Unveiling the angiogenic effects of cannabinoids: Enhancers or inhibitors?

Cannabinoids are compounds found in the cannabis sativa plant. Cannabinoids, such as delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), have potential therapeutic benefits in various medical conditions. Some can activate the cannabinoid receptors type-1 and -2 (CB1 and CB2), that are part of the endocannabinoid system (ECS), alongside the endocannabinoids and their metabolic enzymes. The ECS regulates physiological and cognitive processes and is a potential therapeutic target for a wide range of health conditions like chronic pain, anxiety, and neurodegenerative diseases. Synthetic cannabinoids, are associated with serious health risks, including addiction, psychosis, and death. Nonetheless, some of these molecules are also being explored for pharmacological applications. Angiogenesis is the process of forming new blood vessels from existing ones, crucial for growth, repair, and tissue maintenance. Dysregulation of this process is associated with several diseases, including cancer, diabetic retinopathy and reproductive pathologies, such as preeclampsia. Recent data suggests that cannabinoids may affect angiogenesis. Here, we reviewed their impact on pro-angiogenic factors, extracellular matrix enzymes and inhibitors, immune-inflammatory responses, angiogenic pathways and functional assays, focusing on the main compounds for each cannabinoid class: THC and CBD for phytocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG) for endocannabinoids and WIN-55, JWH-133, XLR-11, LYR-7 and LYR-8, for the synthetic cannabinoids. Despite conflicting reports about the actions of phytocannabinoids and endocannabinoids on angiogenesis, the ability to modulate the angiogenic process is undoubtedly confirmed. This may open a new therapeutical route for angiogenesis-related pathologies. In addition, synthetic cannabinoids present anti-angiogenic actions in several cell models, hinting their potential as anti-angiogenic drugs.

Colchicine reduces the activation of NLRP3 inflammasome in COVID-19 patients.

OBJECTIVE: To evaluate whether colchicine treatment was associated with the inhibition of NLRP3 inflammasome activation in patients with COVID-19. METHODS: We present a post hoc analysis from a double-blinded placebo-controlled randomized clinical trial (RCT) on the effect of colchicine for the treatment of COVID-19. Serum levels of NOD-like receptor protein 3 (NLRP3) inflammasome products-active caspase-1 (Casp1p20), IL-1ß, and IL-18-were assessed at enrollment and after 48-72 h of treatment in patients receiving standard-of-care (SOC) plus placebo vs. those receiving SOC plus colchicine. The colchicine regimen was 0.5 mg tid for 5 days, followed by 0.5 mg bid for another 5 days. RESULTS: Thirty-six patients received SOC plus colchicine, and thirty-six received SOC plus placebo. Colchicine reduced the need for supplemental oxygen and the length of hospitalization. On Days 2-3, colchicine lowered the serum levels of Casp1p20 and IL-18, but not IL-1ß. CONCLUSION: Treatment with colchicine inhibited the activation of the NLRP3 inflammasome, an event triggering the 'cytokine storm' in COVID-19. TRIAL REGISTRATION NUMBERS: RBR-8jyhxh.

Chicken embryo: a useful animal model for drug testing?

The use of chicken embryos (CEs) as an in vivo experimental model for different pharmaceutical purposes is not a novelty. However, in recent years, the number of reports employing CE to evaluate several parameters, such as the toxicity and efficacy of drugs and/or nanosystems, has increased. Therefore, this review discusses the relevance of CE for drug testing, emphasizing the inoculation routes and the embryonic stages. The challenges to be overcome, as well as some practical recommendations to allow CE to be more explored as a promising in vivo model in drug analyses, are also highlighted.

The endocannabinoids anandamide and 2-arachidonoylglycerol modulate the expression of angiogenic factors on HTR8/SVneo placental cells.

