Treatment Effects and Risk Factors Evaluation in Longitudinal Studies : A Statistical Help for Data Analysis

This paper was inspired by the experience of the Authors research group composed by oncologist veterinarians and a biostatistician to evaluate treatments and prognostic factors with the aim to help veterinarians involved in longitudinal studies into evaluating and writing prognostic results. Longitudinal studies are commonly analysed by techniques for survival data, taking into account for the time elapsed from the beginning of observation and the occurrence of an event related to treatment effect or disease course. The presence of incomplete follow-up information for some subjects requires specific descriptive and inferential statistical methods. Two literature datasets were analysed to show statistical models implementation techniques and to discuss statistical issues: I) A multicentre clinical trial on remission maintenance of children with acute Lymphoblastic leukaemia and II) A randomized clinical trial on advanced inoperable lung cancer. Data sets concerned studies on \u201chumans\u201d, nevertheless the peculiar data structure allowed to discuss some aspects which are common to survival analysis studies, regardless on subject\u2019s characteristics. Log-rank test was used to compare survival curves for treatments and the relationship between Log-Rank test and univariate Cox model results was explained. As the evaluation of prognostic impact cannot be based only on p-values, the strength of the association between treatments and prognosis was estimated to take into account for the clinical relevance of results. On the second data set, beside of treatment, other clinical variables were available and a multivariate Cox model was applied. Model implementation was discussed concerning the coding of categorical variables and the relationship between continuous variables and model response. Suggested rules for the maximum number of covariates to be included in order to obtain reliable results were cited. Finally, the predictive ability of the model was discussed based on a measure of the area under ROC curve specific for survival data

Characterization of a population of unique granular lymphocytes in a bitch deciduoma, using a panel of histo- and immunohistochemical markers

The ovaries and uterus were collected after ovariohysterectomy from a 16-month-old Labrador bitch in diestrus that never mated. Discrete swellings were found in the uterine horns, with the macroscopic appearance of normal early pregnancy. At histologic examination, the endometrium, devoid of any conceptus and chorion, showed a marked proliferation, on the basis of which a diagnosis of deciduoma was made. A remarkable population of stromal eosinophilic granular lymphocytes was present, especially in the axis of the endometrial folds. Periodic acid–Schiff and Dolichos biflorus–lectin histochemical reaction and a panel of 10 immunohistochemical markers were used to characterize eosinophilic granular cells. Our findings allowed us to compare these granular cells with the granulated decidual cells, whose presence was until now described only in primates, rodents, or a few other epitheliochorial species. On the basis of our results, the importance of eosinophilic granular cells in a decidualization process is hypothesized to occur also in the bitch

Neonatal mortality in dogs : prognostic value of Doppler ductus venosus waveform evaluation - Preliminary results

Aim: To define the prognostic value of Doppler ultrasonographic morphology of ductus venosus (DV) waveform on canine neonatal mortality. Materials and Methods: Fifty-four healthy pregnant bitches underwent fetal ultrasonographic assessment. The DV waveforms were classified as diphasic (dDVw) or triphasic (tDVw) and compared with neonatal mortality. Results: Ninety-three fetuses were evaluated. Twenty fetuses belonged to litters with neonatal mortality, in which tDVw was observed. Seven fetuses belonged to litters without neonatal mortality, in which tDVw was observed. Fifty-eight fetuses belonged to litters without neonatal mortality, in which only dDVw was observed. Eight fetuses belonged to litters with neonatal mortality, in which only dDVw was observed. The correlation between tDVw and neonatal mortality was statistically significant (odds ratio [OR], 20.7; p<0.0001). Considering only pregnancies with one or two fetuses with the same DV waveform: Two fetuses with tDVw belonged to litters with neonatal mortality; 1 foetus with tDVw belonged to litter without neonatal mortality and 26 fetuses showed dDVw without neonatal mortality. The correlation between tDVw and neonatal mortality even in litters up to two pups was statistically significant (OR, 88.3; p=0.01). Conclusion: Echo-Doppler assessment of DV is feasible in canine fetuses, and the presence tDVw seems to be related to neonatal mortality

Persistent M&#252;llerian Duct Syndrome in a German Shepherd Dog

In mammals, the regression of the m\ufcllerian ducts is regulated by the action of the AMH hormone which is produced by testes during embryonic development. The action of this hormone is mediated by the only known receptor AMHR2. Mutations occurring in the AHM hormone and/or in the AMHR2 receptor gene cause the lack of regression of m\ufcllerian ducts, which may therefore persist even in male embryos carrying a XY chromosomal arrangement. This is known as the persistent m\ufcllerian duct syndrome (PMDS). A female German Shepherd dog was referred to the veterinary clinic because of urinary incontinence. She also showed an anatomical structure that protruded from and enlarged the vulvar labia. From the morphological appearance, one gonad resembled an ovary and the other a testicle. The histological examination instead showed that the gonads were both testes with an underdeveloped parenchyma and without signs of spermatogenetic activity. No alterations were found with regard to the uterus which showed a correctly developed body, cervix, and horns. Genetic analysis, performed on DNA extracted from blood, showed (i) the presence of both X and Y chromosomes, (ii) the absence of chromosome XX/XY chimerism, (iii) a normal SRY gene coding sequence, (iv) a normal AMHR2 gene coding sequence, and (v) a normal AMH gene coding sequence. In this study, we report and characterize a new case of PMDS in a dog excluding that the only mutation hitherto found in the AMHR2 gene is responsible for the observed phenotype

