Nuclear PKR in retinal neurons in the early stage of diabetic retinopathy in streptozotocin­induced diabetic rats.

Retinal neuron apoptosis is a key component of diabetic retinopathy (DR), one of the most common complications of diabetes. Stress due to persistent hyperglycaemia and corresponding glucotoxicity represents one of the primary pathogenic mechanisms of diabetes and its complications. Apoptosis of retinal neurons serves a critical role in the pathogenesis of DR observed in patients with diabetes and streptozotocin (STZ)­induced diabetic rats. Retinal neuron apoptosis occurs one month after STZ injection, which is considered the early stage of DR. The molecular mechanism involved in the suppression of retinal neuron apoptosis during the early stage of DR remains unclear. RNA­dependent protein kinase (PKR) is a stress­sensitive pro­apoptotic kinase. Our previous study indicated that PKR­associated protein X, a stress­sensitive activator of PKR, is upregulated in the early stage of STZ­induced diabetes. In order to assess the role of PKR in DR prior to apoptosis of retinal neurons, immunofluorescence and western blotting were performed to investigate the cellular localization and expression of PKR in the retina in the early stage of STZ­induced diabetes in rats. PKR activity was indirectly assessed by expression levels of phosphorylated eukaryotic translation initiation factor 2α (p­eIF2­α) and the presence of apoptotic cells in the retina was investigated by TUNEL assay. The findings revealed that PKR was localized in the nucleus of retinal ganglion and inner nuclear layer cells from normal and diabetic rats. To the best of our knowledge, the present study is the first to demonstrate nuclear localization of PKR in retinal neurons. Immunofluorescence analysis demonstrated that PKR was expressed in the nuclei of retinal neurons at 3 and 6 days and its expression was decreased at 15 days after STZ treatment. In addition, p­eIF2­α expression and cellular localization followed the trend of PKR, suggesting that this pro­apoptotic kinase was active in the nuclei of retinal neurons. These findings are consistent with the hypothesis that nuclear translocation of PKR may be a mechanism to sequester active PKR, thus preventing upregulation of cytosolic signalling pathways that induce apoptosis in retinal neurons. Apoptotic cells were not detected in the retina in the early stage of DR. A model was proposed to explain the mechanism by which apoptosis of retinal neurons by PKR is suppressed in the early stage of DR. The possible role of mitochondrial RNA (mtRNA) and Alu RNA in this phenomenon is also discussed since it was demonstrated that the cellular stress due to prolonged hyperglycaemia induces the release of mtRNA and transcription of Alu RNA. Moreover, it mtRNA activates PKR, whereas Alu RNA inhibits the activation of this protein kinase.

[Corrigendum] Intratumoral injection of PKR shRNA expressing plasmid inhibits B16-F10 melanoma growth.

It has been brought to our attention that there was an important omission of the word 'shRNA', which should have been featured in a couple of places in the above article. The authors incorrectly claimed that the RNA­dependent protein kinase (PKR) acts as a tumor suppressor; in fact, it is the PKR shRNA that would fulfil the role of being a tumor suppressor. Therefore, the text in the Abstract on p. 2267, line 14, should have read "...these findings suggested that PKR shRNA acts as a tumor suppressor"; likewise, on p. 2272, towards the end of the final sentence of the second paragraph of the Discussion, the text should have read "The results show that PKR silencing inhibits tumor growth, suggesting that PKR shRNA acts as a tumor supressor...". The authors regret that this error was not corrected before the article went to print, and they thank the reader concerned for drawing the matter to our attention. [the original article was published in the Oncology Reports 32: 2267­2273, 2014; DOI: 10.3892/or.2014.3410].

Genetic Polymorphism in the Promoter Region of Serotonin Transporter: Implications for Ethanol Abuse in Children and Adolescents.

