Expression and clinical implications of HLA-G and PD-L1 following kidney transplantation: A cohort study.

Kidney transplantation (KT) is the preferred treatment for end-stage renal diseases. Human leukocyte antigen G (HLA-G) and programmed death-ligand 1 (PD-L1) have notable clinical and therapeutic significance in transplantation because of their roles in promoting tolerance. This study aimed to assess HLA-G and PD-L1 levels at various stages following KT. A cohort of 12 patients was monitored from the pretransplant phase to 12 months post-surgery. Blood samples were taken at specific intervals: before kidney transplantation (T0), and then on the 7th (T7), 30th (T30), 90th (T90), 180th (T180), and 365th days post transplantation. Renal biopsies were performed in patients with graft dysfunction. Plasma levels of soluble HLA-G (sHLA-G) and PD-L1 were quantified using enzyme-linked immunosorbent assays. Additionally, immunohistochemistry was used to detect the presence of both molecules in biopsy samples. Multivariate analysis indicated that episodes of rejection were correlated with decreased expression of sHLA-G (P < .001) and PD-L1 (P < .001). Over the course of the study, the sHLA-G levels also declined (P < .001). Patients who had been transfused had lower PD-L1 levels (P = .03). Furthermore, kidney recipients from related live donors had increased HLA-G expression (P < .001). Our findings suggest that diminished HLA-G and PD-L1 levels correlate with an increased risk of graft rejection. Notably, HLA-G expression significantly decrease after the third-month posttransplantation.

MicroRNA Levels in Cervical Cancer Samples and Relationship with Lesion Grade and HPV Infection.

BACKGROUND: miR-21, miR-214 and miR-let-7a are three validated and well-known miRNAs. miR-21 is described as an "oncomir" while miR-214 and miR-let-7a are described mainly as tumor suppressors. The role of these miRNAs remains unclear in cervical cancer, an important malignancy among women worldwide and responsible for many deaths every year. OBJECTIVE: The objective of this study is to describe the expression profile of miR-21, miR-214 and miR-let-7a in plasma and in cervical scraping from a control group and patients with different grades of cervical lesions and invasive cervical cancer and correlate with HPV infection groups. METHODS: Plasma and cervical scraping were submitted to DNA and RNA extraction. HPV detection and typing were performed by conventional PCR followed by PAGE to amplicons interpretation. The miRNA relative expression in plasma and cervical scraping samples was performed by real time PCR using specific TaqMan probes. RESULTS: miR-21 (p=0.0277) and miR-214 (p=0.0151) were up-regulated in cervical scraping samples of invasive cervical cancer (ICC) group. However, miR-214 was also up-regulated in the LSIL group (p=0.0062). Both miRNAs were not related to HPV infection. However, miR-let-7a was higher in HPV positive plasma samples (p=0.0433) than in HPV negative plasma samples and the correlation analysis confirmed the association between the levels of this miRNA with the presence of HPV (p=0.0407; r=0.3029), but not with lesion grade (p>0.05). CONCLUSION: Our results suggest that miR-21 is related to cervical cancer progression and miR-214 appears to have an ambiguous role in cervical lesions. miR-let-7a may be upregulated at a systemic level in patients with HPV infection.

An improved acetic acid-urea polyacrylamide electrophoresis method to evaluate bovine sperm protamines.

The acetic acid-urea polyacrylamide gel electrophoresis system could separate very similar basic proteins on differences in size and effective charge. This system has been used for many years to analyse histones and their post-translational modifications and widely used in the study of mammal protamines. Two types of protamine have been described, the protamine 1 (P1) and the protamine 2 (P2) family members, which are synthetized by PRM1 and PRM2 genes. The ratio of P1 and P2 is important for predicting fertility in humans and mice. Therefore, the quantification of protamines is a fundamental step in order to establish the ratio between P1 and P2 in these species. In other mammals, studies linking sperm protamination and the protamine ratio with fertility are increasing. So, the use of an effective technique to separate and quantify protamines is important to study sperm P1/P2 ratio. Therefore, this article describes in detail a feasible and useful procedure to isolate bovine sperm protamines, to perform pre-electrophoresis with PEG solution and finally to carry out acid-urea polyacrylamide gel electrophoresis in reverse polarity. This technique allows a clear separation and efficient detection of bovine sperm protamines.

Spermatic profile of captive giant anteaters (Myrmecophaga tridactyla): Knowing more to preserve better.

