Non-contact microdrilling of mouse zona pellucida with an objective-delivered 1.48-microns diode laser.

BACKGROUND and OBJECTIVE: A non-touch laser-induced microdrilling procedure is studied on mouse zona pellucida (ZP). STUDY DESIGN/MATERIALS and METHODS: A 1.48-microns diode laser beam is focused in a 8-microns spot through a 45x objective of an inverted microscope. Mouse zygotes, suspended in a culture medium, are microdrilled by exposing their ZP to a short laser irradiation and allowed to develop in vitro. RESULTS: Various sharp-edged holes can be generated in the ZP with a single laser irradiation. Sizes can be varied by changing irradiation time (3-100 ms) or laser power (22-55 mW). Drilled zygotes present no signs of thermal damage under light and scanning electron microscopy and develop as expected in vitro, except for a distinct eight-shaped hatching behavior. CONCLUSION: The microdrilling procedure can generate standardized holes in mouse ZP, without any visible side effects. The hole formation can be explained by a local photothermolysis of the protein matrix.

The long-term survival of in vitro engineered nervous tissue derived from the specific neural differentiation of mouse embryonic stem cells.

Embryonic stem cells (ESCs) offer attractive prospective as potential source of neurons for cell replacement therapy in human neurodegenerative diseases. Besides, ESCs neural differentiation enables in vitro tissue engineering for fundamental research and drug discovery aimed at the nervous system. We have established stable and long-term three-dimensional (3D) culture conditions which can be used to model long latency and complex neurodegenerative diseases. Mouse ESCs-derived neural progenitor cells generated by MS5 stromal cells induction, result in strictly neural 3D cultures of about 120-mum thick, whose cells expressed mature neuronal, astrocytes and myelin markers. Neurons were from the glutamatergic and gabaergic lineages. This nervous tissue was spatially organized in specific layers resembling brain sub-ependymal (SE) nervous tissue, and was maintained in vitro for at least 3.5 months with great stability. Electron microscopy showed the presence of mature synapses and myelinated axons, suggesting functional maturation. Electrophysiological activity revealed biological signals involving action potential propagation along neuronal fibres and synaptic-like release of neurotransmitters. The rapid development and stabilization of this 3D cultures model result in an abundant and long-lasting production that is compatible with multiple and productive investigations for neurodegenerative diseases modeling, drug and toxicology screening, stress and aging research.

A polymorphic microsatellite in the tumor necrosis factor alpha promoter identifies an allele unique to the NZW mouse strain.

We have amplified a (CA)n:(GT)n microsatellite from the TNF promoters of a panel of mouse strains using the polymerase chain reaction. The length of the microsatellites was polymorphic, with eight alleles observed among 15 inbred strains bearing seven distinct H-2 haplotypes, and four outbred strains. In B10 congenic strains, the TNF allele detected by microsatellite polymorphism segregated with the MHC, and in recombinant haplotypes (NOD, NZW), it segregated with H-2D. The TNF allele found in the NZW strain (H-2z) was distinct from those of all other haplotypes, consistent with the hypothesis that this strain may carry a genetic defect in TNF production.

PKCθ/β and CYLD are antagonistic partners in the NFκB and NFAT transactivation pathways in primary mouse CD3+ T lymphocytes.

In T cells PKCθ mediates the activation of critical signals downstream of TCR/CD28 stimulation. We investigated the molecular mechanisms by which PKCθ regulates NFκB transactivation by examining PKCθ/β single and double knockout mice and observed a redundant involvement of PKCθ and PKCβ in this signaling pathway. Mechanistically, we define a PKCθ-CYLD protein complex and an interaction between the positive PKCθ/β and the negative CYLD signaling pathways that both converge at the level of TAK1/IKK/I-κBα/NFκB and NFAT transactivation. In Jurkat leukemic T cells, CYLD is endoproteolytically processed in the initial minutes of stimulation by the paracaspase MALT1 in a PKC-dependent fashion, which is required for robust IL-2 transcription. However, in primary T cells, CYLD processing occurs with different kinetics and an altered dependence on PKC. The formation of a direct PKCθ/CYLD complex appears to regulate the short-term spatial distribution of CYLD, subsequently affecting NFκB and NFAT repressional activity of CYLD prior to its MALT1-dependent inactivation. Taken together, our study establishes CYLD as a new and critical PKCθ interactor in T cells and reveals that antagonistic PKCθ/β-CYLD crosstalk is crucial for the adjustment of immune thresholds in primary mouse CD3(+) T cells.

Innate immune sensing of modified vaccinia virus Ankara (MVA) is mediated by TLR2-TLR6, MDA-5 and the NALP3 inflammasome.

