DNA methylation in schizophrenia in different patient-derived cell types.

DNA methylation of gene promoter regions represses transcription and is a mechanism via which environmental risk factors could affect cells during development in individuals at risk for schizophrenia. We investigated DNA methylation in patient-derived cells that might shed light on early development in schizophrenia. Induced pluripotent stem cells may reflect a "ground state" upon which developmental and environmental influences would be minimal. Olfactory neurosphere-derived cells are an adult-derived neuro-ectodermal stem cell modified by developmental and environmental influences. Fibroblasts provide a non-neural control for life-long developmental and environmental influences. Genome-wide profiling of DNA methylation and gene expression was done in these three cell types from the same individuals. All cell types had distinct, statistically significant schizophrenia-associated differences in DNA methylation and linked gene expression, with Gene Ontology analysis showing that the differentially affected genes clustered in networks associated with cell growth, proliferation, and movement, functions known to be affected in schizophrenia patient-derived cells. Only five gene loci were differentially methylated in all three cell types. Understanding the role of epigenetics in cell function in the brain in schizophrenia is likely to be complicated by similar cell type differences in intrinsic and environmentally induced epigenetic regulation.

Mouse sperm begin to undergo acrosomal exocytosis in the upper isthmus of the oviduct.

Recent evidence demonstrated that most fertilizing mouse sperm undergo acrosomal exocytosis (AE) before binding to the zona pellucida of the eggs. However, the sites where fertilizing sperm could initiate AE and what stimuli trigger it remain unknown. Therefore, the aim of this study was to determine physiological sites of AE by using double transgenic mouse sperm, which carried EGFP in the acrosome and DsRed2 fluorescence in mitochondria. Using live imaging of sperm during in vitro fertilization of cumulus-oocyte complexes, it was observed that most sperm did not undergo AE. Thus, the occurrence of AE within the female reproductive tract was evaluated in the physiological context where this process occurs. Most sperm in the lower segments of the oviduct were acrosome-intact; however, a significant number of sperm that reached the upper isthmus had undergone AE. In the ampulla, only 5% of the sperm were acrosome-intact. These results support our previous observations that most of mouse sperm do not initiate AE close to or on the ZP, and further demonstrate that a significant proportion of sperm initiate AE in the upper segments of the oviductal isthmus.

PKA-dependent phosphorylation of LIMK1 and Cofilin is essential for mouse sperm acrosomal exocytosis.

Mammalian sperm must acquire their fertilizing ability after a series of biochemical modifications in the female reproductive tract collectively called capacitation to undergo acrosomal exocytosis, a process that is essential for fertilization. Actin dynamics play a central role in controlling the process of exocytosis in somatic cells as well as in sperm from several mammalian species. In somatic cells, small GTPases of the Rho family are widely known as master regulators of actin dynamics. However, the role of these proteins in sperm has not been studied in detail. In the present work we characterized the participation of small GTPases of the Rho family in the signaling pathway that leads to actin polymerization during mouse sperm capacitation. We observed that most of the proteins of this signaling cascade and their effector proteins are expressed in mouse sperm. The activation of the signaling pathways of cAMP/PKA, RhoA/C and Rac1 is essential for LIMK1 activation by phosphorylation on Threonine 508. Serine 3 of Cofilin is phosphorylated by LIMK1 during capacitation in a transiently manner. Inhibition of LIMK1 by specific inhibitors (BMS-3) resulted in lower levels of actin polymerization during capacitation and a dramatic decrease in the percentage of sperm that undergo acrosomal exocytosis. Thus, we demonstrated for the first time that the master regulators of actin dynamics in somatic cells are present and active in mouse sperm. Combining the results of our present study with other results from the literature, we have proposed a working model regarding how LIMK1 and Cofilin control acrosomal exocytosis in mouse sperm.

Stemformatics: visualisation and sharing of stem cell gene expression.