The giant anteater (Myrmecophaga tridactyla) is being threatened by natural habitat destruction and fragmentation, illegal hunting and road kills. In this context, the generation of basic information on the reproductive parameters of this species is vital, aiming to improve reproductive management via, amongst others, assisted reproductive technologies. This study aimed to describe the morphological and functional features of semen collected from captive giant anteaters. Electroejaculation was performed in 13 animals housed in zoos located in São Paulo state, Brazil. Semen samples were collected from 13 animals in 16 procedures. Samples were evaluated for volume, motility, vigor, pH, concentration, sperm morphology, and functional tests. The following mean values were obtained: volume 1.28 ± 0.27 mL; motility 28.3 ± 6.2%; vigor 2.4 ± 0.25; concentration 129.4 ± 36.1 × 106 sperm/mL; pH 7.4 ± 0.2. Total acrosome, head, midpiece, and tail sperm abnormalities were 3.2 ± 0.8%, 25.4 ± 3.6%, 20.7 ± 3.2%, and 14.7 ± 2.6%, respectively. Intact acrosome was found in 83.7 ± 3.1% and intact membrane in 81.1 ± 4.0% of all samples collected. Mitochondrial activity was 66.4 ± 6.0% (Class I), 18.7 ± 2.9% (Class II), 8.0 ± 2.0% (Class III), 3.9 ± 1.0% (Class IV), and 3.0 ± 0.9% (Class V). Sperm DNA fragmentation rate was 13.2 ± 3.7%. These results indicated that electroejaculation is a feasible method for semen collection in giant anteaters, allowing a more detailed description of the semen in this species.

Ceramide synthase TLCD3B is a novel gene associated with human recessive retinal dystrophy.

PURPOSE: Previous studies suggest that ceramide is a proapoptotic lipid as high levels of ceramides can lead to apoptosis of neuronal cells, including photoreceptors. However, no pathogenic variant in ceramide synthases has been identified in human patients and knockout of various ceramide synthases in mice has not led to photoreceptor degeneration. METHODS: Exome sequencing was used to identify candidate disease genes in patients with vision loss as confirmed by standard evaluation methods, including electroretinography (ERG) and optical coherence tomography. The vision loss phenotype in mice was evaluated by ERG and histological analyses. RESULTS: Here we have identified four patients with cone-rod dystrophy or maculopathy from three families carrying pathogenic variants in TLCD3B. Consistent with the phenotype observed in patients, the Tlcd3bKO/KO mice exhibited a significant reduction of the cone photoreceptor light responses, thinning of the outer nuclear layer, and loss of cone photoreceptors across the retina. CONCLUSION: Our results provide a link between loss-of-function variants in a ceramide synthase gene and human retinal dystrophy. Establishment of the Tlcd3b knockout murine model, an in vivo photoreceptor cell degeneration model due to loss of a ceramide synthase, will provide a unique opportunity in probing the role of ceramide in survival and function of photoreceptor cells.

HLA-G 14 bp In/Del and +3142 C/G genotypes are differentially expressed between patients with grade IV gliomas and controls.

Aim: Human Leukocyte Antigen-G (HLA-G) is a non-classical class I molecule that is involved in maternal-fetal immunotolerance. In cancer, this molecule contributes to the tumor escape. The aim of this study was to evaluate the 14 bp In/Del and +3142 C > G polymorphisms of the HLA-G 3' UTR and its relation with plasma and tissue HLA-G expression in patients with grade IV (high-grade) and grade I/II (low-grade) gliomas and controls.Patients and methods: Peripheral blood and tumor biopsies were collected from 85 patients with gliomas and blood samples from 94 controls. Polymorphisms were analyzed from blood DNA. Soluble HLA-G (sHLA-G) was measured by ELISA in plasma of the subjects and the tissue expression by immunohistochemistry on patient's tissue.Results: Higher levels of sHLA-G were observed in grade IV gliomas patients than in controls (p < 0.0001). In grade IV patients, the heterozygous 14pb In/Del, +3142 C/G genotypes and Del/C*In/G haplotype were associated with higher sHLA-G levels (p < 0.0001) when compared with controls. GBM patients were stratified into high and low sHLA-G expression and an association was found between +3142 C allele and high sHLA-G plasmatic levels (p = 0.0095). Tissue HLA-G immunolabel was higher in high-grade than low-grade gliomas (p = 0.0033).Conclusion: This was the first study evaluating HLA-G 3' UTR polymorphisms and expression in patients with gliomas. The 14 bp In/Del and +3142 C/G genotypes and haplotypes showed high influence over sHLA-G expression, suggesting a heterozygous advantage in the tumor context and may contribute to a worse prognosis in glioma patients.

Recurrence/Regrowth in Grade I Meningioma: How to Predict?

The HLA-G and HLA-E molecules, Ki67, progesterone (PR), estrogen (ER) and androgen receptors (AR), p53, COX-2, and HER2 were studied to assess whether the biological behavior of grade I meningiomas is related to their expression. Tissue samples from 96 patients with grade I intracranial meningiomas were analyzed by immunohistochemistry on tissue microarray blocks (TMA) using antibodies specific for HLA-G, HLA-E, Ki67, PR, ER, AR, p53, COX-2, and HER2. Meningiomas were classified as small (≤2 cm, 1.0%), medium (>2 and ≤4 cm, 32.3%), and large (>4 cm, 66.7%). Tumor size was not related to recurrence/regrowth (p = 0.486), but was significantly correlated with peritumoral edema (p = 0.031) and intratumoral calcifications (p = 0.018). Recurrent meningiomas were observed in 14.6% of cases. Immunostaining for each marker was: HLA-G 100%; HLA-E 95.6%; PR 62%; ER 2.1%; AR 6.5%; p53 92.6%; COX-2 100%; HER2 0%; Ki67, mean 2.61 ± 2.29%, median 2.1%. Primary and recurrent meningiomas showed no significant relation with HLA-E and hormone receptors (p > 0.05), except for Ki67, where a higher median was observed in recurrent tumors than in primary (p = 0.014). The larger the tumor, the more severe the peritumoral edema, and the greater the presence of calcifications. Ki67 appears to be a good biomarker of recurrence/regrowth in grade I meningiomas.

