Preliminary Reliability and Validity of the DMRS-SR-30, a Novel Self-Report Measure Based on the Defense Mechanisms Rating Scales.

Defense mechanisms are psychological factors that influence emotional distress and quality of life. There are a number of measures assessing the construct of defense mechanisms, but only few available instruments reflect the gold-standard theoretical hierarchical organization of defenses. We report on the development of a novel 30 item self-report questionnaire, the DMRS-SR-30, based on the parent instrument, the Defense Mechanism Rating Scales (DMRS). This study tested preliminary reliability and validity of the Italian version of the DMRS-SR-30. We first extracted 30 items from the DMRS Q-sort version (DMRS-Q) and adapted them for a self-reported format. We then applied the DMRS quantitative scoring algorithms to provide proportional scores for the 28 individual defenses and summary scores for seven defense levels and overall defensive functioning (ODF) scores. A dynamic interview was used for assessing participant's defense mechanisms with the observer-rated DMRS and DMRS-Q. We examined internal consistency of the scales along with criterion, concurrent, convergent and discriminant validity among participants (N = 94) who completed the DMRS-SR-30, SCL-90, BDI, and IES-R. Results showed very good internal consistency for ODF (Cronbach's alpha = .890) and the high adaptive defense level, whereas some subscales with few items had lower values. Correlation analyses between DMRS-SR-30 and the two DMRS-based observer-rated measures showed very good criterion and concurrent validity for ODF and moderate to high for defense levels subscales. Correlations between the DMRS-SR-30 ODF and SCL-90 GSI, BDI and IES=R (r = -.456, r= -.540, r = -.402, respectively, all p <.001), indicated good convergent validity. Despite the well-known limitations of self-report methods of psychodynamic phenomena, self-report measures are highly practicable for assessing large samples. The DMRS-SR-30 is the first self-assessed measure describing the whole hierarchy of 28 defense mechanisms and providing scores for ODF, defensive categories, defense levels, and individual defenses. Preliminary examination of the Italian version of the DMRS-SR-30 showed promising results of internal consistency, criterion and concurrent validity, and convergent validity and of the measure. Further validation is needed to confirm these findings and explore other aspects of validity and reliability.

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts.

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Extended ex vivo culture of fresh and cryopreserved whole sheep ovaries.

We describe an original perfusion system for the culture of whole ovine ovaries for up to 4 days. A total of 33 ovaries were divided into six groups: control (n=6), not perfused and fixed; Groups SM72 and SM72-FSH (n=6 each), perfused with a simple medium for 72h with or without FSH; Groups CM96 and CM96-FSH (n=6 each), perfused with a complex medium for 96h with or without FSH; Group CM96-FSH-cryo, (n=3) cryopreserved and perfused for 96h with Group CM96-FSH medium. Depending on the medium used, morphological parameters of cultured ovaries differed from fresh organs after 72 (SM72, SM72-FSH) or 96 (CM96, CM96-FSH) h of perfusion. Oestradiol and progesterone were secreted in all groups but FSH had an effect only on Group CM96-FSH, stimulating continued oestradiol secretion 10 times higher than in all other groups. Morphological parameters and hormone secretion of cryopreserved ovaries were not different from fresh controls. This method enables the culture of whole ovaries for up to 4 days, the time required in vivo for 0.5-mm follicles to grow to 2.2mm and then for these follicles to reach the ovulatory size of 4mm or more. It could be used as a research tool or to complement current techniques for preserving female fertility.

Intercellular bridges are essential for human parthenogenetic cell survival.

Parthenogenetic cells, obtained from in vitro activated mammalian oocytes, display multipolar spindles, chromosome malsegregation and a high incidence of aneuploidy, probably due to the lack of paternal contribution. Despite this, parthenogenetic cells do not show high rates of apoptosis and are able to proliferate in a way comparable to their biparental counterpart. We hypothesize that a series of adaptive mechanisms are present in parthenogenetic cells, allowing a continuous proliferation and ordinate cell differentiation both in vitro and in vivo. Here we identify the presence of intercellular bridges that contribute to the establishment of a wide communication network among human parthenogenetic cells, providing a mutual exchange of missing products. Silencing of two molecules essential for intercellular bridge formation and maintenance demonstrates the key function played by these cytoplasmic passageways that ensure normal cell functions and survival, alleviating the unbalance in cellular component composition.

Direct comparative analysis of conventional and directional freezing for the cryopreservation of whole ovaries.

OBJECTIVE: To compare conventional slow equilibrium cooling and directional freezing for cryopreservation of whole ovaries. DESIGN: Experimental animal study. SETTING: Academic research environment. ANIMAL(S): Adult ewes. INTERVENTION(S): Eighty-one ovaries were randomly assigned to fresh control, conventional freezing (CF), and directional freezing (DF) group. Ovaries of CF and DF groups were perfused via the ovarian artery with Leibovitz L-15 medium, 10% fetal bovine serum, and 1.5 M dimethyl sulfoxide for 5 minutes. Each ovary was inserted into a glass test tube containing 10 mL of the same solution and cooled to -100°C or -70°C, respectively. Ovaries were stored in liquid nitrogen for a minimum of 2 weeks. MAIN OUTCOME MEASURE(S): Structural integrity of cortical and medulla regions, vascular integrity, follicle in vitro development, cell proliferation, and DNA damage and repair. RESULT(S): All examined parameters indicate that the structure of DF ovaries remains largely intact and comparable to fresh controls, whereas significant damages were observed in CF ovaries. CONCLUSION(S): Directional freezing allows good preservation of whole ovaries, with most of the parameters taken into consideration almost identical to those recorded in fresh control samples. This encourages a reconsideration of the possible use of whole-ovary cryopreservation as a viable alternative to cortical fragments.