OBJECTIVES: To provide a review of published literature regarding genetic polymorphism of serotonin transporter gene, named as 5-HTTLPR, and its potential role as a susceptibility marker for ethanol abuse in childhood and adolescence. METHODS: A literature review of several databases was conducted with the following keywords: 5-HTTLPR, children or adolescents or teenagers, susceptibility, alcohol or ethanol, abuse or misuse. RESULTS: Alcohol interacts with serotonergic synaptic transmission in several ways, and the reduced availability of serotonin transporters might foster brain dysfunction, driving to alcohol abuse. The initial use of ethanol in children and adolescents is determined primarily by environmental influences, whereas the establishment of drinking patterns is strongly controlled by genetic factors. Functional polymorphic variants in the promoter region of the 5-HTTLPR gene have age-dependent effects in alcohol abuse. This polymorphism, mapped to the 5' region of the SLC6A4, is a variable number of tandem repeats (VNTR) and involves a direct repeat of 20-23 base pairs GC-rich sequences, comprising a short (S) allele, consisting of 14 repeats, and a long (L) allele, with 16 repeats. Additional variants have been described, although their influences on childhood and adolescence ethanol use are not clear. CONCLUSION: The influence of the 5-HTTLPR allelic variants in children and adolescent misuse of alcohol might be considered for clinical management, preventing long-term behavior problem. Identifying genetic markers associated to the potential alcohol misuse or abuse could be useful in guiding management and formulating effective coping strategies.

OBJECTIFS: Offrir une revue de la littérature publiée sur le polymorphisme génétique du gène transporteur de la sérotonine, nommé 5-HTTLPR, et son rôle potentiel de marqueur de la susceptibilité à l'abus d'éthanol dans l'enfance et l'adolescence. MÉTHODES: Une revue de la littérature dans plusieurs bases de données a été menée à l'aide des mots clés suivants: 5-HTTLPR, enfants ou adolescents ou teenagers, susceptibilité, alcool ou éthanol, abus ou excès. RÉSULTATS: L'alcool interagit de plusieurs façons avec la transmission synaptique sérotoninergique, et la disponibilité réduite des transporteurs de la sérotonine peut favoriser une dysfonction cérébrale, qui mène à l'abus d'alcool. L'utilisation initiale d'éthanol chez les enfants et les adolescents est déterminée principalement par des influences environnementales, alors que l'établissement de modèles de consommation d'alcool est fortement contrôlé par des facteurs génétiques. Les variantes polymorphiques fonctionnelles de la région promotrice du gène 5-HTTLPR ont des effets selon l'âge sur l'abus d'alcool. Ce polymorphisme, localisé à la région 5' de SLC6A4, est un nombre variable de répétitions en tandem (NVRT) et implique une répétition directe de séquences de 20­23 paires de base riches en GC, comprenant un allèle court (C), consistant en 14 répétitions, et un allèle long (L), avec 16 répétitions. Les variantes additionnelles ont été décrites, bien que leurs influences sur l'utilisation d'éthanol dans l'enfance et l'adolescence ne soient pas définies. CONCLUSION: L'influence des variantes alléliques de 5-HTTLPR sur l'excès d'alcool chez les enfants et les adolescents pourrait être considérée pour la prise en charge clinique, et la prévention de problèmes de comportement à long terme, L'identification des marqueurs génétiques associés à l'excès ou l'abus d'alcool potentiel pourrait être utile pour guider la prise en charge et formuler des stratégies d'adaptation efficaces.

Knockdown of chemokine receptor CXCR4 gene by RNA interference: Effects on the B16-F10 melanoma growth.

The incidence of malignant melanoma has increased greatly in recent decades presenting a high mortality rate despite intensive efforts in this area of research. Recent studies indicate that the chemokine receptor 4 (CXCR4) plays a critical role in cancer. Thus, it has been reported that CXCL12 binding to CXCR4 initiates various downstream signaling pathways that result in a plethora of responses involved in cell proliferation and metastasis. Recently, we demonstrated that CXCR4 silencing by RNA interference (RNAi) significantly reduced the number of pulmonary metastatic nodules. In the present study, we examined the effect of the intratumoral injection of CXCR4 short hairpin (shRNA) expressing plasmids on the growth of B16­F10 melanoma in mice. In vitro transfection of these tumor cells with CXCR4 shRNA expressing plasmid (CXCR4 shRNA) significantly reduced the levels of CXCR4 mRNA (85%) and CXCR4 protein (70%) compared with the control. We showed that the tumor growth was significantly reduced (66%) in mice inoculated with transfected B16­F10 melanoma cells when compared with the control group. We also found that the intratumoral injection of CXCR4 shRNA expressing plasmids results in a significant inhibition (70%) of B16­F10 melanoma growth. This finding supports the hypothesis that a direct administration of RNAi­based therapeutics into the target tumor is a promising approach for overcoming the hurdles of systemic delivery. The present study is the first demonstration that CXCR4 plays a critical role in B16-F10 melanoma growth. Currently there is great interest in the development of antagonists for therapeutic targeting CXCR4 expression. Considering our results and the fact that CXCR4 is highly conserved between human and mouse, this experimental model of cancer may be useful for the discovery of new CXCR4 antagonists with clinical implications.