HLA-G Ins/Del polymorphism and +3142C/G SNP are not related to neuromyelitis optica spectrum disorder (NMOSD) development, disability status or anti-aquaporin 4 presence in Brazilian patients.

We analyzed the association of polymorphisms from the 3' untranslated region of the HLA-G gene in 70 neuromyelitis optica spectrum disorder (NMOSD) patients and 162 healthy controls. No associations were found between the polymorphisms in NMOSD when compared to healthy controls, serology of the anti-AQP4 NMOSD biomarker and Expanded Disability Status Scale (EDSS). In conclusion, the 3' untranslated region 14 bp Ins/Del and +3142C/G polymorphisms seem not to be associated with NMOSD susceptibility, autoantibody production, nor a neurological deficit in patients.

Trends for in-hospital metrics in the treatment of intracranial aneurysms in Minas Gerais, Brazil.

Background: Aneurysmal subarachnoid hemorrhage (ASAH) and unruptured aneurysm (URA) are particularly important because of the high mortality rates, and physical and cognitive impairment, which affect the economically active population. The present work aims at describing the scenario regarding in-hospital indicators related to the following therapeutic approaches: vascular microsurgery (VMS) and endovascular therapy (EVT) in the state of Minas Gerais, Brazil, in order to gather information to construct hypotheses and plan actions. Methods: The study has an ecological design and it is also analytical for the time trends of 2008-2014. Trends for VMS and EVT therapies and mortality are estimated using linear regression, followed by the Prais-Winsten procedure. Data were obtained through Hospital Information System (Sistema de Informações Hospitalares - SIH) of Brazilian Public Health System (Sistema Único de Saúde - SUS). Results: In 2014, 601 procedures were performed, and out of these, 373 (62%) were triggered by SAH and 228 (38%) by URA. The frequency of procedures performed in males and in females results in a ratio of 1:2 for SAH procedures and 1:3 for URA procedures. A heterogeneous distribution of therapeutic approaches was seen among the hospital studied, suggesting a technological preference, which determines the therapeutic approach. Conclusion: Technological bias was observed for aneurysm treatment in Minas Gerais's hospitals. EVT is seen to have a growing trend to detriment of VMS (ß1 = 0.024; p = 0.025), with a stable mortality for both therapeutics in both pathologies (SAH and URA). EVT has been associated with a shorter hospital stay, with higher expenses for both ASAH and URA treatment. EVT showed an inverse correlation with in-hospital fatality for ASAH treatment.

Sexually Transmitted Infections Detected by Multiplex Real Time PCR in Asymptomatic Women and Association with Cervical Intraepithelial Neoplasia.

OBJECTIVE: To determine the frequency of sexually transmitted infections (STIs) in asymptomatic women and the association of STIs with cervical intraepithelial neoplasia (CIN). METHODS: A cross-sectional study was performed, enrolling women examined in a general gynecology clinic and in a colposcopy referral center from October 2014 to October 2015. The colposcopy group consisted of 71 women, and the general gynecology group consisted of 55 women. Cervical samples were collected for cervical cytology and a multiplex real-time polymerase chain reaction (PCR) was developed to detect human papillomavirus (HPV) and the STIs caused by the following microorganisms: Chlamydia trachomatis, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum, and Neisseria gonorrhoeae. A multivariate analysis was performed by logistic regression, considering the significance level of 0.05. RESULTS: The general frequency of STIs was: 46.8% (HPV); 27.8% (C. trachomatis); 28.6% (M. genitalium); 0.8% (M. hominis); 4.8% (U. urealyticum); and 4.8% (N. gonorrhoeae). The significant risk factors for CIN were: HPV infection (odds ratio [OR] = 2.53; p = 0.024); C. trachomatis (OR = 3.04; p = 0.009); M. genitalium (OR = 2.37; p = 0.04); and HPV and C. trachomatis coinfection (OR = 3.11; p = 0.023). After the multivariate analysis, a significant association was found between HPV and CIN (OR = 2.48; 95% confidence interval [95%CI]: 1.04-5.92; p = 0.04); and between C. trachomatis and CIN (OR = 2.69; 95%CI: 1.11-6.53; p = 0.028). CONCLUSION: The frequency of STIs was high in asymptomatic patients. Infections by HPV and C. trachomatis were independently associated with the presence of CIN. The high frequency of STIs in asymptomatic women suggests the need for routine screening of these infections.