Asymptomatic unilateral ovarian leiomioma in a German shepherd bitch

This report shows for the first time clinical imaging (ultrasound and computed tomography), histological and immunohistochemical findings of an ovarian leiomyoma, coincidentally diagnosed in an asymptomatic unmated nulliparous ten year-old German shepherd bitch concurrently suffering from multiple mammary tumors. A thorough examination allowed the differentiation of ovarian leiomyoma from other spindle cell tumors. An accurate description of the diagnostic procedures useful in the managing of ovarian leiomyoma could provide valuable information to veterinary practitioners. Indeed, despite its rarity and nonspecific symptoms, ovarian leiomyoma may also affect the dog with an unknown potential risk

A first glance on the epigenome of Capra hircus

DNA methylation and microRNAs (miRNA) are two important forms of epigenetic modifications that play an important role in gene regulation in animals. Methylation at the carbon 5 position of cytosine residues is a fundamental layer of cellular differentiation through the control of transcriptional potential. MiRNA are small noncoding RNA molecules that regulate gene expression. Complete DNA methylomes for several organisms are now available; at the present, methylome of the domestic goat is unexplored. There is also still limited knowledge about miRNAs expression profiles in small ruminant species. Therefore, to contribute information on epigenetic modification in Capra hircus, we analysed the methylome and the miRNA population of three tissues (hypothalamus, pituitary and ovary) from 3 adult Saanen goats. We used Methylated DNA binding domain sequencing with enrichment of methylated DNA fragments and next generation sequencing. We produced least 23 million reads per sample, which were aligned to the goat reference genome. Further analyses were performed to identify peaks corresponding to hyper-methylated regions. We sequenced miRNAs expressed in the three tissues with Illumina high-throughput sequencing. Reads were mapped on the Capra hircus reference genome and both known and novel miRNAs, and miRNA target sites were identified using information collected in miRBase and using specific bioinformatic tools. This study produced a comprehensive miRNA profile related to the biology of goat. Furthermore, this is the first work dealing with methylome in Capra hircus: our preliminary results could provide new information for a deeper comprehension of epigenetic mechanisms of this species

Nefopam, an analogue of orphenadrine, protects against both NMDA receptor-dependent and independent veratridine-induced neurotoxicity

Nefopam hyghochloride is a potent analgesic compound commercialized in most Western Europe for 20 years, which possesses a profile distinct from that of opioids or anti-inflammatory drugs. Previous evidence suggested a central action of nefopam but the detailed mechanisms remain unclear. While, nefopam structure resembles that of orphenadrine, an uncompetitive NMDA receptor antagonist, here we report that differently from orphenadrine, nefopam (100 microM) failed to protect cultured cerebellar neurons from excitotoxicity following direct exposure of neurons to glutamate. Moreover, nefopam failed to displace MK-801 binding to hippocampal membranes. Nefopam effectively prevented NMDA receptor-mediated early appearance (30 min) of toxicity signs induced by the voltage sensitive sodium channel (VSSC) activator veratridine. The later phase (24 h) of neurotoxicity by veratridine occurring independently from NMDA receptor activation, was also prevented by nefopam. Nefopam effect was not mimicked by the GABA receptor agonist muscimo

The methylome of the hypothalamus of prepubertal and pubertal goats

Puberty is the fulfillment of fertility, a process involving physiological and morphological development. It is well known that the increased hypothalamic secretion of the gonadotropin-releasing hormone (GnRH) is essential for the activation of this process, even if the elements coordinating the timing of puberty have not been fully identified1,2. Recent studies provide proof that there is an epigenetic regulation of female puberty, and DNA methylation, the most studied epigenetic modification, plays a major role in it3. We analyzed DNA methylation patterns of 5 Alpine goats at their prepubertal stage and 5 that reached puberty in order to highlight differences in their methylome. Detection of methylated regions across the goat genome involved a Methyl Binding Domain (MBD) enrichment followed by deep sequencing (Hiseq2000 Illumina). The software ChIPseeqer4 permitted the identification of peaks corresponding to hyper-methylated regions. We have observed a higher methylation level in prepubertal goats. The distribution of the methylation peaks across the genome and within CpG islands per chromosome per group of animals has been analyzed. Furthermore, we have investigated differential methylation in genes associated with puberty. Specifically, Cbx7, coding for a core component of the Polycomb group silencing complex, and GnRHR, the gene coding for GnRH receptor, showed a higher number of peaks into two intragenic fragments within prepubertal goats. These results, accompanied by transcriptome analysis, provide a foundation for elucidating the role of DNA methylation in the complex mechanisms that drive puberty in goat species

Sox9 Duplications Are a Relevant Cause of Sry-Negative XX Sex Reversal Dogs

Sexual development in mammals is based on a complicated and delicate network of genes and hormones that have to collaborate in a precise manner. The dark side of this pathway is represented by pathological conditions, wherein sexual development does not occur properly either in the XX and the XY background. Among them a conundrum is represented by the XX individuals with at least a partial testis differentiation even in absence of SRY. This particular condition is present in various mammals including the dog. Seven dogs characterized by XX karyotype, absence of SRY gene, and testicular tissue development were analysed by Array-CGH. In two cases the array-CGH analysis detected an interstitial heterozygous duplication of chromosome 9. The duplication contained the SOX9 coding region. In this work we provide for the first time a causative mutation for the XXSR condition in the dog. Moreover this report supports the idea that the dog represents a good animal model for the study of XXSR condition caused by abnormalities in the SOX9 locus