The interest on the endocannabinoid system (ECS) in human reproduction has grown due to its involvement in placenta development, which led to growing concerns over pregnant cannabis consumer's impact on pregnancy outcome. The endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) modulate placental trophoblast proliferation and apoptosis. However, their role on other placentation events such as angiogenesis and invasion are unknown. Using the human extravillous trophoblast HTR-8/SVneo cells, a well-accepted model of first trimester extravillous trophoblast (EVT), this study aims to investigate whether AEA and 2-AG can modulate the expression of angiogenesis- and invasion-related factors. Transcript analysis of angiogenic factors of the vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP) protein family demonstrated the ability of AEA to increase VEGF-C and VEGFR3 expression via cannabinoid receptors CB1 and CB2 while the placental growth factor (PlGF) was increased through CB1. Moreover, an increase in VEGFR1, sFLT1, VEGFR2, MMP-2 and TIMP-1 independent of cannabinoid receptor activation was verified. However, 2-AG only increased PlGF transcript through CB1/CB2 activation. Both endocannabinoids stimulated HTR8/SVneo endothelial-like tube formation. As for the wound healing assay, only 2-AG was able to increase the percentage of wound closure. Moreover, the data demonstrated that both AEA and 2-AG, via cannabinoid receptors, activated the STAT3 signaling pathway. Distinct effects were observed on transcription factor HIF-1α and AKT phosphorylation that decreased with both endocannabinoids. Although different angiogenic and migration factors are affected the results obtained in this work showcase once more the ability of the endocannabinoids to modulate key processes in placental physiology.

Descriptive electromyography signals analysis of equine longissimus dorsi, rectus abdominis and gluteus medius muscles during maneuvers used to activate the core / [Análise descritiva do sinal eletromiográfico dos músculos longuíssimo dorsal, reto abdominal e glúteo médio de equinos durante a realização de manobras utilizadas para ativação do core]

Maneuvers to activate the equine's core can make a difference in their physical and psychic health. Although these activities are recommended and practiced, there is little research proving their effectiveness. This article aims to describe, through surface electromyography, the occurrences, durations and sequences activity of longissimus dorsi, rectus abdominis and gluteus medius during thoracolumbar flexion (TLF), lumbar and lumbosacral flexion (LLSF), global flexion (GF), which is the combination of TLF and LLSF, and tail traction (TT). Seven healthy adult horses of three different breeds performed five repetitions of these movements for five seconds (sec). Electromyographic activity was captured with non-invasive superficial sensors positioned in the skin regions covering these muscles. The sequence was performed once per animal, muscle activity captured by surface electromyography, data from two replicates of each animal were selected, analyzed on matLab software and data tabulation were described during each maneuver. These maneuvers provoked punctual and transient activation of muscles mentioned above, confirming the ability to activate equine core muscles. However, responses were not standardized, which means there were variations of occurrence, duration and sequence, suggesting that for practical application of those maneuvers it is necessary to perform more repetitions with longer durations to activate more muscles.(AU)

Manobras para ativação da musculatura do core equino podem ser diferenciais para saúde física e psíquica dos animais, sendo recomendadas e praticadas, mas existem poucas pesquisas comprovando a eficácia delas. Este artigo tem como objetivo descrever, por meio da eletromiografia de superfície, as ocorrências, as durações e as sequências temporais da atividade muscular do longuíssimo dorsal, do reto abdominal e do glúteo médio durante a realização das manobras de flexão toracolombar, flexão lombar e lombossacra, flexão global (toracolombar e lombossacra) e tração de cauda. Para isso, sete equinos adultos e hígidos de três raças realizaram esses quatro movimentos clássicos para ativar o core equino, com cinco repetições e manutenção do estímulo reflexivo por cinco segundos. Durante a realização, a atividade eletromiográfica foi capturada com a utilização de sensores superficiais posicionados de forma não invasiva em regiões cutâneas referentes a cada músculo. O protocolo completo de manobras foi realizado uma vez por cada animal enquanto a atividade muscular era capturada. Posteriormente, duas repetições de cada animal foram triadas e submetidas ao software matLab para análise. Com base na tabulação dos dados, foram descritas as variáveis eletromiográficas de presença ou ausência de ativação muscular, a duração média dos picos eletromiográficos superiores ao RMS (root mean square) e a sequência da atividade muscular observada durante cada manobra. Essas manobras provocaram ativações pontuais e transitórias nos três músculos, o que confirma a capacidade de excitar músculos do core equino. Contudo, as respostas não foram padronizadas, sugerindo que, na prática dessa atividade, devem-se realizar mais repetições com durações superiores a cinco segundos, a fim de se buscarem maiores ativações.(AU)

The major endocannabinoid anandamide (AEA) induces apoptosis of human granulosa cells.