Genome-scale technologies are increasingly adopted by the stem cell research community, because of the potential to uncover the molecular events most informative about a stem cell state. These technologies also present enormous challenges around the sharing and visualisation of data derived from different laboratories or under different experimental conditions. Stemformatics is an easy to use, publicly accessible portal that hosts a large collection of exemplar stem cell data. It provides fast visualisation of gene expression across a range of mouse and human datasets, with transparent links back to the original studies. One difficulty in the analysis of stem cell signatures is the paucity of public pathways/gene lists relevant to stem cell or developmental biology. Stemformatics provides a simple mechanism to create, share and analyse gene sets, providing a repository of community-annotated stem cell gene lists that are informative about pathways, lineage commitment, and common technical artefacts. Stemformatics can be accessed at stemformatics.org.

Variability in the generation of induced pluripotent stem cells: importance for disease modeling.

In the field of disease modeling, induced pluripotent stem cells (iPSCs) have become an appealing choice, especially for diseases that do not have an animal model. They can be generated from patients with known clinical features and compared with cells from healthy controls to identify the biological bases of disease. This study was undertaken to determine the variability in iPSC lines derived from different individuals, with the aim of determining criteria for selecting iPSC lines for disease models. We generated and characterized 18 iPSC lines from eight donors and considered variability at three levels: (a) variability in the criteria that define iPSC lines as pluripotent cells, (b) variability in cell lines from different donors, and (c) variability in cell lines from the same donor. We found that variability in transgene expression and pluripotency marker levels did not prevent iPSCs from fulfilling all other criteria for pluripotency, including teratoma formation. We found low interindividual and interclonal variability in iPSCs that fulfilled the most stringent criteria for pluripotency, with very high correlation in their gene expression profiles. Interestingly, some cell lines exhibited reprogramming instability, spontaneously regressing from a fully to a partially reprogrammed state. This was associated with a low percentage of cells expressing the pluripotency marker stage-specific embryonic antigen-4. Our study shows that it is possible to define a similar "ground state" for each cell line as the basis for making patient versus control comparisons, an essential step in order to identify disease-associated variability above individual and cell line variability.

NRF2 activation restores disease related metabolic deficiencies in olfactory neurosphere-derived cells from patients with sporadic Parkinson's disease.

BACKGROUND: Without appropriate cellular models the etiology of idiopathic Parkinson's disease remains unknown. We recently reported a novel patient-derived cellular model generated from biopsies of the olfactory mucosa (termed olfactory neurosphere-derived (hONS) cells) which express functional and genetic differences in a disease-specific manner. Transcriptomic analysis of Patient and Control hONS cells identified the NRF2 transcription factor signalling pathway as the most differentially expressed in Parkinson's disease. RESULTS: We tested the robustness of our initial findings by including additional cell lines and confirmed that hONS cells from Patients had 20% reductions in reduced glutathione levels and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] metabolism compared to cultures from healthy Control donors. We also confirmed that Patient hONS cells are in a state of oxidative stress due to higher production of H(2)O(2) than Control cultures. siRNA-mediated ablation of NRF2 in Control donor cells decreased both total glutathione content and MTS metabolism to levels detected in cells from Parkinson's Disease patients. Conversely, and more importantly, we showed that activation of the NRF2 pathway in Parkinson's disease hONS cultures restored glutathione levels and MTS metabolism to Control levels. Paradoxically, transcriptomic analysis after NRF2 pathway activation revealed an increased number of differentially expressed mRNAs within the NRF2 pathway in L-SUL treated Patient-derived hONS cells compared to L-SUL treated Controls, even though their metabolism was restored to normal. We also identified differential expression of the PI3K/AKT signalling pathway, but only post-treatment. CONCLUSIONS: Our results confirmed NRF2 as a potential therapeutic target for Parkinson's disease and provided the first demonstration that NRF2 function was inducible in Patient-derived cells from donors with uniquely varied genetic backgrounds. However, our results also demonstrated that the response of PD patient-derived cells was not co-ordinated in the same way as in Control cells. This may be an important factor when developing new therapeutics.

Induced pluripotent stem cells: a new technology to study human diseases.