OBJETIVO: Determinar a frequência de infecções sexualmente transmissíveis (ISTs) em mulheres assintomáticas e a associação destas infecções com a neoplasia intraepitelial cervical (NIC). MéTODOS: Foi realizado um estudo transversal recrutando mulheres atendidas em uma clínica ginecológica geral e em um centro de referência para colposcopia, de outubro de 2014 a outubro de 2015. O grupo de colposcopia consistiu de 71 mulheres, e o grupo de ginecologia geral consistiu de 55 mulheres. Amostras cervicais foram coletadas para citologia cervical e uma reação em cadeia de polimerase (RCP) multiplex em tempo real para detecção do vírus do papiloma humano (HPV) e das ISTs provocadas pelos seguintes micro-organismos: Chlamydia trachomatis, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum e Neisseria gonorrhoeae. Foi realizada uma análise multivariada por regressão logística, considerando-se o nível de significância de 0,05. RESULTADOS: A frequência geral de ISTs foi: 46,8% (HPV); 27,8% (C. trachomatis); 28,6% (M. genitalium); 0,8% (M. hominis); 4,8% (U. urealyticum); e 4,8% (N. gonorrhoeae). Os fatores de risco significantes para NIC foram: infecção pelo HPV (razão de probabilidades [RP] = 2,53; p = 0,024); C. trachomatis (RP = 3,04; p = 0,009); M. genitalium (RP = 2,37; p = 0,04); e coinfecção por HPV e C. trachomatis (RP = 3,11; p = 0,023). Após a análise multivariada, foi encontrada uma associação significante entre HPV e NIC (RP = 2.48; intervalo de confiança de 95% [IC95%]: 1,04­5,92; p = 0,04) e entre C. trachomatis e NIC (RP = 2,69; IC95%: 1,11­6,53; p = 0,028). CONCLUSõES: A frequência de ISTs foi alta em mulheres assintomáticas. Infecções por HPV e C. trachomatis foram independentemente associadas com a presença de NIC. A alta frequência de ISTs em mulheres assintomáticas sugere a necessidade de rastreamento rotineiro dessas infecções.

Sexually transmitted infections detected by multiplex real time PCR in asymptomatic women and association with cervical Intraepithelial neoplasia / Infecções sexualmente transmissíveis detectadas por PCR multiplex em tempo real em mulheres assintomáticas e associação com neoplasia intraepitelial cervical

Abstract Objective To determine the frequency of sexually transmitted infections (STIs) in asymptomatic women and the association of STIs with cervical intraepithelial neoplasia (CIN). Methods A cross-sectional studywas performed, enrollingwomen examined in a general gynecology clinic and in a colposcopy referral center fromOctober 2014 to October 2015. The colposcopy groupconsisted of 71women, and the general gynecologygroupconsisted of 55 women. Cervical samples were collected for cervical cytology and a multiplex realtime polymerase chain reaction (PCR) was developed to detect human papillomavirus (HPV) and the STIs caused by the following microorganisms: Chlamydia trachomatis, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum, and Neisseria gonorrhoeae. A multivariate analysis was performed by logistic regression, considering the significance level of 0.05. Results The general frequency of STIs was: 46.8% (HPV); 27.8% (C. trachomatis); 28.6% (M. genitalium); 0.8% (M. hominis); 4.8% (U. urealyticum); and 4.8% (N. gonorrhoeae). The significant risk factors for CIN were: HPV infection (odds ratio [OR] = 2.53; p = 0.024); C. trachomatis (OR = 3.04; p = 0.009); M. genitalium (OR = 2.37; p = 0.04); and HPV and C. trachomatis coinfection (OR = 3.11; p = 0.023). After the multivariate analysis, a significant associationwas found betweenHPVand CIN(OR = 2.48; 95% confidence interval [95%CI]: 1.04-5.92; p = 0.04); and between C. trachomatis and CIN (OR = 2.69; 95%CI: 1.11-6.53; p = 0.028). Conclusion The frequency of STIs was high in asymptomatic patients. Infections by HPV and C. trachomatis were independently associated with the presence of CIN. The high frequency of STIs in asymptomatic women suggests the need for routine screening of these infections.