The endocannabinoid system (ECS) plays a crucial role in human reproduction. Changes in anandamide (AEA) levels affect reproductive events and has already been suggested as biomarker of reproductive potential of male and female gametes. Although cannabinoid-receptor 1 (CB1) was already identified in human granulosa cells (hGCs) the ECS was not characterized on granulosa cells line COV434 nor the effects of AEA on GCs viability and function depicted. Therefore, the aim of this study was to characterize the ECS elements and explore the effects of AEA on both COV434 and hGCs. Our results revealed that hGCs express the full enzymatic machinery responsible for AEA metabolism as well as cannabinoid receptors. In addition, AEA induced a reduction in both COV434 and hGCs viability in a concentration and time-dependent manner. Nevertheless, the effects of AEA in cell viability was independent of either CB1 or CB2 receptors. There was no ROS release in both cell models; however, AEA induced morphological changes, presenting chromatin condensation at 72 h, and variation on mitochondrial membrane potential. Moreover, AEA induced an increase in caspase -3/-7 activities in both cell models, but in hGCs there was also an increase in caspase 8 activity. This study supports the idea that ECS balance is crucial for folliculogenesis and oocyte quality as dysregulated AEA levels may compromise female fertility.

HPV infection as a risk factor for atherosclerosis: A connecting hypothesis.

Atheromatous plaques occurring in large arteries are common and life-threatening lesions. Multiple factors are involved in the pathogenesis of atheromatous plaques, such as hyperlipidaemia and hypercholesterolaemia, high blood pressure and chronic systemic inflammation. Recent findings have suggested that infection with high-risk human papillomavirus (HPV) may increase the risk of developing atheromatous plaques. However, HPV is considered a tissue-specific virus with a strong tropism towards squamous epithelial cells, and the mechanisms whereby it may promote the development of atheromas remain unclear. Here, we propose a connecting hypothesis to explain the possible causative role of HPV on atheroma development. We hypothesize that HPV infection may promote atheroma formation in infected patients by enhancing systemic inflammation or by directly targeting blood vessels via nucleic acids carried by extracellular vesicles such as exosomes. The pro-inflammatory effects of HPV and the release of extracellular vesicles by HPV-transformed cells are well documented in scientific literature. Possible experimental approaches to test this hypothesis are also discussed, especially experiments employing transgenic mice bearing HPV16 transgenes. If correct, this hypothesis would have major implications for the prevention of cardiovascular diseases, especially due to the preventable nature of HPV infection through vaccination.

The fundamental role of the endocannabinoid system in endometrium and placenta: implications in pathophysiological aspects of uterine and pregnancy disorders.

BACKGROUND: The endocannabinoid system (ECS) consists of the cannabinoid receptors CB1 and CB2, the main endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) and their metabolic enzymes N-acylphosphatidylethanolamine-specific phospholipase D, fatty acid amide hydrolase, diacylglycerol lipase and monoacylglycerol lipase. This system is involved in the modulation of essential physiological processes. Its role in the reproductive system has become significantly important in recent years, given its major role in events such as gametogenesis, decidualisation, implantation and placentation. OBJECTIVE AND RATIONALE: In this paper, we review the literature and summarize the role of the ECS elements in reproduction and their potential as early markers for diagnosis of reproductive disorders or as pharmacological targets for treatment. SEARCH METHODS: Original research and review papers published from 1964 to June 2019 were selected in terms of relevance, reliability and quality by searching PubMed, MEDLINE and Web of Science, using the following search terms: endocannabinoid system and endometriosis; endocannabinoid system and ectopic pregnancy; endocannabinoid system and miscarriage; endocannabinoid system and pre-eclampsia; endocannabinoid system and endometrial cancer; endocannabinoid system and reproduction; endocannabinoid, endometrium; placenta; N-acylethanolamines; anandamide; 2-arachidonoylglycerol; and cannabinoids. OUTCOMES: This review demonstrates relevant information concerning ECS alterations in endometriosis, ectopic pregnancy, miscarriage, pre-eclampsia and endometrial cancer. We highlight the importance of the endocannabinoids in endometrial and placental physiology and pathophysiology, from studies in vitro and in vivo and in clinical observations. The most studied of the endogenous cannabinoids is AEA. The levels of AEA were increased in plasma of patients with endometriosis and miscarriage, as well as in the fallopian tube of women with ectopic pregnancy and in endometrial biopsies of endometrial cancer. Changes in the pattern of expression of the cannabinoid receptor CB1 were also observed in endometrial biopsies of endometriosis, fallopian tube and decidua of patients with ectopic pregnancy and pre-eclamptic placenta. Moreover, alterations in CB2 expression have been reported in association with endometrial cancer. In general, studies on the cannabinoid signalling through CB2 and on the biological activities of the other major endocannabinoid, namely 2-AG, as well as its metabolic enzymes are scarce and avidly required. WIDER IMPLICATIONS: The pathophysiological mechanisms involved in the described endometrial and placental pathologies are still unclear and lack the means for an early diagnosis. Based on current evidence, though alterations in ECS are demonstrated at tissue level, it is difficult to associate plasmatic changes in AEA with specific endometrial and placental diseases. Thus, pairing alterations in AEA levels with 2-AG and/or other endocannabinoid-like molecules may provide more accurate and early diagnoses. In addition, patients may benefit from new therapies that target the ECS and endocannabinoid signalling.