Induced pluripotent stem cells (iPS cells) are somatic cells that have been reprogrammed to a pluripotent state by the introduction of specific factors. They can be generated from cells of different origins such as fibroblasts, keratinocytes, hepatocytes and blood. iPS cells are similar to embryonic stem cells in several aspects such as morphology, expression of pluripotency markers and the capacity to develop teratomas; tumors containing cells of the three germ layers. As pluripotent stem cells they can be differentiated into several lineages including neuronal, cardiac and blood cells. Recently, several groups have successfully generated patient-specific iPS cells from donors suffering different disorders and differentiated them into the cell type affected by the disease. These new human cell-based models cannot only be used to study the dynamics of diseases but also as systems to screen new drugs. Moreover, iPS cells promise to be good candidates for regenerative medicine.

Disease-specific, neurosphere-derived cells as models for brain disorders.

There is a pressing need for patient-derived cell models of brain diseases that are relevant and robust enough to produce the large quantities of cells required for molecular and functional analyses. We describe here a new cell model based on patient-derived cells from the human olfactory mucosa, the organ of smell, which regenerates throughout life from neural stem cells. Olfactory mucosa biopsies were obtained from healthy controls and patients with either schizophrenia, a neurodevelopmental psychiatric disorder, or Parkinson's disease, a neurodegenerative disease. Biopsies were dissociated and grown as neurospheres in defined medium. Neurosphere-derived cell lines were grown in serum-containing medium as adherent monolayers and stored frozen. By comparing 42 patient and control cell lines we demonstrated significant disease-specific alterations in gene expression, protein expression and cell function, including dysregulated neurodevelopmental pathways in schizophrenia and dysregulated mitochondrial function, oxidative stress and xenobiotic metabolism in Parkinson's disease. The study has identified new candidate genes and cell pathways for future investigation. Fibroblasts from schizophrenia patients did not show these differences. Olfactory neurosphere-derived cells have many advantages over embryonic stem cells and induced pluripotent stem cells as models for brain diseases. They do not require genetic reprogramming and they can be obtained from adults with complex genetic diseases. They will be useful for understanding disease aetiology, for diagnostics and for drug discovery.

Role for PADI6 and the cytoplasmic lattices in ribosomal storage in oocytes and translational control in the early mouse embryo.

The mechanisms that mediate the establishment of totipotency during the egg-to-embryo transition in mammals remain poorly understood. However, it is clear that unique factors stored in the oocyte cytoplasm are crucial for orchestrating this complex cellular transition. The oocyte cytoplasmic lattices (CPLs) have long been predicted to function as a storage form for the maternal contribution of ribosomes to the early embryo. We recently demonstrated that the CPLs cannot be visualized in Padi6-/- oocytes and that Padi6-/- embryos arrest at the two-cell stage. Here, we present evidence further supporting the association of ribosomes with the CPLs by demonstrating that the sedimentation properties of the small ribosomal subunit protein, S6, are dramatically altered in Padi6-/- oocytes. We also show that the abundance and localization of ribosomal components is dramatically affected in Padi6-/- two-cell embryos and that de novo protein synthesis is also dysregulated in these embryos. Finally, we demonstrate that embryonic genome activation (EGA) is defective in Padi6-/- two-cell embryos. These results suggest that, in mammals, ribosomal components are stored in the oocyte CPLs and are required for protein translation during early development.

Proteomic profiling of murine oocyte maturation.

