CHARACTERIZATION OF Kiss1 NEURONS USING TRANSGENIC MOUSE MODELS

Humans and mice with loss-of-function mutations of the genes encoding kisspeptins (Kiss1) or kisspeptin receptor (Kiss1r) are infertile due to hypogonadotropic hypogonadism. Within the hypothalamus, Kiss1 mRNA is expressed in the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (Arc). In order to better study the different populations of kisspeptin cells we generated Kiss1-Cre transgenic mice. We obtained one line with Cre activity specifically within Kiss1 neurons (line J2-4), as assessed by generating mice with Cre-dependent expression of green fluorescent protein or beta-galactosidase. Also, we demonstrated Kiss1 expression in the cerebral cortex and confirmed previous data showing Kiss1 mRNA in the medial nucleus of amygdala and anterodorsal preoptic nucleus. Kiss1 neurons were more concentrated towards the caudal levels of the Arc and higher leptin-responsivity was observed in the most caudal population of Arc Kiss1 neurons. No evidence for direct action of leptin in AVPV Kiss1 neurons was observed. Me lanocortin fibers innervated subsets of Kiss1 neurons of the preoptic area and Arc, and both populations expressed melanocortin receptors type 4 (MC4R). Specifically in the preoptic area, 18-28% of Kiss1 neurons expressed MC4R. In the Arc, 90% of Kiss1 neurons were glutamatergic, 50% of which also were GABAergic. In the AVPV, 20% of Kiss1 neurons were glutamatergic whereas 75% were GABAergic. The differences observed between the Kiss1 neurons in the preoptic area and the Arc likely represent neuronal evidence for their differential roles in metabolism and reproduction. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Male and female odors induce Fos expression in chemically defined neuronal population

Olfactory information modulates innate and social behaviors in rodents and other species. Studies have shown that the medial nucleus of the amygdala (MEA) and the ventral premammillary, nucleus (PMV) are recruited by conspecific odor stimulation. However, the chemical identity of these neurons is not determined. We exposed sexually inexperienced male rats to female or male odors and assessed Fos immunoreactivity (Fos-ir) in neurons expressing NADPH diaphorase activity (NADPHd, a nitric oxide synthase), neuropeptide Urocortin 3, or glutamic acid decarboxylase rnRNA (GAD-67, a GABA-synthesizing enzyme) in the MEA and PMV. Male and female odors elicited Fos-ir in the MEA and PMV neurons, but the number of Fos-immunoreactive neurons was higher following female odor exposure, in both nuclei. We found no difference in odor induced Fos-ir ill the MEA and PMV comparing fed and fasted animals. Ill the MEA, NADPHd neurons colocalized Fos-ir only in response to female odors. In addition, Urocortin 3 neurons comprise a distinct population and they do not express Fos-ir after conspecific odor stimulation. We found that 80% of neurons activated by male odors coexpressed GAD-67 mRNA. Following female odor, 50% of Fos neurons coexpressed GAD-67 rnRNA. The PMV expresses very little GAD-67, and virtually no colocalization with Fos was observed. We found intense NADPHd activity in PMV neurons, some of which coexpressed Fos-ir after exposure to both odors. The majority of the PMV neurons expressing NADPHd colocalized cocaine-and amphetamine-regulated transcript (CART). Our findings suggest that female and male odors engage distinct neuronal populations in the MEA, thereby inducing contextualized behavioral responses according to olfactory cues. In the PMV, NADPHd/CART neurons respond to male and female odors, suggesting a role in neuroendocrine regulation in response to olfactory cues. (C) 2009 Elsevier Inc. All rights reserved.