Decidual NK cell-derived conditioned medium from miscarriages affects endometrial stromal cell decidualisation: endocannabinoid anandamide and tumour necrosis factor-α crosstalk.

STUDY QUESTION: What are the effects of endocannabinoid anandamide (AEA) in uterine natural killer (unK) cells from miscarriage decidua, regarding their cytokine profile and endometrial stromal cell (ESC) crosstalk? SUMMARY ANSWER: uNK-conditioned media from miscarriage samples present high TNF-α levels which inhibit ESC decidualisation. WHAT IS KNOWN ALREADY: AEA plasma levels are higher in women who have suffered a miscarriage. Moreover, AEA inhibits ESC proliferation and differentiation, although the levels and impact on the uNK cell cytokine profile at the feto-maternal interface remain elusive. STUDY DESIGN, SIZE, DURATION: This laboratory-based study used human primary uNK cells which were isolated from first-trimester decidua (gestational age, 5-12 weeks) derived from 8 women with elective pregnancy termination and 18 women who suffered a miscarriage. PARTICIPANTS/MATERIALS, SETTING, METHODS: The first-trimester placental tissues were assayed for AEA levels by UPLC-MS/MS and respective enzymatic profile by western blot. The uNK cells were isolated and maintained in culture. The expression of angiogenic markers in uNK cells was examined by quantitative PCR (qPCR). The uNK-conditioned medium was analysed for IFN-γ, TNF-α and IL-10 production by enzyme-linked immunosorbent assay, and the impact on ESC differentiation was assessed by measuring decidual markers Prl, Igfbp-1 and Fox01 mRNA expression using qPCR. MAIN RESULTS AND THE ROLE OF CHANCE: AEA levels were higher in miscarriage decidua compared with decidua from elective terminations. The uNK cell-conditioned medium from the miscarriage samples exhibited high TNF-α levels and interfered with the decidualisation of ESCs. Exacerbated inflammation and elevated TNF-α levels at the feto-maternal interface may trigger AEA signalling pathways that, in turn, may impact decidualisation and the angiogenic ability of uNK cells. LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Primary uNK cell responses are based on a simple in vitro model. Thus, in complex microenvironments, such as the feto-maternal interface, the mechanisms may not be exactly the same. Also, the inflammatory events of miscarriage that, in this study, have happened prior to processing of the samples may cause different responses to that observed. In addition, the magnitude of the inflammatory response, required to trigger the AEA pathways that impact decidualisation and the uNK angiogenic ability in vivo, is still unclear. WIDER IMPLICATIONS OF THE FINDINGS: The endocannabinoid AEA is a modulator of reproductive competence. AEA not only may contribute to neuroendocrine homeostasis but also can take part in uterine changes occurring during early pregnancy. STUDY FUNDING/COMPETING INTEREST(S): The work was supported by UID/MULTI/04378/2019 with funding from Fundação para a Ciência e a Tecnologia (FCT)/MCTES through national funds and PORTUGAL 2020 Partnership Agreement, NORTE-01-0145-FEDER-000024. S.C. Cunha acknowledges FCT for the IF/01616/2015 contract. There are no conflicts of interest.