In an effort to better understand oocyte function, we utilized two-dimensional (2D) electrophoresis and mass spectrometry to identify proteins that are differentially expressed during murine oocyte maturation. Proteins from 500 germinal vesicle (GV) and metaphase II-(MII) arrested oocytes were extracted, resolved on 2D electrophoretic gels, and stained with silver. Analysis of the gels indicated that 12 proteins appeared to be differentially expressed between the GV and MII stage. These proteins were then cored from the 2D gels and identified by mass spectrometry as: transforming acidic coiled-coil protein 3 (TACC3), heat shock protein 105 (HSP105), programmed cell death six-interacting protein (PDCD6IP), stress-inducible phosphoprotein (STI1), importin alpha2, adenylsuccinate synthase (ADDS), nudix, spindlin, lipocalin, lysozyme, translationally controlled tumor protein (TCTP), and nucleoplasmin 2 (NPM2). Interestingly, PDCD6IP, importin alpha2, spindlin, and NPM2 appear slightly larger in mass and more acidic on the MII oocyte gel compared to the GV oocyte gel, suggesting that they may be post-translationally modified during oocyte maturation. Given NPM2 is an oocyte-restricted protein, we chose to further investigate its properties during oocyte maturation and preimplantation development. Real-Time RT-PCR showed that NPM2 mRNA levels rapidly decline at fertilization. Indirect immunofluorescence analysis showed that, with the exception of cortical localization in MII-arrested oocytes, NPM2 is localized to the nucleus of both GV stage oocytes and all stages of preimplantation embryos. We then performed one-dimensional (1D) western blot analysis of mouse oocytes and preimplantation embryos and found that, as implicated by the 2D gel comparison, NPM2 undergoes a phosphatase-sensitive electrophoretic mobility shift during the GV to MII transition. The slower migrating NPM2 form is also present in pronuclear embryos but by the two-cell stage, the majority of NPM2 exists as the faster migrating form, which persists to the blastocyst stage.

Testis-specific lactate dehydrogenase (LDH-C4; Ldh3) in murine oocytes and preimplantation embryos.

LDH-C(4) (Ldh3) is a member of the lactate dehydrogenase family of isozymes that catalyze the terminal reaction in the glycolytic pathway. In mammals, 3 genes, ldha, ldhb, and Ldhc, encode the subunits that assemble into catalytically active homo- and heterotetramers. Differential expression of these genes determines the lactate dehydrogenase (LDH) isozyme composition of tissues, and, as is well known, A subunits predominate in skeletal muscle and B subunits are abundantly produced in brain and heart, with the Ldh2 isozyme the most abundant form in oocytes. The C peptide can be detected first in pachytene spermatocytes and constitutes the primary LDH of spermatozoa. Originally the Ldhc gene (Ldh3 in terminology applied to murine cells) was considered to be testis specific on the basis of immunochemical, enzymatic, and molecular analyses. Here we report the detection of this isozyme in the murine oocyte and early embryo. Our results indicate that Ldh3 mRNA is transcribed in oocytes and cannot be detected in fertilized eggs. Ldh3 protein, however, persists to the blastocyst stage of embryonic development localizing mainly to the cortex region of oocytes, eggs, zygotes, and embryonic blastomeres.

Effect of gonadotropin-releasing hormone agonist and antagonist on proliferation and apoptosis of human luteinized granulosa cells.

The GnRH agonist leuprolide acetate (LA) inhibited DNA synthesis in epidermal growth factor-stimulated human granulosa luteinized cell cultures. This effect was blocked by the prior addition of a GnRH antagonist antide (ANT), and this compound per se was able to produce a stimulatory effect of DNA synthesis on basal conditions. Leuprolide acetate produced an increase in the percentage of apoptotic cells, and when these two factors were co-incubated, ANT blocked the apoptotic effect produced by LA.

Mouse cPLA2gamma, a novel oocyte and early embryo-abundant phospholipase A2 gamma-like protein, is targeted to the nuclear envelope during germinal vesicle breakdown.

This report documents the characterization of a novel mouse oocyte protein which was originally identified by microsequence analysis of a 67.8 kDa protein spot (pI 5.7) on a Coomassie-stained two-dimensional (2D) gel of murine egg proteins. Tandem mass spectroscopic analysis of the peptides obtained from the cored protein yielded sequences that appeared to match only ovary, egg, and preimplantation embryo cDNAs. We then cloned the novel gene by RACE-PCR, and analysis of the deduced cDNA sequence found that this maternal product was approximately 56% identical to human cytosolic phospholipase A2gamma (cPLA2gamma). Based on this sequence homology, we named the molecule mouse cytosolic phospholipase A2gamma (cPLA2gamma). As with human cPLA2gamma, mouse cPLA2gamma contains a lipase consensus sequence and lacks the calcium binding domain that is found in other PLA2 proteins. However, mouse cPLA2gamma is different from human cPLA2gamma in that mouse cPLA2gamma expression is restricted to the ovary and that the protein does not contain the myristoylation and prenylation lipid-anchoring motifs that are present in human cPLA2gamma. Within oocytes, mouse cPLA2gamma localizes mainly to the oocyte cortex and to the nucleoplasm. Interestingly, during germinal vesicle breakdown, mouse cPLA2gamma aggregates dynamically relocate from the oocyte cortex to the nuclear envelope, suggesting a possible role for this putative egg-restricted phospholipase A2gamma in membrane remodeling. Furthermore, mouse cPLA2gamma protein continues to be expressed in the embryo until the 4-8-cell stage of development, suggesting that mouse cPLA2gamma may function as a previously uncharacterized maternal effect gene.