Chemical identity and connections of medial preoptic area neurons expressing melanin-concentrating hormone during lactation

Lactation is an energy-demanding process characterized by massive food and water consumption, cessation of the reproductive cycle and induction of maternal behavior. During lactation, melanin-concentrating hormone (MCH) mRNA and peptide expression are increased in the medial preoptic area (MPO) and in the anterior paraventricular nucleus of the hypothalamus. Here we show that MCH neurons in the MPO coexpress the GABA synthesizing enzyme GAD-67 mRNA. We also show that MCH neurons in the MPO of female rats are innervated by neuropeptides that control energy homeostasis including agouti-related protein (AgRP), alpha-melanocyte stimulating hormone (alpha MSH) and cocaine- and amphetamine-regulated transcript (CART). Most of these inputs originate from the arcuate nucleus neurons. Additionally, using injections of retrograde tracers we found that CART neurons in the ventral premammillary nucleus also innervate the MPO. We then assessed the projections of the female MPO using injections of anterograde tracers. The MPO densely innervates hypothalamic nuclei related to reproductive control including the anteroventral periventricular nucleus, the ventrolateral subdivision of the ventromedial nucleus (VMHvl) and the ventral premammillary nucleus (PMV). We found that the density of MCH-ir fibers is increased in the VMHvl and PMV during lactation. Our findings suggest that the expression of MCH in the MPO may be induced by changing levels of neuropeptides involved in metabolic control. These MCH/GABA neurons may, in turn, participate in the suppression of cyclic reproductive function and/or sexual behavior during lactation through projections to reproductive control sites. (C) 2009 Elsevier B.V. All rights reserved.

Cyan fluorescent protein expression in ganglion and amacrine cells in a thy1-CFP transgenic mouse retina.

PURPOSE: To characterize cyan fluorescent protein (CFP) expression in the retina of the thy1-CFP (B6.Cg-Tg(Thy1-CFP)23Jrs/J) transgenic mouse line. METHODS: CFP expression was characterized using morphometric methods and immunohistochemistry with antibodies to neurofilament light (NF-L), neuronal nuclei (NeuN), POU-domain protein (Brn3a) and calretinin, which immunolabel ganglion cells, and syntaxin 1 (HPC-1), glutamate decarboxylase 67 (GAD(67)), GABA plasma membrane transporter-1 (GAT-1), and choline acetyltransferase (ChAT), which immunolabel amacrine cells. RESULTS: CFP was extensively expressed in the inner retina, primarily in the inner plexiform layer (IPL), ganglion cell layer (GCL), nerve fiber layer, and optic nerve. CFP fluorescent cell bodies were in all retinal regions and their processes ramified in all laminae of the IPL. Some small, weakly CFP fluorescent somata were in the inner nuclear layer (INL). CFP-containing somata in the GCL ranged from 6 to 20 microm in diameter, and they had a density of 2636+/-347 cells/mm2 at 1.5 mm from the optic nerve head. Immunohistochemical studies demonstrated colocalization of CFP with the ganglion cell markers NF-L, NeuN, Brn3a, and calretinin. Immunohistochemistry with antibodies to HPC-1, GAD(67), GAT-1, and ChAT indicated that the small, weakly fluorescent CFP cells in the INL and GCL were cholinergic amacrine cells. CONCLUSIONS: The total number and density of CFP-fluorescent cells in the GCL were within the range of previous estimates of the total number of ganglion cells in the C57BL/6J line. Together these findings suggest that most ganglion cells in the thy1-CFP mouse line 23 express CFP. In conclusion, the thy1-CFP mouse line is highly useful for studies requiring the identification of ganglion cells.

Frequency of islet cell autoantibodies (IA-2 and GAD) in young Brazilian type 1 diabetes patients

Type 1 diabetes, as an autoimmune disease, presents several islet cell-specific autoantibodies such as islet cell antibody (ICA), anti-insulin, anti-glutamic acid decarboxylase (GAD) and the antibody (Ab) against tyrosine phosphatase (PTP)-like protein known as ICA-512 (IA-2). In order to determine the frequency of the anti-GAD and anti-IA-2 autoantibodies in Brazilian type 1 diabetes patients we studied 35 diabetes mellitus (DM) type 1 patients with recent-onset disease (£12 months) and 37 type 1 diabetes patients with long-duration diabetes (>12 months) who were compared to 12 children with normal fasting glucose. Anti-GAD65 and anti-IA-2 autoantibodies were detected with commercial immunoprecipitation assays. The frequency of positive results in recent-onset DM type 1 patients was 80.0% for GADAb, 62.9% for IA-2Ab and 82.9% for GADAb and/or IA-2Ab. The long-duration type 1 diabetes subjects presented frequencies of 54.1% for GADAb and IA-2Ab, and 67.5% for GAD and/or IA-2 antibodies. The control group showed no positive cases. Anti-GAD and IA-2 assays showed a high frequency of positivity in these Brazilian type 1 diabetes patients, who presented the same prevalence as a Caucasian population.