Thermographic assessment of saddles used in Mangalarga Marchador horses / Avaliação termográfica de selas utilizadas em cavalos da raça Mangalarga Marchador

Poor saddle-fitting is one of the main causes of back pain in horses. Mangalarga Marchador is a popular breed in Brazil, being used mainly for pleasure riding and sports. This study aimed to thermographically assess saddles used in horses of this breed. Thermographic images were obtained from 18 saddles of animals from different categories during a Mangalarga Marchador National Exposition. The evaluation was based on three parameters: contact area symmetry, dorsal midline interaction and total skin contact area (25%, 50%, 75% or 100%). Contact area asymmetry was observed in 83.3% of saddles. Dorsal midline interaction was observed with the same frequency. Only 22.2% of saddles assessed in the present study had panels with contact areas greater than 50%. Based on the results of this essay it can be concluded that thermography is a useful tool for the evaluation of saddle contact area with the back of horses and that there is a high frequency of fitting unconformities in saddles used in Mangalarga Marchador horses.(AU)

O ajuste inadequado da sela é um dos principais causadores de lombalgias em equinos. A raça Mangalarga Marchador está entre as mais populares do Brasil, sendo muito utilizada para cavalgadas e prática de esportes. O presente estudo teve como objetivo avaliar termograficamente as selas utilizadas em cavalos dessa raça. Para tal, foram realizadas imagens termográficas de 18 selas de animais de diversas categorias durante uma Exposição Nacional do Cavalo Mangalarga Marchador. A avaliação foi realizada baseando-se em três parâmetros: simetria da área de contato, interação com a linha média dorsal e área total de contato com a pele (25%, 50%, 75% ou 100%). Em 83,3% das selas avaliadas foi observada assimetria da área de contato. A interação com a linha média dorsal foi observada com a mesma frequência. Apenas 22,2% das selas avaliadas no presente estudo tinham suadouros com área de contato maior que 50%. Com base nos resultados obtidos neste trabalho, conclui-se que a termografia é uma ferramenta útil na avaliação do contato das selas com o dorso dos cavalos e que existe alta frequência de inconformidades no ajuste de selas utilizadas na raça Mangalarga Marchador.(AU)

Effects of cannabis tetrahydrocannabinol on endocannabinoid homeostasis in human placenta.

Cannabis use has become a hot topic in several countries due to the debate about its legalization for medical purposes. However, data are limited regarding adverse events, safety and potential impact on reproductive health. Cannabis consumption during pregnancy has been associated with gestational disorders such as preterm birth, intrauterine growth restriction, low birth weight and increased risk of miscarriage, though the underlying biochemical mechanisms are still unknown. Given that the endocannabinoid system (ECS) is involved in several reproductive processes, we tested the hypothesis that the negative outcomes may result from the impact on the ECS homeostasis caused by the main psychoactive compound of cannabis, Δ9-tetrahydrocannabinol (THC). We demonstrate that THC (10-40 µM) impairs placental endocannabinoid system by disrupting the endocannabinoid anandamide (AEA) levels and the expression of AEA synthetic and degrading enzymes N-arachidonoylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD) and fatty acid amide hydrolase (FAAH), respectively. Although, no alterations in cannabinoid receptors CB1 and CB2 expression were observed. Thus, long-term local AEA levels are associated with a shift in the enzymatic profile to re-establish ECS homeostasis. In chronic cannabis users, high AEA levels in placenta may disturb the delicate balance of trophoblast cells turnover leading to alterations in normal placental development and foetal growth.

Laurus nobilis (laurel) aqueous leaf extract's toxicological and anti-tumor activities in HPV16-transgenic mice.

Cancers induced by human papillomavirus (HPV) infection remain a significant public health threat, fueling the study of new therapies. Laurel (Laurus nobilis) compounds and extracts recently showed in vitro activity against HPV-transformed cell lines. This work aims to evaluate the in vivo efficacy and hepatic toxicity of a laurel extract in a transgenic mouse model of HPV16-induced cancer. The extract was administered in drinking water (20 mg per animal per day) for three consecutive weeks, using four experimental groups (n = 10) (group I: HPV16-/- without treatment, group II: treated HPV16-/-, group III: HPV16+/- without treatment and group IV: treated HPV16+/-). Following the treatment period, animals were sacrificed and skin samples were used to classify skin lesions histologically. Toxicological parameters included hematological and biochemical blood markers, splenic and hepatic histology and hepatic oxidative stress. The extract did not prevent the progression of HPV16-induced cutaneous lesions in this model. The treated wild-type animals showed mild hepatitis, while transgenic animals suffered weight loss. However, there were no changes concerning hematological, biochemical and hepatic oxidative stress markers.