Gonadotropin-releasing hormone antagonist antide inhibits apoptosis of preovulatory follicle cells in rat ovary.

Analogs of GnRH, including agonists (GnRH-a) and antagonists (GnRH-ant), have been widely used to inhibit gonadotropin pituitary release. Aside from the effect of GnRH analogs on the pituitary-gonadal axis, studies have shown that GnRH has extrapituitary effects, particularly on rat and human ovaries. In the present study, we evaluated the direct in vivo effects of the GnRH-a, leuprolide acetate (LA), or the GnRH-ant, Antide (Ant), either singly or together, on ovarian follicular development in prepubertal eCG-treated rats. LA significantly decreased ovarian weight, whereas Ant increased ovarian weight compared with controls; however, coinjection of both compounds had no effect. In addition, LA increased the number of preantral follicles (PFs) and atretic follicles, and decreased the number of early antral follicles (EAFs) and preovulatory follicles (POFs). Coinjection of Ant interfered with this LA effect. Ant alone increased the number of POFs compared with that of controls. Analysis of apoptosis has shown that LA increases the percentage of apoptotic cells in PFs, EAFs, and POFs; however, Ant prevented this effect. In addition, Ant alone decreased the percentage of apoptotic cells in EAFs and POFs. Data have shown that Ant per se inhibited BAX translocation from cytosol to mitochondria and retained cytochrome C in the mitochondria, whereas LA induced cytochrome C release. We conclude that Ant inhibits apoptosis in preovulatory follicles through a decrease of BAX translocation to mitochondria, suggesting that GnRH may act as a physiological intraovarian modulator factor that is able to interfere with follicular development through an increase in apoptotic events mediated by an imbalance among the BCL-2 family members.

Inhibin a increases apoptosis in early ovarian antral follicles of diethylstilbestrol-treated rats.

The purpose of this study was to evaluate the role of inhibin A in follicular development and apoptosis-related mechanisms in preantral and early antral follicles from prepubertal diethylstilbestrol (DES)-treated rats. Granulosa cells isolated from the ovaries of 23- to 25-day-old rats were cultured in serum-free medium containing FSH (20 ng/ml), transforming growth factor beta (5 ng/ml), and estradiol (50 ng/ml) in the presence or absence of different concentrations of recombinant human inhibin A. (3)H-Thymidine incorporation was decreased in the presence of Inh, but no significant changes were observed in progesterone and estradiol levels in culture medium. An increase in low molecular weight DNA fragmentation indicative of apoptosis and an increase in the levels of Bax protein with no changes in Bcl-2 protein levels were evident in early antral follicles incubated for 24 h with Inh. For each animal, Inh (0.5 micro g/ovary) was injected intrabursally in one ovary, and the contralateral ovary served as a control. Ovarian histology revealed an inhibitory effect of Inh treatment on the follicular development induced by DES. At 24 h after Inh injection, the number of preantral follicles was increased compared with controls, whereas the number of early antral follicles was decreased. In addition, in vivo Inh treatment caused an increase in the percentage of apoptotic cells in preantral and early antral follicles. These results suggest that inhibin produced by the dominant follicle may act as a paracrine factor inhibiting the growth of neighboring follicles, thus participating in the mechanism of follicular selection.