How reliable is Ki-67 immunohistochemistry in grade 2 breast carcinomas? A QA study of the Swiss Working Group of Breast- and Gynecopathologists

Adjuvant chemotherapy decisions in breast cancer are increasingly based on the pathologist's assessment of tumor proliferation. The Swiss Working Group of Gyneco- and Breast Pathologists has surveyed inter- and intraobserver consistency of Ki-67-based proliferative fraction in breast carcinomas. Methods Five pathologists evaluated MIB-1-labeling index (LI) in ten breast carcinomas (G1, G2, G3) by counting and eyeballing. In the same way, 15 pathologists all over Switzerland then assessed MIB-1-LI on three G2 carcinomas, in self-selected or pre-defined areas of the tumors, comparing centrally immunostained slides with slides immunostained in the different laboratoires. To study intra-observer variability, the same tumors were re-examined 4 months later. Results The Kappa values for the first series of ten carcinomas of various degrees of differentiation showed good to very good agreement for MIB-1-LI (Kappa 0.56–0.72). However, we found very high inter-observer variabilities (Kappa 0.04–0.14) in the read-outs of the G2 carcinomas. It was not possible to explain the inconsistencies exclusively by any of the following factors: (i) pathologists' divergent definitions of what counts as a positive nucleus (ii) the mode of assessment (counting vs. eyeballing), (iii) immunostaining technique, and (iv) the selection of the tumor area in which to count. Despite intensive confrontation of all participating pathologists with the problem, inter-observer agreement did not improve when the same slides were re-examined 4 months later (Kappa 0.01–0.04) and intra-observer agreement was likewise poor (Kappa 0.00–0.35). Conclusion Assessment of mid-range Ki-67-LI suffers from high inter- and intra-observer variability. Oncologists should be aware of this caveat when using Ki-67-LI as a basis for treatment decisions in moderately differentiated breast carcinomas.

Cleft palate in mice with a targeted mutation in the γ-aminobutyric acid-producing enzyme glutamic acid decarboxylase 67

The functions of neurotransmitters in fetal development are poorly understood. Genetic observations have suggested a role for the inhibitory amino acid neurotransmitter γ-aminobutyric acid (GABA) in the normal development of the mouse palate. Mice homozygous for mutations in the β-3 GABAA receptor subunit develop a cleft secondary palate. GABA, the ligand for this receptor, is synthesized by the enzyme glutamic acid decarboxylase. We have disrupted one of the two mouse Gad genes by gene targeting and also find defects in the formation of the palate. The striking similarity in phenotype between the receptor and ligand mutations clearly demonstrates a role for GABA signaling in normal palate development.

Histologic Correlation Of Expression Of Ki-67 In Squamous Cell Carcinoma Of The Glottis According To The Degree Of Cell Differentiation.

Squamous cell carcinoma is the most common neoplasm of the larynx and glottis, and its prognosis depends on the size of the lesion, level of local invasion, cervical lymphatic spread, and presence of distant metastases. Ki-67 (MKI67) is a protein present in the core, whose function is related to cell proliferation. To evaluate the expression of marker Ki-67 in squamous cell carcinoma of the larynx and glottis and its correlation to pathological findings. Experimental study with immunohistochemistry analysis of Ki-67, calculating the percentage of the cell proliferation index in glottic squamous cell carcinomas. Sixteen cases were analyzed, with six well-differentiated and 10 poorly/moderately differentiated tumors. There was a correlation between cell proliferation index and degree of cell differentiation, with higher proliferation in poorly/moderately differentiated tumors. The cell proliferation index, as measured by Ki-67, may be useful in the characterization of histological degree in glottic squamous cell tumors.