The association between extended spectrum beta-lactamase (ESBL) and ampicillin C (AmpC) beta-lactamase genes with multidrug resistance in Escherichia coli isolates recovered from turkeys in Brazil.

1. The aim of this study was to analyse the association between Escherichia coli isolates recovered from turkeys and the expression of beta-lactamase genes, such as extended spectrum beta-lactamase (ESBL) and ampicillin class C (AmpC). The phenotype of the resistance profile was examined using the association between amoxicillin and ceftiofur resistance. 2. Results showed that 84% from the turkey isolates harboured 4 or 5 genes associated with the CoIV plasmid. In an antibiogram test, 82% of the isolates were multidrug-resistant, the highest levels of resistance being against erythromycin (99%) and amoxicillin (76.1%). ESBL-positive groups were 31% positive for the ctx-m-2 gene, 6.8% were positive for ctx-m-8 and 70% harboured the tem wild gene. 3. All positive isolates from the AmpC beta-lactamase-positive group harboured the cmy-2 gene. The presence of the cmy-2 gene was associated with both the CTX-group genes and resistance to ceftiofur. 4. There was a high prevalence of avian pathogenic E. coli in suspected cases of colibacillosis in turkeys and a high antimicrobial resistance index. The results highlighted the risk of ceftiofur resistance and the presence of both ESBL and AmpC beta-lactamase E. coli in the turkey production chain.

Searching for the best real-time RT-PCRs to detect Zika virus infections: the importance of comparing several protocols.

Clinical manifestations of Zika, dengue, and chikungunya virus infections are very similar, making it difficult to reach a diagnosis based only on clinical grounds. In addition, there is an intense cross-reactivity between antibodies directed to Zika virus and other flaviviruses, and an accurate Zika diagnosis is best achieved by real-time RT-PCR. However, some real-time RT-PCR show better performance than others. To reach the best possible Zika diagnosis, the analytic sensitivity of some probe-based real-time RT-PCR amplifying Zika virus RNA was evaluated in spiked and clinical samples. We evaluated primers and probes to detect Zika virus, which had been published before, and tested sensitivity using serum spiked and patient samples by real-time RT-PCR. When tested against spiked samples, the previously described primers showed different sensitivity, with very similar results when samples from patients (serum and urine) were analyzed. Real-time RT-PCR designed to amplify Zika virus NS1 showed the best analytical sensitivity for all samples.

Ozone therapy prevents the onset of dysplasia in HPV16-transgenic mice-A pre-clinical efficacy and safety analysis.

Infection with high-risk human papillomavirus (HPV), most often HPV16, is associated with the development of anogenital and oropharyngeal cancers. Recently, ozone therapy was reported to have considerable efficacy against rabbit VX2 tumors, induced by the cottontail rabbit papillomavirus. The present study aims to determine whether similar results can be obtained in HPV16-transgenic mice, possibly paving the way for new therapeutic options against HPV-induced cancers. HPV16-transgenic and wild-type, female, 20 weeks-old mice were injected intraperitoneally with medical O3/O2 (80░mL/kg, at O3 50░µg/mL), once a day, for 5 consecutive days. The animals were sacrificed at 25 weeks-old, and skin samples were analyzed histologically to study tumour progression. Blood and internal organ samples were used to study toxicological parameters. 85.7% of untreated transgenic mice showed dysplastic skin lesions, compared with 28.6% of O3-treated mice. This was associated with a marked reduction of dermal inflammation associated with those lesions. No significant changes were observed in any toxicological parameters. These preliminary results support the hypothesis that O3 therapy is effective against papillomavirus-induced lesions, particularly against those induced by the most common high-risk virus, HPV16. Further studies are needed to confirm the mechanisms underlying these effects.

Cannabinoid-induced cell death in endometrial cancer cells: involvement of TRPV1 receptors in apoptosis.