Effects of a gonadotropin-releasing hormone agonist on rat ovarian follicle apoptosis: regulation by epidermal growth factor and the expression of Bcl-2-related genes.

The purpose of the present study was to evaluate the in vivo effect of the GnRH analogue leuprolide acetate (LA) on follicular development and apoptosis-related mechanisms in preovulatory ovarian follicles (POF) obtained from prepubertal eCG-treated rats. Serum progesterone and estradiol levels were measured, and a significant decrease in circulating estradiol levels was observed in the LA group, whereas serum progesterone levels remained unchanged. Ovarian histology revealed an inhibitory effect of LA treatment on the follicular development induced by eCG. After 48 h of LA treatment, the numbers of atretic and preantral follicles were increased as compared with controls, whereas the number of antral follicles had decreased. Cells undergoing DNA fragmentation were quantified by performing in situ 3' end labeling of DNA with digoxygenin-dUTP on ovarian sections. LA treatment caused an increase in the percentage of apoptotic cells in preantral and antral follicles. DNA isolated from these POF incubated 24 h in serum-free medium exhibited the typical apoptotic DNA degradation pattern. Treatment of follicles with epidermal growth factor (EGF) suppressed the spontaneous onset of DNA fragmentation, and a similar effect was observed in LA follicles. POF obtained from LA-treated rats showed no changes in Bcl-2 or Bax protein levels. However, a reduction in the Bcl-xL:Bcl-xS ratio was observed, with a greater decrease in Bcl-xL compared with Bcl-xS during the incubation, suggesting a lower stability of the Bcl-xL isoform in the LA group. These results indicate that in vivo GnRH agonist treatment produces an increase in the apoptosis process in POF from eCG-treated rats, and this effect is reversed in vitro by EGF. This GnRH analogue also reduced the stability of the Bcl-xL protein, thus interfering with follicular development by an as yet unknown mechanism.

La inhibina (Inh) A producida por células de granulosa humanas actuaría como factor autocrino-paracino regulando el mecanismo de luteinización folicular / Inh A produced by the human granulosa cells would act as autocrinous-paracrinous factor regulating the follicular luteinization mechanism

Las Inh gonadales ejercen un efecto local en la regulación de la foliculogénesis. En éste trabajo se midió la producción de inhibina popr células de granulosa humana en cultivo por un enzimoinmunoensayo específico para las formas A y B encontrándose mayores niveles de Inh A que B (2,89ñ0,88 vs 0,025ñ0,008 ng/1 por 10 cels. n:6, p<0.05). Se detrminó la concentración de inhibinas, Progesterona, Estradiol, FSH y hCG en fluido folicular (FF) obtenido el día de la aspiración ovocitaria en 23 pacientes sometidas a FIV. Se encontró que la concentración de Inh A fue mayor que la de Inh B (6,87ñ1,27 vs 1,24ñ0,23 ng/mg proteína). Se observó una correlación positiva entre la concentración intrafolicular de Inh A y Progesterona (r=0,47 p<0.05), Estradiol (r=0,67 p<0.05) y FSH (r=0,74 p<0.05). También se observó correlación positiva entre los niveles de E y FSH en FF (r=0.56 p<0.05). La Inh B en FF no correlacionó, con ninguno de los parámetros estudiados. Se ensayó el efecto in vitro de la Inh. A en cultivo de células de granulosa inmaduras de rata, que posee la ventaja de tener mayor capacidad proliferativa que las humanas obtenidas por FIV. Se estimuló con FSH (20 ng/ml), Estradiol (500 ng/ml) y TGFß (5 ng/ml) durante 48 hs. Se observó inhibición (p<0,05) de la proliferación medida por incorporación de timidina. (Control: 5543ñ146; Inh 4 ng/ml: 4767ñ174; Inh 40 ng/ml: 3480ñ117 cpm). Esto fue confirmado por MTS (Absorbancia=Control: 1,3ñ0,2; Inh 40 ng/ml: 0,5ñ0,02; p<0,05). Concluímos: a) las células de granulosa humanas provenientes de FIV producen más Inh A que B cultivo, ésta misma relación se observa en FF, b) el contenido de Inh A en FF se correlaciona con la concentración de FSH y esteroides foliculares y c) la Inh A inhibe la proliferación de células de granulosa en cultivo. Estos resultados sugieren que la Inh A actuaría como factor autócrino/parácrino regulando el mecanismo de selección y/o luteinización folicular