Resumo Objetivo Determinar a frequência de infecções sexualmente transmissíveis (ISTs) em mulheres assintomáticas e a associação destas infecções com a neoplasia intraepitelial cervical (NIC). Métodos Foi realizado um estudo transversal recrutando mulheres atendidas em uma clínica ginecológica geral e em um centro de referência para colposcopia, de outubro de 2014 a outubro de 2015. O grupo de colposcopia consistiu de 71 mulheres, e o grupo de ginecologia geral consistiu de 55 mulheres. Amostras cervicais foram coletadas para citologia cervical e uma reação em cadeia de polimerase (RCP) multiplex em tempo real para detecção do vírus do papiloma humano (HPV) e das ISTs provocadas pelos seguintes micro-organismos: Chlamydia trachomatis, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum e Neisseria gonorrhoeae. Foi realizada uma análise multivariada por regressão logística, considerando-se o nível de significância de 0,05. Resultados A frequência geral de ISTs foi: 46,8% (HPV); 27,8% (C. trachomatis); 28,6% (M. genitalium); 0,8% (M. hominis); 4,8% (U. urealyticum); e 4,8% (N. gonorrhoeae). Os fatores de risco significantes para NIC foram: infecção pelo HPV (razão de probabilidades [RP] = 2,53; p = 0,024); C. trachomatis (RP = 3,04; p = 0,009); M. genitalium (RP = 2,37; p = 0,04); e coinfecção por HPV e C. trachomatis (RP = 3,11; p = 0,023). Após a análise multivariada, foi encontrada uma associação significante entre HPV e NIC (RP = 2.48; intervalo de confiança de 95% [IC95%]: 1,04-5,92; p = 0,04) e entre C. trachomatis e NIC (RP = 2,69; IC95%: 1,11-6,53; p = 0,028). Conclusões A frequência de ISTs foi alta em mulheres assintomáticas. Infecções por HPV e C. trachomatis foram independentemente associadas com a presença de NIC. A alta frequência de ISTs em mulheres assintomáticas sugere a necessidade de rastreamento rotineiro dessas infecções.

Levels of MMP-9 in patients with intracranial aneurysm: Relation with risk factors, size and clinical presentation.

BACKGROUND: Intracranial aneurysms are arterial anomalies affecting 2% to 3% of the general population in the world and these ruptures are associated with a high mortality. Some risk factors, such as age, gender, smoking, alcohol, hypertension and familial history are associated with the number of aneurysms and their size. In addition, inflammatory processes within the blood vessels of the brain can activate matrix metalloproteinase-9 (MMP-9), which degrades various components of the extracellular matrix, such as elastin. Thereby, this work has aimed at evaluating the relationship between plasma MMP-9 levels and the risk factors that are associated with intracranial aneurysm, as well as investigating the aneurysm statuses (ruptured and unruptured) and comparing them with the control volunteers. METHODS: Between August 2014 to June 2016, blood samples were collected from 282 patients (204 ruptured and 78 unruptured saccular intracranial aneurysms) and 286 control volunteers. The MMP-9 plasma levels were measured by ELISA. Statistical analyzes were performed with SPSS software when using parametric or nonparametric tests, after the normality tests. RESULTS: Higher levels of MMP-9 were found in the aneurysm groups as a whole and when they were stratified by rupture status, then compared with the control group (p < 0.0001). When stratifying them by diameter, those smaller than 7 mm presented high levels of MMP-9 (p < 0.0001), especially in the ruptured ones. As for risk factors, hypertension and smoking were the most important. However, hypertension was mostly associated with the ruptured aneurysms (p < 0.0001). CONCLUSIONS: High levels of MMP-9 were found in smaller ruptured and unruptured intracranial aneurysms (<7 mm) with strongest statistical associations than other sizes, especially when associated with smoking and hypertension.

Molecular Screening of 43 Brazilian Families Diagnosed with Leber Congenital Amaurosis or Early-Onset Severe Retinal Dystrophy.

Leber congenital amaurosis (LCA) is a severe disease that leads to complete blindness in children, typically before the first year of life. Due to the clinical and genetic heterogeneity among LCA and other retinal diseases, providing patients with a molecular diagnosis is essential to assigning an accurate clinical diagnosis. Using our gene panel that targets 300 genes that are known to cause retinal disease, including 24 genes reported to cause LCA, we sequenced 43 unrelated probands with Brazilian ancestry. We identified 42 unique variants and were able to assign a molecular diagnosis to 30/43 (70%) Brazilian patients. Among these, 30 patients were initially diagnosed with LCA or a form of early-onset retinal dystrophy, 17 patients harbored mutations in LCA-associated genes, while 13 patients had mutations in genes that were reported to cause other diseases involving the retina.

Factors and molecules that could impact cell differentiation in the embryo generated by nuclear transfer.

Somatic cell nuclear transfer is a technique to create an embryo using an enucleated oocyte and a donor nucleus. Nucleus of somatic cells must be reprogrammed in order to participate in normal development within an enucleated egg. Reprogramming refers to the erasing and remodeling of cellular epigenetic marks to a lower differentiation state. Somatic nuclei must be reprogrammed by factors in the oocyte cytoplasm to a rather totipotent state since the reconstructed embryo must initiate embryo development from the one cell stage to term. In embryos reconstructed by nuclear transfer, the donor genetic material must respond to the cytoplasmic environment of the cytoplast and recapitulate this normal developmental process. Enucleation is critically important for cloning efficiency because may affect the ultrastructure of the remaining cytoplast, thus resulting in a decline or destruction of its cellular compartments. Nonetheless, the effects of in vitro culturing are yet to be fully understood. In vitro oocyte maturation can affect the abundance of specific transcripts and are likely to deplete the developmental competence. The epigenetic modifications established during cellular differentiation are a major factor determining this low efficiency as they act as epigenetic barriers restricting reprogramming of somatic nuclei. In this review we discuss some factors that could impact cell differentiation in embryo generated by nuclear transfer.