Brief demethylation step allows the conversion of adult human skin fibroblasts into insulin-secreting cells.

The differentiated state of mature cells of adult organisms is achieved and maintained through the epigenetic regulation of gene expression, which consists of several mechanisms including DNA methylation. The advent of induced pluripotent stem cell technology enabled the conversion of adult cells into any other cell type passing through a stable pluripotency state. However, indefinite pluripotency is unphysiological, inherently labile, and makes cells prone to culture-induced alterations. The direct conversion of one cell type to another without an intermediate pluripotent stage is also possible but, at present, requires the viral transfection of appropriate transcription factors, limiting its therapeutic potential. The aim of this study was to investigate whether it is possible to achieve the direct conversion of an adult cell by exposing it to a demethylating agent immediately followed by differentiating culture conditions. Adult human skin fibroblasts were exposed for 18 h to the DNA methyltransferase inhibitor 5-azacytidine, followed by a three-step protocol for the induction of endocrine pancreatic differentiation that lasted 36 d. At the end of this treatment, 35 ± 8.9% fibroblasts became pancreatic converted cells that acquired an epithelial morphology, produced insulin, and then released the hormone in response to a physiological glucose challenge in vitro. Furthermore, pancreatic converted cells were able to protect recipient mice against streptozotocin-induced diabetes, restoring a physiological response to glucose tolerance tests. This work shows that it is possible to convert adult fibroblasts into insulin-secreting cells, avoiding both a stable pluripotent stage and any transgenic modification.

Characterization of the constitutive pig ovary heat shock chaperone machinery and its response to acute thermal stress or to seasonal variations.

Reduced oocyte competence causes the lower fertility reported in domestic sows during the warm months of the year. Somatic cells express heat shock proteins (HSPs) to protect themselves from damage caused by thermal stress. HSPs are classified as molecular chaperones and control the correct folding of newly synthesized or damaged proteins. The present work performed a comprehensive survey of the different components of the heat shock chaperone machinery in the pig ovary, which included the HSP40, HSP70, HSP90, and HSP110 families, as well as heat shock factors (HSF) 1 and 2. Pig ovarian follicles constitutively expressed different members of these families; therefore, we examined their ability to respond to heat stress. In order to take into account the role of the complex follicular architecture, whole pig ovaries were exposed to 41.5°C for 1 h. This exposure significantly disrupted oocyte maturation and determined the upregulation of the HSP70, HSP40, HSPH1, HSPA4, HSPA4L, HSF1, and HFS2 genes, whereas expression levels of HSP90A and HSP90B, as well as those of genes unrelated to heat stress were not altered. Unexpectedly HSP and HSF expression levels changed only in oocytes but not in cumulus cells. Cumulus-oocyte complexes isolated from ovaries collected in summer showed the same pattern as those collected in winter. We conclude that the HSP chaperone machinery is constitutively fully operational in the pig ovary. However, following thermal stimuli or seasonal variations, cumulus cell HS-related gene expression remains unchanged, and only oocytes activate a response, suggesting why this mechanism is insufficient to preserve their competence both in vitro and in vivo.

Centrosome amplification and chromosomal instability in human and animal parthenogenetic cell lines.

Parthenotes have been proposed as a source of embryonic stem cells but they lack the centriole which is inherited through the sperm in all mammalian species, except for rodents. We investigated the centrosome of parthenotes and parthenogenetic embryonic stem cells using parthenogenetic and biparental pig pre-implantation embryos, human and pig parthenogenetic and biparental embryonic stem cells, sheep fibroblasts derived from post implantation parthenogenetic and biparental embryos developed in vivo. We also determined the level of aneuploidy in parthenogenetic cells. Oocytes of all species were activated using ionomycin and 6-dimethylaminopurine (6-DMAP). Over 60% of parthenogenetic blastomeres were affected by an excessive number of centrioles. Centrosome amplification, was observed by microscopical and ultrastructural analysis also in parthenogenetic cell lines of all three species. Over expression of PLK2 and down regulation of CCNF, respectively involved in the stimulation and inhibition of centrosome duplication, were present in all species. We also detected down regulation of spindle assembly checkpoint components such as BUB1, CENPE and MAD2. Centrosome amplification was accompanied by multipolar mitotic spindles and all cell lines were affected by a high rate of aneuploidy. These observations indicate a link between centrosome amplification and the high incidence of aneuploidy and suggest that parthenogenetic stem cells may be a useful model to investigate how aneuploidy can be compatible with cell proliferation and differentiation.