Solar twins in M 67: evolutionary status and lithium abundance

Aims. We determine the age and mass of the three best solar twin candidates in open cluster M 67 through lithium evolutionary models. Methods. We computed a grid of evolutionary models with non-standard mixing at metallicity [Fe/H] = 0.01 with the Toulouse-Geneva evolution code for a range of stellar masses. We estimated the mass and age of 10 solar analogs belonging to the open cluster M 67. We made a detailed study of the three solar twins of the sample, YPB637, YPB1194, and YPB1787. Results. We obtained a very accurate estimation of the mass of our solar analogs in M 67 by interpolating in the grid of evolutionary models. The three solar twins allowed us to estimate the age of the open cluster, which is 3.87(-0.66)(+0.55) Gyr, which is better constrained than former estimates. Conclusions. Our results show that the 3 solar twin candidates have one solar mass within the errors and that M 67 has a solar age within the errors, validating its use as a solar proxy. M 67 is an important cluster when searching for solar twins.

Coherent Contributions to Isospin Mixing in the Mirror Pair (67)As and (67)Se

Isospin symmetry breaking has been investigated in mass A = 67 mirror nuclei through the experimental determination of the E1 strengths of analog electromagnetic transitions. Lifetimes of excited states have been measured in (67)Se and (67)As with the centroid shift method. Through the comparison of the B(E1) strengths of the mirror 9/2(+) -> 7/2(-) transitions, the isovector and the isoscalar components of the electromagnetic transition amplitude were extracted. The presence of a large isoscalar component provides evidence for coherent contributions to isospin mixing, probably involving the isovector giant monopole resonance.

Nucleophosmin, p53, and Ki-67 expression patterns on an oral squamous cell carcinoma tissue microarray

Oral cancer is the eighth most prevalent cancer worldwide. It causes significant mortality and morbidity rates, which have motivated the search for prognostic factors to better tailor the individual management of oral squamous cell carcinoma patients. Nucleophosmin is a multifunctional protein that is involved in many cellular activities, such as, regulation of the tumor suppressor genes TP53 and p14(ARF). and is associated with proliferative and growth suppressive roles in the cell. Nucleophosmin is overexpressed in many solid tumors in human, including tumors of the colon, liver, stomach, ovary, and prostate. In this study, we analyzed the expression of nucleophosmin, Ki-67, and p53 by immunohistochemistry in oral squamous cell carcinomas. Less than 10% of nuclear staining was observed in 90.3%, 50.6%, and 65.3% of the cases for nucleophosmin, p53, and Ki-67, respectively. Expression of p53 was not significantly associated with any of the clinicopathologic parameters analyzed. Increased expression of Ki-67 was associated with the presence of lymph node metastasis (P < .0001), advanced stages of disease (P = .0030), tumors occurring in the floor of mouth (P = .0018), and moderately/well-differentiated tumors (P = .0287). Local recurrence was associated with higher expression of nucleophosmin (P = .0233), and disease-free survival rate was significantly better in patients with low expression of nucleophosmin. Multivariate analysis suggested that expression of nucleophosmin could be an independent prognostic factor for oral squamous cell carcinoma patients. (C) 2010 Elsevier Inc. All rights reserved.