Reduced expression of the murine HLA-G homolog Qa-2 is associated with malignancy, epithelial-mesenchymal transition and stemness in breast cancer cells.

Qa-2 is believed to mediate a protective immune response against cancer; however, little is known about the role of Qa-2 in tumorigenesis. Here, we used 4T1 breast cancer cells to study the involvement of Qa-2 in tumor progression in a syngeneic host. Qa-2 expression was reduced during in vivo tumor growth and in cell lines derived from 4T1-induced tumors. Tumor-derived cells elicited an epithelial-mesenchymal transition associated with upregulation of Zeb1 and Twist1/2 and enhanced tumor initiating and invasive capacities. Furthermore, these cells showed increased stem characteristics, as demonstrated by upregulation of Hes1, Sox2 and Oct3/4, and enrichment of CD44high/CD24median/low cells. Remarkably, Qa-2 cell-surface expression was excluded from the CD44high/CD24median/low subpopulation. Tumor-derived cells showed increased Src activity, and treatment of these cells with the Src kinase inhibitor PP2 enhanced Qa-2 but reduced Sox2 and CD44high/CD24median/low expression levels, suggesting that Src signaling, while positively associated with stemness, negatively regulates Qa-2 expression in breast cancer. Finally, overexpression of the Qa-2 family member Q7 on the cell surface slowed down in vivo tumor growth and reduced the metastatic potential of 4T1 cells. These results suggest an anti-malignant role for Qa-2 in breast cancer development, which appears to be absent from cancer stem cells.

Leveraging splice-affecting variant predictors and a minigene validation system to identify Mendelian disease-causing variants among exon-captured variants of uncertain significance.

The genetic heterogeneity of Mendelian disorders results in a significant proportion of patients that are unable to be assigned a confident molecular diagnosis after conventional exon sequencing and variant interpretation. Here, we evaluated how many patients with an inherited retinal disease (IRD) have variants of uncertain significance (VUS) that are disrupting splicing in a known IRD gene by means other than affecting the canonical dinucleotide splice site. Three in silico splice-affecting variant predictors were leveraged to annotate and prioritize variants for splicing functional validation. An in vitro minigene system was used to assay each variant's effect on splicing. Starting with 745 IRD patients lacking a confident molecular diagnosis, we validated 23 VUS as splicing variants that likely explain disease in 26 patients. Using our results, we optimized in silico score cutoffs to guide future variant interpretation. Variants that alter base pairs other than the canonical GT-AG dinucleotide are often not considered for their potential effect on RNA splicing but in silico tools and a minigene system can be utilized for the prioritization and validation of such splice-disrupting variants. These variants can be overlooked causes of human disease but can be identified using conventional exon sequencing with proper interpretation guidelines.

Qa-2 expression levels is related with tumor-infiltrating lymphocytes profile during solid Ehrlich tumor development.

The Qa-2 has been described as Human Leucocyte Antigen G (HLA-G) murine homolog. This homology is well accepted to gene and protein structure, in different pathology process and embryos implantation. However, in some neoplasm, this homology is questioned, where Qa-2 has been proposed as an immunogenic molecule, associated to tumor rejection. In this way, the aim of this study was to describe the pattern of Qa-2 expression and its relationship with the profile of tumor-infiltrating lymphocytes in solid Ehrlich tumor. The Ehrlich tumor growth was evaluated in Balb/c female mice in different tumor stages. The inflammatory infiltration features were determined by histopathology and, both lymphocyte type and tissue Qa-2 expression by immunohistochemistry. ELISA kit was used to determine soluble Qa-2 in the serum from the animals. We observed that Qa-2 in neoplastic cells increases in intermediate tumor development stages, while, serum Qa-2 increases in the late stage. Qa-2 increasing is correlated with CD3+ increase. Our results suggest that Qa-2 has a role opposite to HLA-G in Ehrlich solid carcinoma, and may be modulating the immune response by attracting the inflammatory infiltrate, especially T CD8+ Lymphocytes.

Evaluation of the p16 and Ki-67 Biomarkers as Predictors of the Recurrence of Premalignant Cervical Cancer Lesions after LEEP Conization / Avaliação dos biomarcadores p16 e Ki-67 como preditores de recidivas de lesões pré-cancerígenas do colo do útero após conização por cirurgia de alta frequência