Modified vaccinia virus Ankara (MVA) is an attenuated double-stranded DNA poxvirus currently developed as a vaccine vector against HIV/AIDS. Profiling of the innate immune responses induced by MVA is essential for the design of vaccine vectors and for anticipating potential adverse interactions between naturally acquired and vaccine-induced immune responses. Here we report on innate immune sensing of MVA and cytokine responses in human THP-1 cells, primary human macrophages and mouse bone marrow-derived macrophages (BMDMs). The innate immune responses elicited by MVA in human macrophages were characterized by a robust chemokine production and a fairly weak pro-inflammatory cytokine response. Analyses of the cytokine production profile of macrophages isolated from knockout mice deficient in Toll-like receptors (TLRs) or in the adapter molecules MyD88 and TRIF revealed a critical role for TLR2, TLR6 and MyD88 in the production of IFNbeta-independent chemokines. MVA induced a marked up-regulation of the expression of RIG-I like receptors (RLR) and the IPS-1 adapter (also known as Cardif, MAVS or VISA). Reduced expression of RIG-I, MDA-5 and IPS-1 by shRNAs indicated that sensing of MVA by RLR and production of IFNbeta and IFNbeta-dependent chemokines was controlled by the MDA-5 and IPS-1 pathway in the macrophage. Crosstalk between TLR2-MyD88 and the NALP3 inflammasome was essential for expression and processing of IL-1beta. Transcription of the Il1b gene was markedly impaired in TLR2(-/-) and MyD88(-/-) BMDM, whereas mature and secreted IL-1beta was massively reduced in NALP3(-/-) BMDMs or in human THP-1 macrophages with reduced expression of NALP3, ASC or caspase-1 by shRNAs. Innate immune sensing of MVA and production of chemokines, IFNbeta and IL-1beta by macrophages is mediated by the TLR2-TLR6-MyD88, MDA-5-IPS-1 and NALP3 inflammasome pathways. Delineation of the host response induced by MVA is critical for improving our understanding of poxvirus antiviral escape mechanisms and for designing new MVA vaccine vectors with improved immunogenicity.

Microglia/macrophages migrate through retinal epithelium barrier by a transcellular route in diabetic retinopathy: role of PKCζ in the Goto Kakizaki rat model.

Diabetic retinopathy is associated with ocular inflammation, leading to retinal barrier breakdown, macular edema, and visual cell loss. We investigated the molecular mechanisms involved in microglia/macrophages trafficking in the retina and the role of protein kinase Cζ (PKCζ) in this process. Goto Kakizaki (GK) rats, a model for spontaneous type 2 diabetes were studied until 12 months of hyperglycemia. Up to 5 months, sparse microglia/macrophages were detected in the subretinal space, together with numerous pores in retinal pigment epithelial (RPE) cells, allowing inflammatory cell traffic between the retina and choroid. Intercellular adhesion molecule-1 (ICAM-1), caveolin-1 (CAV-1), and PKCζ were identified at the pore border. At 12 months of hyperglycemia, the significant reduction of pores density in RPE cell layer was associated with microglia/macrophages accumulation in the subretinal space together with vacuolization of RPE cells and disorganization of photoreceptors outer segments. The intraocular injection of a PKCζ inhibitor at 12 months reduced iNOS expression in microglia/macrophages and inhibited their migration through the retina, preventing their subretinal accumulation. We show here that a physiological transcellular pathway takes place through RPE cells and contributes to microglia/macrophages retinal trafficking. Chronic hyperglycemia causes alteration of this pathway and subsequent subretinal accumulation of activated microglia/macrophages.

MHC class II hierarchy of superantigen presentation predicts efficiency of infection with mouse mammary tumor virus.

Superantigens (SAgs) encoded by infectious mouse mammary tumor viruses (MMTVs) play a crucial role in the viral life cycle. Their expression by infected B cells induces a proliferative immune response by SAg-reactive T cells which amplifies MMTV infection. This response most likely ensures stable MMTV infection and transmission to the mammary gland. Since T cell reactivity to SAgs from endogenous Mtv loci depends on MHC class II molecules expressed by B cells, we have determined the ability of MMTV to infect various MHC congenic mice. We show that MHC class II I-E+ compared with I-E- mouse strains show higher levels of MMTV infection, most likely due to their ability to induce a vigorous SAg-dependent immune response following MMTV encounter. Inefficient infection is observed in MHC class II I-E- mice, which have been shown to present endogenous SAgs poorly. Therefore, during MMTV infection the differential ability of MHC class II molecules to form a functional complex with SAg determines the magnitude of the proliferative response of SAg-reactive T cells. This in turn influences the degree of T cell help provided to infected B cells and therefore the efficiency of amplification of MMTV infection.

Mouse fetal trisomy 13 and hypotrophy of the spinal cord: effect on calbindin-D28k and calretinin expressed by neurons of the spinal cord and dorsal root ganglia.