Ki-67 expression in non-tumour epithelium adjacent to oral cancer as risk marker for multiple oral tumours

Objective: The aim of this study was to determine whether the differential assessment of epithelial proliferation is useful to diagnose premalignant fields and assess the risk of multiple tumours. Material and methods: We analysed 83 oral carcinomas with associated non-tumour epithelium classified as distant or close according to its distance (> or < 1 cm) from the invasion point, and as squamous hyperplasia, mild, moderate, severe dysplasia or carcinoma in situ. Twenty-five healthy oral mucosa samples were used as controls. An immunohistochemical technique was applied using Mib-1. Ki-67 in premalignant epithelium was assessed in basal layer, parabasal layer, medium and upper third. Results: Parabasal expression was significantly higher or showed a tendency to be higher in close and distant epithelia with any histological grade than in the controls. Parabasal Ki-67 significantly differed between distant epithelia associated with multiple vs single tumours (P < 0.001) and between distant epithelia associated with multiple tumours vs controls (P < 0.001). This difference was not observed between distant epithelia associated with single tumours and controls (P = 0.175). The cut-off point that differentiated epithelia associated with multiple tumours was > 50% of Ki-67 + parabasal cells in distant epithelia, which yielded 0.88 sensitivity and 0.79 specificity. Conclusions: The concept of a precancerous field may be linked to an increase in the proliferative activity of parabasal cells.

A guerrilha esquecida : memórias do Caparaó (1966-67), o primeiro foco guerrilheiro contra a ditadura militar no Brasil

Este estudo tem como objetivo discutir a memória histórica do primeiro foco guerrilheiro no Brasil, organizado pelo Movimento Nacional Revolucionário (MNR), em 1966-1967, com o apoio de Leonel Brizola e de Cuba, na região do Caparaó, divisa dos Estados de Minas Gerais e Espírito Santo. Assim, o estudo se propõe a analisar o pouco conhecido episódio da formação, do idealismo e do desfecho que envolveu a Guerrilha do Caparaó, apresentando as memórias dos guerrilheiros (sendo em sua grande maioria ex-militares), dos agentes da repressão, dos setores conservadores da sociedade e dos habitantes das comunidades do entorno do atual Parque Nacional do Caparaó, demonstrando, assim, as diferentes percepções e representações sobre a referida Guerrilha. Para tanto, será exposto todo o contexto histórico da época em questão. Como metodologia, utilizaremos o conceito de memória, que, em suas ramificações, abrangerá a história oral, além de análises bibliográficas, pesquisas em jornais do período (A Gazeta, O Globo, Jornal do Brasil, A Última Hora, Tribuna da Imprensa, Correio da Manhã, O Estado de São Paulo, Estado de Minas, O Diário da Tarde), em revistas (O Cruzeiro, Opinião e Revista Capixaba) e em documentos da Delegacia de Ordem Política e Social (DOPS) dos Arquivos Públicos dos estados do Espírito Santo e Minas Gerais, e documentos do Serviço Nacional de Informação (SNI), do Arquivo Nacional.

Valor prognóstico da correlação imunoistoquímica do Ki-67 e p53 em carcinomas epidermóides da laringe

Os fatores prognósticos histológicos podem colaborar para determinar a evolução desta neoplasia. OBJETIVO: Correlacionar a expressão imunoistoquímica do p53 e Ki-67 com idade, grau histológico, comprometimento de linfonodos e estadiamento patológico de pacientes com carcinomas epidermóides de laringe. MÉTODO: Foram avaliados trinta casos consecutivos de carcinomas epidermóides de laringe, submetidos à técnica de imunoistoquímica para verificar a expressão dos anticorpos p53 e Ki-67. RESULTADO: A idade média foi de 56,2 anos e a imunoexpressão dos marcadores foi mais observada no grupo com mais de 50 anos de idade, especialmente a do anticorpo Ki-67 (p=0,032). Não houve relação do p53 e Ki-67 com o comprometimento de linfonodos. O Ki-67 se expressou em 70% nos casos de alto grau histológico e 80% nos de baixo, enquanto o p53 em 70% apenas nos de alto grau. O estadiamento patológico mostrou que no grupo dos carcinomas avançados a expressão do p53 foi de 61,5%, enquanto o Ki-67 mostrou positividade para os casos precoces (100%) e avançados (73,1%). CONCLUSÃO: Não houve diferenças significantes entre a imunoexpressão do p53 e do Ki-67 em carcinomas epidermóides de laringe, exceto no grupo de pacientes com mais de 50 anos, onde a expressão do Ki-67 foi significativamente maior.