Macrorheology of cystic fibrosis, chronic obstructive pulmonary disease & normal sputum

<p>Abstract</p> <p>Background</p> <p>Prior microrheologic assessments of selected, microlitre plugs of cystic fibrosis (CF) sputum suggest no intrinsic rheologic abnormality. However, such analyses may not be representative of CF sputum as a whole. We therefore reassessed this question using whole sputum macrorheology. Additionally, we wished to further explore the relationships between sputum rheology, inflammation and infection.</p> <p>Methods</p> <p>Dynamic oscillatory macrorheometry was performed on whole expectorated sputum from stable adults with CF (n = 18) and COPD (n = 12) and induced sputum from normal controls (n = 7). Concomitant sputum inflammatory mediator levels were measured in CF and COPD samples. Sputum collected from CF subjects (n = 6) at commencement and completion of intravenous antibiotic therapy for an infective exacerbation was also assessed.</p> <p>Results</p> <p>CF sputum neutrophil elastase activity (NE) was significantly related to degree of sputum purulence (p = 0.049) and correlated significantly with measures of sputum viscoelasticity (r = 0.696, p = 0.008 for storage modulus G' at 9 Hz). There were significant differences in viscoelasticity between subject groups when samples were compared irrespective of appearance/degree of sputum purulence. However, the macrorheology of mucoid CF sputum did not differ from normal sputum (eg median (range) G' at 9 Hz 2.25 (0.79, 3.26) vs 2.04 (1.4,4.6) Pa, p = 1). In contrast, mucoid COPD samples demonstrated significantly greater viscoelasticity (G' at 9 Hz 4.5 (2.4, 23) Pa) than sputum from both CF (p = 0.048) & normal subjects (p = 0.009). Antibiotic therapy during exacerbations was associated with significant reductions in CF sputum viscoelasticity, with mean (SD) G' at 9 Hz decreasing from 28.5 (11.5) Pa at commencement to 6.4 (4.6) Pa on day 7 (p = 0.01).</p> <p>Conclusion</p> <p>The macrorheologic properties of whole, mucoid CF sputum are not different from normal, confirming the results of prior microrheologic studies. Instead, CF sputum viscoelasticity is related to secondary infection, decreases with intravenous antibiotic therapy and correlates with inflammation. In contrast, COPD sputum demonstrates inherently greater viscoelasticity, providing a novel target for potential therapeutic interventions.</p

Rhabdovirus Matrix Protein Structures Reveal a Novel Mode of Self-Association

The matrix (M) proteins of rhabdoviruses are multifunctional proteins essential for virus maturation and budding that also regulate the expression of viral and host proteins. We have solved the structures of M from the vesicular stomatitis virus serotype New Jersey (genus: Vesiculovirus) and from Lagos bat virus (genus: Lyssavirus), revealing that both share a common fold despite sharing no identifiable sequence homology. Strikingly, in both structures a stretch of residues from the otherwise-disordered N terminus of a crystallographically adjacent molecule is observed binding to a hydrophobic cavity on the surface of the protein, thereby forming non-covalent linear polymers of M in the crystals. While the overall topology of the interaction is conserved between the two structures, the molecular details of the interactions are completely different. The observed interactions provide a compelling model for the flexible self-assembly of the matrix protein during virion morphogenesis and may also modulate interactions with host proteins

Intercellular communication in spheroids

This chapter has shown that the response of spheroid cells to gap junctional communication may lead to certain metabolic and cell physiological changes. It has also become apparent that the functions of the gap junctions are very complex. They may, for example, be related to the fundamental effects of cAMP and/or Ca 2+. These lines of evidence should be pursued further. However, further insight into these functions may also be gained from a study of the structure and function of the gap-junctional proteins, as well as from a genetic approach (e.g., Willecke et al. 1982, 1983). In this context, the spheroids are of particular importance as test systems, since they perfectly simulate the three dimensional arrangement of cells encountered in a tissue. Indeed, the results presented in the sections "Biophysical and Biochemical Effects Associated with Intercellular Communications" and "Intercellular Communication and Radiosensitivity" have revealed clear cut differences between cells growing as spheroids or as monolayers in response to communication dependent processes, which indicate that the response of the monolayers could be somewhat trivial. The advantage of multicellular spheroid systems with three-dimensional growth over monolayer cultures is unquestionable. Cells growing in three-dimensional multicell spheroids may re-establish their regulatory activities and, therefore, match the in vivo conditions more closely. Multicell spheroids allow in vitro investigations on differentiating systems and on interactions between normal and malignant cells, thus substituting costly in vivo experiments