Trisomy 13 was detected in 10% of mouse embryos obtained from pregnant females which were doubly heterozygous for Robertsonian chromosomes involving chromosome 13. The developing dorsal root ganglia and spinal cords were examined in trisomy 13 and littermate control mice between days 12 and 18 of gestation (E12-18). The overall size of the dorsal root ganglia and number of ganglion cells within a given ganglion were not altered, but the number of neurons immunoreactive for calbindin and calretinin was reduced. The trisomic spinal cord was reduced in size with neurons lying in a tightly compact distribution in the gray matter. In trisomic fetuses, the extent of the neuropil of the spinal cord was reduced, and may represent a diminished field of interneuronal connectivity, due to reduced arborization of dendritic processes of the neurons present, particularly of calbindin-immunostained neurons. Furthermore, the subpopulation of calretinin-immunoreactive neurons and axons was also reduced in developing trisomic gray and white matter, respectively. Thus, overexpression of genes on mouse chromosome 13 exerts a deleterious effect on the development of neuropil, affecting both dendritic and axonal arborization in the trisomy 13 mouse. The defect of calbindin or calretinin expression by subsets of dorsal root ganglion or spinal cord neurons may result from deficient cell-to-cell interactions with targets which are hypoplastic.

EuroPhenome: a repository for high-throughput mouse phenotyping data.

The broad aim of biomedical science in the postgenomic era is to link genomic and phenotype information to allow deeper understanding of the processes leading from genomic changes to altered phenotype and disease. The EuroPhenome project (http://www.EuroPhenome.org) is a comprehensive resource for raw and annotated high-throughput phenotyping data arising from projects such as EUMODIC. EUMODIC is gathering data from the EMPReSSslim pipeline (http://www.empress.har.mrc.ac.uk/) which is performed on inbred mouse strains and knock-out lines arising from the EUCOMM project. The EuroPhenome interface allows the user to access the data via the phenotype or genotype. It also allows the user to access the data in a variety of ways, including graphical display, statistical analysis and access to the raw data via web services. The raw phenotyping data captured in EuroPhenome is annotated by an annotation pipeline which automatically identifies statistically different mutants from the appropriate baseline and assigns ontology terms for that specific test. Mutant phenotypes can be quickly identified using two EuroPhenome tools: PhenoMap, a graphical representation of statistically relevant phenotypes, and mining for a mutant using ontology terms. To assist with data definition and cross-database comparisons, phenotype data is annotated using combinations of terms from biological ontologies.

IFN-gamma-dependent transcription of MHC class II IA is impaired in macrophages from aged mice

To determine the effect of aging on IFN-gamma-induced MHC class II antigen expression, we produced bone marrow¿derived macrophages in vitro. In these conditions, we analyzed the effect of aging on the genomic expression of macrophages without the influence of other cell types that may be affected by aging. Although macrophages from young and aged mice showed an identical degree of differentiation, after incubation with IFN-gamma, the expression at the cell surface of the IA complex and the levels of IAbeta protein and mRNA were lower in aged macrophages. Moreover, the transcription of the IAbeta gene was impaired in aged macrophages. The amount of transcription factors that bound to the W and X, but not to the Y, boxes of the IAbeta promoter gene was lower in aged macrophages. Similar levels of CIITA mRNA were found after IFN-gamma treatment of both young and aged macrophages. This shows that neither the initial cascade that starts after the interaction of IFN-gamma with the receptor nor the second signals involved in the expression of CIITA are impaired in aged macrophages. These data indicate that aging is associated with low levels of MHC class II gene induction by IFN-gamma because of impaired transcription.

BMP-2 and TGF-beta1 differentially control expression of type II procollagen and alpha 10 and alpha 11 integrins in mouse chondrocytes.

Bone morphogenetic protein (BMP)-2 and transforming growth factor (TGF)-beta1 are multifunctional cytokines both proposed as stimulants for cartilage repair. Thus it is crucial to closely examine and compare their effects on the expression of key markers of the chondrocyte phenotype, at the gene and protein level. In this study, the expression of alpha 10 and alpha 11 integrin subunits and the IIA/IIB spliced forms of type II procollagen have been monitored for the first time in parallel in the same in vitro model of mouse chondrocyte dedifferentiation/redifferentiation. We demonstrated that TGF-beta1 stimulates the expression of the non-chondrogenic form of type II procollagen, IIA isoform, and of a marker of mesenchymal tissues, i.e. the alpha 11 integrin subunit. On the contrary, BMP-2 stimulates the cartilage-specific form of type II procollagen, IIB isoform, and a specific marker of chondrocytes, i.e. the alpha 10 integrin subunit. Collectively, our results demonstrate that BMP-2 has a better capability than TGF-beta1 to stimulate chondrocyte redifferentiation and reveal that the relative expressions of type IIB to type IIA procollagens and alpha 10 to alpha 11 integrin subunits are good markers to define the differentiation state of chondrocytes. In addition, adenoviral expression of Smad6, an inhibitor of BMP canonical Smad signaling, did not affect expression of total type II procollagen or the ratio of type IIA and type IIB isoforms in mouse chondrocytes exposed to BMP-2. This result strongly suggests that signaling pathways other than Smad proteins are involved in the effect of BMP-2 on type II procollagen expression.

