rather than habitats seem to decide primarily the level of resistance against various stress conditions.