Abstract Objective To evaluate the expressions of biomarkers p16 and Ki-67 in low-grade (LG) or high-grade (HG) lesions, and to relate them to risk factors and the recurrence of these lesions. Methods A retrospective case-control study of 86 patients with LG and HG lesions who underwent a loop electrosurgical excision procedure (LEEP) between 1999 and 2004. The control group was composed of 69 women with no recurrence, and the study group, of 17 patients with recurrence. All patients were followed-up over a two-year period after surgery, and screened every six months, including cytology and colposcopy. Biopsy samples collected from LEEP were submitted to immunohistochemical analysis for p16 and Ki-67. The statistical analysis was performed using the Statistical Package for the Social Sciences software (SPSS, IBM-SPSS, Inc., Chicago, IL, US), with a significant p < 0.05. Results The biomarkers p16 and Ki-67, separately or combined, showed no relation to recurrence on the total analysis. However, evaluating specifically HG lesions, the positive expression (2+ and 3 + ) of p16/Ki-67 was associated with recurrence (0.010). In addition, p16 isolated was also more expressive in HG lesions (2+ and 3 + , p= 0.018), but it was unrelated to recurrence. Conclusion Proteins p16 and Ki-67, both isolated and combined, are not reliable primary markers for the recurrence of cervical lesions in the majority of LG lesions. However, analyzing only the group with prior diagnosis of HG lesions, the expressions of p16 and of p16/Ki-67 were associated with recurrence, and they may be useful in monitoring these cases.

Resumo Objetivo Avaliar as positividades dos biomarcadores p16 e Ki-67 em lesões de baixo grau (BG) ou de alto grau (AG), e relacioná-las com os fatores de risco e com a recidiva dessas lesões. Métodos Estudo retrospectivo caso-controle, com 86 pacientes com lesões de BG e AG, submetidas à conização por cirurgia de alta frequência entre 1999 e 2004. O grupo de controle foi constituído de 69 mulheres sem recidivas, e o grupo de estudo, de 17 pacientes que recidivaram. Todas as pacientes foram acompanhadas durante dois anos após a cirurgia, com controle a cada seis meses, incluindo citologia e colposcopia. As peças provenientes de cirurgia de alta frequência (CAF) foram submetidas a imunohistoquímica para p16 e Ki-67. A análise estatística foi realizada com o programa Statistical Package for the Social Sciences (SPSS, IBM-SPSS, Inc., Chicago, IL, EUA), com p significante quando < 0,05. Resultados Isoladamente ou em conjunto, p16 e Ki-67 não se relacionaram com as recidivas quando analisados na totalidade dos casos. Entretanto, avaliando especificamente as lesões de AG, a positividade (2+ e 3 + ) do conjunto p16/Ki-67 foi relacionada com recidiva (0,010). No mais, p16, isoladamente, foi também mais expresso nas lesões de AG (2+ e 3 + , p= 0,018), mas sem relação com recidiva. Conclusão Quando testadas na totalidade dos casos, as proteínas p16 e Ki-67, separadas ou em conjunto, se mostraram ineficientes como marcadores primários de recidiva de lesões precursoras. Entretanto, quando avaliadas somente no grupo diagnóstico prévio de lesão de AG, as expressões das proteínas p16 e p16/Ki-67 têm relação com a recidiva, e podem ser úteis no acompanhamento desses casos.

Evaluation of the p16 and Ki-67 Biomarkers as Predictors of the Recurrence of Premalignant Cervical Cancer Lesions after LEEP Conization.

Objective To evaluate the expressions of biomarkers p16 and Ki-67 in low-grade (LG) or high-grade (HG) lesions, and to relate them to risk factors and the recurrence of these lesions. Methods A retrospective case-control study of 86 patients with LG and HG lesions who underwent a loop electrosurgical excision procedure (LEEP) between 1999 and 2004. The control group was composed of 69 women with no recurrence, and the study group, of 17 patients with recurrence. All patients were followed-up over a two-year period after surgery, and screened every six months, including cytology and colposcopy. Biopsy samples collected from LEEP were submitted to immunohistochemical analysis for p16 and Ki-67. The statistical analysis was performed using the Statistical Package for the Social Sciences software (SPSS, IBM-SPSS, Inc., Chicago, IL, US), with a significant p ​​< 0.05. Results The biomarkers p16 and Ki-67, separately or combined, showed no relation to recurrence on the total analysis. However, evaluating specifically HG lesions, the positive expression (2+ and 3+) of p16/Ki-67 was associated with recurrence (0.010). In addition, p16 isolated was also more expressive in HG lesions (2+ and 3 + , p = 0.018), but it was unrelated to recurrence. Conclusion Proteins p16 and Ki-67, both isolated and combined, are not reliable primary markers for the recurrence of cervical lesions in the majority of LG lesions. However, analyzing only the group with prior diagnosis of HG lesions, the expressions of p16 and of p16/Ki-67 were associated with recurrence, and they may be useful in monitoring these cases.