Waddlia chondrophila enters and multiplies within human macrophages

Waddlia chondrophila is an obligate intracellular bacterium of the Chlamydiales order. W. chondrophila has been isolated twice from aborted bovine foetuses and a serological study supported the abortigenic role of W. chondrophila in bovine species. Recently, we observed a strong association between the presence of anti-Waddlia antibodies and human miscarriage. To further investigate the pathogenic potential of W. chondrophila in humans, we studied the entry and the multiplication of this Chlamydia-like organism in human macrophages. Confocal and electron microscopy confirmed that W. chondrophila is able to enter human monocyte-derived macrophages. Moreover, W. chondrophila multiplied readily within macrophages. The proportion of infected macrophages increased from 13% at day 0 to 96% at day 4, and the mean number of bacteria per macrophage increased by 3logs in 24h. Intracellular growth of W. chondrophila was associated with a significant cytopathic effect. Thus, W. chondrophila may enter and grow rapidly within human macrophages, inducing lysis of infected cells. Since macrophages are one of the major components of the innate immune response, these findings indirectly suggest the possible human pathogenicity of W. chondrophila.

Peritoneal carcinomatosis due to ovarian cancer: comparison between MDCT, MRI and 18F-FDG PET/CT

Purpose: To compare MDCT, MRI and 18F-FDG PET/CT for the detection of peritoneal carcinomatosis due to ovarian cancerMethods and Materials: Fifteen women (mean age 65±) with clinical suspicion of ovarian cancer and peritoneal carcinomatosis underwent MDCT, MRI and 18F-FDG PET/CT, simultaneously and shortly performed before surgery (delay 8.1± days). According to the peritoneal cancer index nine abdominopelvic regions were defined. We applied four scores of lesion size on MDCT and MR images, while the maximal standard uptake value (SUVmax) was measured on 18F-FDG PET/CT. Three sites of lymphadenopathy and posterobasal pleural carcinomatosis were also analyzed. First, one radiologist blindly and separately read MDCT and MR images, while one nuclear physician blindly read PET/CT images grading each lesion according to four diagnostic certitudes. Secondly, all the images were reviewed jointly and compared with histopathology. Receiver operating characteristics (ROC) analysis was performed.Results: Peritoneal implants were proven in ten women (75%). Altogether, 228 abdominopelvic sites were compared. Sensitivity and specificity for MDCT was 90.2% and 90.6%, for MRI 93.5% and 86.3%, and for 18F-FDG PET/CT 92.7% and 95.7%, respectively. ROC area under the curve were 0.93 for MDCT and MRI, and 0.96 for 18F-FDG PET/CT respectively. No significant differences (p=0.11) were found between the three modalities.Conclusion: Although MRI revealed to be the most sensitive and 18F-FDG PET/CT the most specific modality, no significant differences were shown between the three techniques.

Positional bias of general and tissue-specific regulatory motifs in mouse gene promoters

Background: The arrangement of regulatory motifs in gene promoters, or promoterarchitecture, is the result of mutation and selection processes that have operated over manymillions of years. In mammals, tissue-specific transcriptional regulation is related to the presence ofspecific protein-interacting DNA motifs in gene promoters. However, little is known about therelative location and spacing of these motifs. To fill this gap, we have performed a systematic searchfor motifs that show significant bias at specific promoter locations in a large collection ofhousekeeping and tissue-specific genes.Results: We observe that promoters driving housekeeping gene expression are enriched inparticular motifs with strong positional bias, such as YY1, which are of little relevance in promotersdriving tissue-specific expression. We also identify a large number of motifs that show positionalbias in genes expressed in a highly tissue-specific manner. They include well-known tissue-specificmotifs, such as HNF1 and HNF4 motifs in liver, kidney and small intestine, or RFX motifs in testis,as well as many potentially novel regulatory motifs. Based on this analysis, we provide predictionsfor 559 tissue-specific motifs in mouse gene promoters.Conclusion: The study shows that motif positional bias is an important feature of mammalianproximal promoters and that it affects both general and tissue-specific motifs. Motif positionalconstraints define very distinct promoter architectures depending on breadth of expression andtype of tissue.