La inhibina (Inh) A producida por células de granulosa humanas actuaría como factor autocrino-paracino regulando el mecanismo de luteinización folicular / Inh A produced by the human granulosa cells would act as autocrinous-paracrinous factor regulating the follicular luteinization mechanism

Las Inh gonadales ejercen un efecto local en la regulación de la foliculogénesis. En éste trabajo se midió la producción de inhibina popr células de granulosa humana en cultivo por un enzimoinmunoensayo específico para las formas A y B encontrándose mayores niveles de Inh A que B (2,89ñ0,88 vs 0,025ñ0,008 ng/1 por 10 cels. n:6, p<0.05). Se detrminó la concentración de inhibinas, Progesterona, Estradiol, FSH y hCG en fluido folicular (FF) obtenido el día de la aspiración ovocitaria en 23 pacientes sometidas a FIV. Se encontró que la concentración de Inh A fue mayor que la de Inh B (6,87ñ1,27 vs 1,24ñ0,23 ng/mg proteína). Se observó una correlación positiva entre la concentración intrafolicular de Inh A y Progesterona (r=0,47 p<0.05), Estradiol (r=0,67 p<0.05) y FSH (r=0,74 p<0.05). También se observó correlación positiva entre los niveles de E y FSH en FF (r=0.56 p<0.05). La Inh B en FF no correlacionó, con ninguno de los parámetros estudiados. Se ensayó el efecto in vitro de la Inh. A en cultivo de células de granulosa inmaduras de rata, que posee la ventaja de tener mayor capacidad proliferativa que las humanas obtenidas por FIV. Se estimuló con FSH (20 ng/ml), Estradiol (500 ng/ml) y TGFß (5 ng/ml) durante 48 hs. Se observó inhibición (p<0,05) de la proliferación medida por incorporación de timidina. (Control: 5543ñ146; Inh 4 ng/ml: 4767ñ174; Inh 40 ng/ml: 3480ñ117 cpm). Esto fue confirmado por MTS (Absorbancia=Control: 1,3ñ0,2; Inh 40 ng/ml: 0,5ñ0,02; p<0,05). Concluímos: a) las células de granulosa humanas provenientes de FIV producen más Inh A que B cultivo, ésta misma relación se observa en FF, b) el contenido de Inh A en FF se correlaciona con la concentración de FSH y esteroides foliculares y c) la Inh A inhibe la proliferación de células de granulosa en cultivo. Estos resultados sugieren que la Inh A actuaría como factor autócrino/parácrino regulando el mecanismo de selección y/o luteinización folicular (AU)

Penfigoide cicatrizal localizado (variedad Brunsting-Perry) / Localized cicatricial pemphigoid (Brunsting-Perry)

Se presenta un paciente de sexo masculino de 74 años con diagnóstico clínico e inmunopatológico de penfigoide cicatrizal localizado, cuyas lesiones excedieron a la localización clásicamente descripta (nuca y dorso a nivel de la cintura escapular). Fue tratado inicialmente con sulfonas y luego con deflazacort, manteniéndose en la actualidad libre de lesiones. Se revisan las características clínicas, patológicas e inmunológicas de la entidad (AU)

Penfigoide cicatrizal localizado (variedad Brunsting-Perry) / Localized cicatricial pemphigoid (Brunsting-Perry)

Se presenta un paciente de sexo masculino de 74 años con diagnóstico clínico e inmunopatológico de penfigoide cicatrizal localizado, cuyas lesiones excedieron a la localización clásicamente descripta (nuca y dorso a nivel de la cintura escapular). Fue tratado inicialmente con sulfonas y luego con deflazacort, manteniéndose en la actualidad libre de lesiones. Se revisan las características clínicas, patológicas e inmunológicas de la entidad