Objetivo Avaliar as positividades dos biomarcadores p16 e Ki-67 em lesões de baixo grau (BG) ou de alto grau (AG), e relacioná-las com os fatores de risco e com a recidiva dessas lesões. Métodos Estudo retrospectivo caso-controle, com 86 pacientes com lesões de BG e AG, submetidas à conização por cirurgia de alta frequência entre 1999 e 2004. O grupo de controle foi constituído de 69 mulheres sem recidivas, e o grupo de estudo, de 17 pacientes que recidivaram. Todas as pacientes foram acompanhadas durante dois anos após a cirurgia, com controle a cada seis meses, incluindo citologia e colposcopia. As peças provenientes de cirurgia de alta frequência (CAF) foram submetidas a imuno-histoquímica para p16 e Ki-67. A análise estatística foi realizada com o programa Statistical Package for the Social Sciences (SPSS, IBM-SPSS, Inc., Chicago, IL, EUA), com p significante quando < 0,05. Resultados Isoladamente ou em conjunto, p16 e Ki-67 não se relacionaram com as recidivas quando analisados na totalidade dos casos. Entretanto, avaliando especificamente as lesões de AG, a positividade (2+ e 3 + ) do conjunto p16/Ki-67 foi relacionada com recidiva (0,010). No mais, p16, isoladamente, foi também mais expresso nas lesões de AG (2+ e 3 + , p = 0,018), mas sem relação com recidiva. Conclusão Quando testadas na totalidade dos casos, as proteínas p16 e Ki-67, separadas ou em conjunto, se mostraram ineficientes como marcadores primários de recidiva de lesões precursoras. Entretanto, quando avaliadas somente no grupo diagnóstico prévio de lesão de AG, as expressões das proteínas p16 e p16/Ki-67 têm relação com a recidiva, e podem ser úteis no acompanhamento desses casos.

In vitro culture of ovine mammary gland cells expressing beta-lactoglobulin and beta-casein / Células da glândula mamária ovina cultivadas in vitro expressam beta-lactoglobulina e beta-caseina

The expression of milk proteins in vitro is essential to exploit the mammary gland cells as a biological model. Enzymatic tissue disaggregation has been widely used to establish mammary cell culture, but its effect in long-term ovine mammary cell culture is not completely elucidated. This study aimed at comparing mechanical/enzymatic and mechanical dissociation methods to establish ovine mammary cell culture. We compared cellular differentiation induced by lactating ewe serum or fetal bovine serum based on the gene expression levels of milk proteins (beta-lactoglobulin, alpha s1-casein, and betacasein). Mechanically dissociated cells were positive immunostaining for cytokeratin 8.13, such as mammary epithelial cells. These cells are responsible for milk protein expression and they are low immunostaining for vimentin, mesenchymal marker. Mechanical/enzymatic dissociation cells were positive for vimentin. The fastest cell growth (cell/hour) was observed in the mechanical dissociation group cultured with 10% fetal bovine serum medium. Mechanically and mechanically/enzymatically derived cells were able to express beta-casein and beta-lactoglobulin, but not alpha s1-casein. The relative expression of beta-lactoglobulin was not affected by the tissue dissociation method or culture media, beta-casein relative expression was down regulated in mechanically dissociated cells cultured in the presence of lactating ewe serum, (P = 0.019). Beta-casein relative expression was also down regulated in mechanically/enzymatically dissociated cells cultured with fetal bovine serum (P = 0.021). In the present conditions, we conclude that mechanical dissociation followed by culture with 10% of fetal bovine serum was the most efficient method to induce milk proteins' mRNA expression by ovine mammary epithelial cells in vitro.(AU)

A expressão in vitro de proteínas do leite é essencial para explorar as células da glândula mamária como um modelo biológico. A desagregação tecidual via enzimática é amplamente utilizada para o estabelecimento cultivo de células mamárias. No entanto, seu efeito a longo prazo no cultivo de células da glândula mamária ovina ainda não é bem elucidado. Este estudo tem como objetivo comparar dois métodos de dissociação tecidual, mecânico/enzimático e mecânico, para estabelecer cultivo celular de glândula mamária ovina. A indução da diferenciação celular, por adição de soro de ovelha lactante ou soro fetal bovino, foi avaliada pelos níveis de expressão de proteínas do leite (beta-lactoglobulina, alpha s1-caseína e beta-caseína). Células mecanicamente dissociadas foram positivamente marcadas para a presença de citoqueratina 8.13, marcador para células epiteliais mamárias. Essas células são as responsáveis pela produção das proteínas do leite e são pouco marcadas para a presença de vimentina, marcador para células de origem mesenquimal. Já as células obtidas da dissociação mecânica/ enzimática foram positivamente marcadas para presença de vimentina. A maior velocidade de crescimento (células/hora) foi observado para o grupo com dissociação mecânica cultivado em meio com 10% de soro fetal bovino. As células obtidas tanto da dissociação mecânica quanto mecânica/enzimática foram capazes de expressar beta-lactoglobulina e beta-caseína, mas não alfa s1-caseína. A expressão relativa de beta-lactoglobulina não foi afetada pelo método de dissociação ou meio de cultivo. A expressão relativa da beta-caseína foi negativamente regulada para células mecanicamente dissociadas e cultivadas na presença de soro de ovelha lactante (P = 0,019). A expressão relativa da beta-caseína também foi negativamente regulada para células dissociadas de forma mecânica/enzimática e cultivadas com soro fetal bovino (P = 0,021). Nas condições do presente estudo, concluímos que o método de dissociação mecânica seguido pelo cultivo em meio com 10% de soro fetal bovino foi o método mais eficiente para induzir a expressão mRNA de proteínas do leite por células epiteliais mamárias ovinas in vitro.(AU)