PAR6, A Potential Marker for the Germ Cells Selected to Form Primordial Follicles in Mouse Ovary

Partitioning-defective proteins (PAR) are detected to express mainly in the cytoplast, and play an important role in cell polarity. However, we showed here that PAR6, one kind of PAR protein, was localized in the nuclei of mouse oocytes that formed primordial follicles during the perinatal period, suggesting a new role of PAR protein. It is the first time we found that, in mouse fetal ovaries, PAR6 appeared in somatic cell cytoplasm and fell weak when somatic cells invaded germ cell cysts at 17.5 days post coitus (dpc). Meanwhile, the expression of PAR6 was observed in cysts, and became strong in the nuclei of some germ cells at 19.5 dpc and all primordial follicular oocytes at 3 day post parturition (dpp), and then obviously declined when the primordial follicles entered the folliculogenic growth phase. During the primordial follicle pool foundation, the number of PAR6 positive germ cells remained steady and was consistent with that of formed follicles at 3 dpp. There were no TUNEL (apoptosis examination) positive germ cells stained with PAR6 at any time studied. The number of follicles significantly declined when 15.5 dpc ovaries were treated with the anti-PAR6 antibody and PAR6 RNA interference. Carbenoxolone (CBX, a known blocker of gap junctions) inhibited the expression of PAR6 in germ cells and the formation of follicles. Our results suggest that PAR6 could be used as a potential marker of germ cells for the primordial follicle formation, and the expression of PAR6 by a gap junction-dependent process may contribute to the formation of primordial follicles and the maintenance of oocytes at the diplotene stage

Dendritic cell-specific delivery of Flt3L by coronavirus vectors secures induction of therapeutic antitumor immunity

Efficacy of antitumor vaccination depends to a large extent on antigen targeting to dendritic cells (DCs). Here, we assessed antitumor immunity induced by attenuated coronavirus vectors which exclusively target DCs in vivo and express either lymphocyte- or DC-activating cytokines in combination with a GFP-tagged model antigen. Tracking of in vivo transduced DCs revealed that vectors encoding for Fms-like tyrosine kinase 3 ligand (Flt3L) exhibited a higher capacity to induce DC maturation compared to vectors delivering IL-2 or IL-15. Moreover, Flt3L vectors more efficiently induced tumor-specific CD8(+) T cells, expanded the epitope repertoire, and provided both prophylactic and therapeutic tumor immunity. In contrast, IL-2- or IL-15-encoding vectors showed a substantially lower efficacy in CD8(+) T cell priming and failed to protect the host once tumors had been established. Thus, specific in vivo targeting of DCs with coronavirus vectors in conjunction with appropriate conditioning of the microenvironment through Flt3L represents an efficient strategy for the generation of therapeutic antitumor immunity

Comparative maturation of cynomolgus monkey oocytes in vivo and in vitro

BACKGROUND: In vitro maturation (IVM) of oocytes followed by fertilization in vitro (IVF) and embryo transfer offers an alternative to conventional IVF treatment that minimises drug administration and avoids ovarian hyperstimulation. However, the technique is less efficient than maturation in vivo. In the present study, a non-human primate model was used to address the hypothesis that the number of oocytes is increased and their nuclear and cytoplasmic maturity after IVM are improved when maturation is initiated in vivo by priming with hCG. METHODS: Young, adult cynomolgus monkeys were given recombinant human (rh) gonadotropins to stimulate the development of multiple follicles, and oocytes were aspirated 0, 12, 24, or 36 h after injection of an ovulatory dose of rhCG. The nuclear status of oocytes was determined at the time of recovery and after culture for a total elapsed time of 40–44 hours after hCG. RESULTS: Priming with hCG significantly increased the number of oocytes harvested, especially after delaying aspiration for 24 h or longer. Nuclear maturation after the full period in culture was also enhanced by priming: 71.5, 83.6, and 94.6% of oocytes collected at 0, 12, and 24 h hCG had progressed to MII by the end of the culture period, compared to 87.8% of oocytes that were retrieved at 36 h. A large proportion of oocytes reaching the MII stage had either or both abnormal spindles (>40%) and misaligned chromosomes (>60%), judging by immunofluorescence microscopy, but these abnormalities were independent of culture time. The mitochondria were evenly distributed throughout the cytoplasm at all stages of maturation. Importantly, there was no microscopic evidence that the duration of culture had any injurious effects on the cells. CONCLUSION: In conclusion, the evidence supports this non-human primate as a model for human IVM and the practice of priming with hCG to promote developmental potential