In vivo knockdown of CXCR4 using jetPEI/CXCR4 shRNA nanoparticles inhibits the pulmonary metastatic potential of B16­F10 melanoma cells.

Metastasis is a key factor that limits survival in the majority of patients with cancer. Thus, numerous efforts have been made to elucidate the molecular mechanisms involved in this phenomenon. B16­F10 melanoma cells have been demonstrated to be highly metastatic to the lungs in mice. The aim of the current study was to investigate the role of CXC motif chemokine receptor 4 (CXCR4) in the metastatic potential of B16­F10 melanoma cells in mice. In vitro transfection of B16­F10 tumor cells with CXCR4 short hairpin RNA (shRNA) expressing plasmids (CXCR4 shRNA) significantly reduced the expression levels of CXCR4 mRNA (80%) and protein (68%), compared with the control. In addition, these results demonstrated that pulmonary metastasis was significantly inhibited (85%) in mice inoculated with CXCR4 shRNA­transfected B16­F10 melanoma cells. The polycation­based nanoparticle (jetPEI) was used to investigate the effect of CXCR4 knockdown in vivo on the metastatic potential of B16­F10 melanoma cells. The number of pulmonary metastatic nodules was significantly reduced (50%) in animals that received a retro­orbital injection of jetPEI­CXCR4­1 shRNA. The current study demonstrated that CXCR4 serves a role in the metastatic potential of B16­F10 melanoma cells. Currently there is a great interest in the development of antagonists for the therapeutic targeting of CXCR4 expression. Taking the results of the current study and the fact that CXCR4 is highly conserved between humans and mice into account, this experimental model of metastasis with B16­F10 melanoma cells may aid in the discovery of CXCR4 antagonists with clinical implications.

Intratumoral injection of PKR shRNA expressing plasmid inhibits B16-F10 melanoma growth.

The RNA-dependent protein kinase (PKR) is a serine/threonine kinase that is involved in the regulation of important cell processes such as apoptosis, signal transduction, cell proliferation and differentiation. However, the role played by PKR in cancer remains controversial. RNA interference (RNAi) has currently become an important technique in understanding gene function. Previously, we showed that PKR shRNA downregulates PKR expression in B16-F10 melanoma cells and reduces the metastatic potential of these tumor cells. In the present study, we examined the effect of the intratumoral injection of PKR shRNA­expressing plasmid on the growth of B16-F10 melanoma in mice. The results showed that this treatment significantly reduced tumor growth. Thus, these findings suggested that PKR acts as a tumor suppressor, a finding that is consistent with our previous study on the experimental model of metastasis. Moreover, the results suggested that this effect may be mediated by the transcription factor NF-κB. The present study confirmed the hypothesis that the direct administration of RNAi-based therapeutics in the target tumor is a promising approach for overcoming the obstacles of systemic delivery. The results also suggested that the intratumoral injection of PKR shRNA­expressing vector is a novel therapeutic approach for human solid tumors such as cutaneous melanoma and breast cancer, since PKR is overexpressed in these tumors.

Expression and cellular localization of microRNA-29b and RAX, an activator of the RNA-dependent protein kinase (PKR), in the retina of streptozotocin-induced diabetic rats.

PURPOSE: The apoptosis of retinal neurons plays a critical role in the pathogenesis of diabetic retinopathy (DR), but the molecular mechanisms underlying this phenomenon remain unclear. The purpose of this study was to investigate the cellular localization and the expression of microRNA-29b (miR-29b) and its potential target PKR associated protein X (RAX), an activator of the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway, in the retina of normal and diabetic rats. METHODS: Retinas were obtained from normal and diabetic rats within 35 days after streptozotocin (STZ) injection. In silico analysis indicated that RAX is a potential target of miR-29b. The cellular localization of miR-29b and RAX was assessed by in situ hybridization and immunofluorescence, respectively. The expression levels of miR-29b and RAX mRNA were evaluated by quantitative reverse transcription PCR (qRT-PCR), and the expression of RAX protein was evaluated by western blot. A luciferase reporter assay and inhibition of endogenous RAX were performed to confirm whether RAX is a direct target of miR-29b as predicted by the in silico analysis. RESULTS: We found that miR-29b and RAX are localized in the retinal ganglion cells (RGCs) and the cells of the inner nuclear layer (INL) of the retinas from normal and diabetic rats. Thus, the expression of miR-29b and RAX, as assessed in the retina by quantitative RT-PCR, reflects their expression in the RGCs and the cells of the INL. We also revealed that RAX protein is upregulated (more than twofold) at 3, 6, 16, and 22 days and downregulated (70%) at 35 days, whereas miR-29b is upregulated (more than threefold) at 28 and 35 days after STZ injection. We did not confirm the computational prediction that RAX is a direct target of miR-29b. CONCLUSIONS: Our results suggest that RAX expression may be indirectly regulated by miR-29b, and the upregulation of this miRNA at the early stage of STZ-induced diabetes may have a protective effect against the apoptosis of RGCs and cells of the INL by the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway.

Measurement of cytotoxic activity in experimental cancer.

Several tumor cell systems have been developed for investigating the cytotoxic activity of different substances. The Ehrlich ascytic tumor (EAT) has been studied extensively and used to increase our comprehension of immunotherapy (Yamamoto and Naraparaju. Cancer Res 57:2187-2192, 1997). In this study, we evaluated the ability of an enzymatic method to assess cytotoxic tumor activity in vitro. To prepare cytotoxic cells, lymphoid cells isolated from the lymph nodes of C57BL/6 mice were activated with interleukin-2 (IL-2). The cytotoxic activity to EAT cells was assessed by an enzymatic test using lactate dehydrogenase (LDH), an enzyme widely distributed in mammalian tissues which, in the presence of NAD/NADH, converts lactate to pyruvate. IL-2 treatment of lymph node cells induced a greater percentage of lysis (75.47%) than effector cells that were not treated with IL-2 (42.38%). This suggests that IL-2 directly activates effector cells and not tumor cells. The results demonstrate that the LDH release-measurement method can be utilized to assay cytotoxic cell-mediated activity in which murine tumor cells act as the target. Levels of IL-2 appear to have a direct correlation to cellular immunity and treatment with these substances may strengthen immune function and decrease the pace of disease development.

A importância dos exames laboratoriais no monitoramento da doença de Graves e artrite reumatóide / The laboratorial tests and its importance in the clinical course of therapeutic monitoring in Graves' disease and rheumatoid arthritis

A auto-imunidade se manifesta quando ocorre uma falha nos mecanismos de auto-tolerância responsáveis pela discriminação entre o próprio e o não próprio, desencadeando uma resposta imune adaptativa específica contra os auto-antígenos. A doença de Graves (DG), uma doença auto-imune associada com atividade excessiva da tireóide, podendo vir associada a outras doenças auto-imunes endócrinas, como Diabetes mellitus tipo 1, e não endócrinas como Lupus Eritematoso Sistêmico (LES) ou Artrite Reumatóide (AR). As doenças auto-imunes são caracterizadas pela etiologia multifatorial, onde fatores genéticos, endócrinos, imunológicos, infecciosos, ambientais e emocionais contribuem para o desencadeamento e agravamento dos processos lesivos. Tem-se pesquisado cada vez mais a influência de fatores genéticos sobre o desenvolvimento de doenças auto-imunes e é neste contexto que se observa a forte associação do HLA-DR, tanto com DG como com AR. O presente trabalho tem como objetivo descrever as alterações laboratoriais observadas em uma paciente do sexo feminino, 22 anos, branca, com o diagnóstico das duas doenças auto-imunes de evolução crônica, a DG e AR, correlacionar os resultados laboratoriais com os sinais e sintomas clínicos apresentados e avaliar os exames laboratoriais realizados para o monitoramento clínico e terapêutico.