Mitochondrial Dysfunction and Apoptosis in Cumulus Cells of Type I Diabetic Mice

Impaired oocyte quality has been demonstrated in diabetic mice; however, the potential pathways by which maternal diabetes exerts its effects on the oocyte are poorly understood. Cumulus cells are in direct contact with the oocyte via gap junctions and provide essential nutrients to support oocyte development. In this study, we investigated the effects of maternal diabetes on the mitochondrial status in cumulus cells. We found an increased frequency of fragmented mitochondria, a decreased transmembrane potential and an aggregated distribution of mitochondria in cumulus cells from diabetic mice. Furthermore, while mitochondrial biogenesis in cumulus cells was induced by maternal diabetes, their metabolic function was disrupted as evidenced by lower ATP and citrate levels. Moreover, we present evidence suggesting that the mitochondrial impairments induced by maternal diabetes, at least in part, lead to cumulus cell apoptosis through the release of cytochrome c. Together the deleterious effects on cumulus cells may disrupt trophic and signaling interactions with the oocyte, contributing to oocyte incompetence and thus poor pregnancy outcomes in diabetic females

Recent developments in genetics and medically assisted reproduction : from research to clinical applications

Two leading European professional societies, the European Society of Human Genetics and the European Society for Human Reproduction and Embryology, have worked together since 2004 to evaluate the impact of fast research advances at the interface of assisted reproduction and genetics, including their application into clinical practice. In September 2016, the expert panel met for the third time. The topics discussed highlighted important issues covering the impacts of expanded carrier screening, direct-to-consumer genetic testing, voiding of the presumed anonymity of gamete donors by advanced genetic testing, advances in the research of genetic causes underlying male and female infertility, utilisation of massively parallel sequencing in preimplantation genetic testing and non-invasive prenatal screening, mitochondrial replacement in human oocytes, and additionally, issues related to cross-generational epigenetic inheritance following IVF and germline genome editing. The resulting paper represents a consensus of both professional societies involved.Peer reviewe

GDF9 is Transiently Expressed in Oocytes before Follicle Formation in the Human Fetal Ovary and is Regulated by a Novel NOBOX Transcript

During human fetal ovary development, the process of primordial follicle formation is immediately preceded by a highly dynamic period of germ cell and somatic cell reorganisation. This is regulated by germ-cell specific transcription regulators, by the conserved RNA binding proteins DAZL and BOLL and by secreted growth factors of the TGFβ family, including activin βA: these all show changing patterns of expression preceding follicle formation. In mice, the transcription factor Nobox is essential for follicle formation and oocyte survival, and NOBOX regulates the expression of GDF9 in humans. We have therefore characterised the expression of GDF9 in relation to these known key factors during follicle formation in the human fetal ovary. mRNA levels of GDF9, BMP15 and NOBOX were quantified by qRT-PCR and showed dramatic increases across gestation. GDF9 protein expression was localised by immunohistochemistry to the same population of germ cells as those expressing activin βA prior to follicle formation but did not co-localise with either BOLL or DAZL. A novel NOBOX isoform was identified in fetal ovary that was shown to be capable of up-regulating the GDF9 promoter in reporter assays. Thus, during oogenesis in humans, oocytes go through a dynamic and very sharply demarcated sequence of changes in expression of these various proteins, even within individual germ cell nests, likely to be of major functional significance in determining selective germ cell survival at this key stage in ovarian development. Transcriptional variation may contribute to the range of age of onset of POI in women with NOBOX mutations