Among a variety of phytocannabinoids, Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are the most promising therapeutic compounds. Besides the well-known palliative effects in cancer patients, cannabinoids have been shown to inhibit in vitro growth of tumor cells. Likewise, the major endocannabinoids (eCBs), anandamide (AEA) and 2-arachidonoylglycerol (2-AG), induce tumor cell death. The purpose of the present study was to characterize cannabinoid elements and evaluate the effect of cannabinoids in endometrial cancer cell viability. The presence of cannabinoid receptors, transient receptor potential vanilloid 1 (TRPV1), and endocannabinoid-metabolizing enzymes were determined by qRT-PCR and Western blot. We also examined the effects and the underlying mechanisms induced by eCBs and phytocannabinoids in endometrial cancer cell viability. Besides TRPV1, both EC cell lines express all the constituents of the endocannabinoid system. We observed that at concentrations higher than 5 µM, eCBs and CBD induced a significant reduction in cell viability in both Ishikawa and Hec50co cells, whereas THC did not cause any effect. In Ishikawa cells, contrary to Hec50co, treatment with AEA and CBD resulted in an increase in the levels of activated caspase -3/-7, in cleaved PARP, and in reactive oxygen species generation, confirming that the reduction in cell viability observed in the MTT assay was caused by the activation of the apoptotic pathway. Finally, these effects were dependent on TRPV1 activation and intracellular calcium levels. These data indicate that cannabinoids modulate endometrial cancer cell death. Selective targeting of TPRV1 by AEA, CBD, or other stable analogues may be an attractive research area for the treatment of estrogen-dependent endometrial carcinoma. Our data further support the evaluation of CBD and CBD-rich extracts for the potential treatment of endometrial cancer, particularly, that has become non-responsive to common therapies.

Salmonella Gallinarum field isolates and its relationship to vaccine strain SG9R.

1. The aim of the present study was to determine if the 9R-strain of the Salmonella Gallinarum live vaccine was responsible for having fowl typhoid outbreaks in chicken flocks from both chicken and turkey breeders as well as to verify the antimicrobial resistance of the isolates from the outbreaks. 2. The triplex polymerase chain reaction, standard antimicrobial test, beta-lactamase genes identification and Ion Torrent PMG whole-genome sequence were used in the field isolates and in the vaccine strain of S. Gallinarum. 3. The 60 tested isolates were not from vaccine origin and manifested high resistance to drugs from macrolide and quinolone groups. Whole-genome sequencing (WGS) and single nucleotide polymorphism analysis on selected isolates for core genes from Salmonella enterica confirmed the wild origin of these isolates and showed two possible sources of S. Gallinarum in the studied outbreaks. 4. S. Gallinarum isolated from fowl typhoid outbreaks in the studied period were not caused by the use of the SG9R live vaccine. The source of strains sequenced was diverse.

The endocannabinoid system expression in the female reproductive tract is modulated by estrogen.

The endocannabinoid system (ECS) is involved in several physiological events that resulted in a growing interest in its modulation. Moreover, the uterine levels of anandamide (AEA), the major endocannabinoid, must be tightly regulated to create proper embryo implantation conditions. However, there are no evidences about the regulation of AEA in uterus by estrogen. Thus, the aim of this study is to elucidate whether estradiol benzoate (EB) and tamoxifen (TAM) administration to ovariectomized (OVX) rats can induce changes in the expression of cannabinoid receptors and AEA-metabolic enzymes in uterus by evaluating gene transcription and protein levels by qPCR, Western blot and immunohistochemistry. Moreover, the plasmatic and uterine levels of AEA and of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α), the major cyclooxygenase-2 (COX-2) products, were determined by UPLC-MS/MS. The immunohistochemistry showed that cannabinoid receptors, as well as AEA-metabolic enzymes are mainly located in the epithelial cells of both lumen and glands and, to a lesser extent, in the muscle cells. Moreover, EB administration to OVX rats significantly increased CB1, CB2, NAPE-PLD, FAAH and COX-2 expression and transcription. These effects were absent in TAM and TAM+EB treatments showing that this response is estrogen receptor dependent. Additionally, although uterine levels of AEA remained unchanged in EB or TAM treated animals, they showed a rise with EB treatment in plasma. The latter also produced a decrease in uterine PGE2 levels. In summary, these data collectively indicate that the expression of ECS components, as well as, the AEA and PGE2 levels in rat uterus is modulated by EB. Thus, estradiol may have a direct regulatory role in the modulation of ECS in female reproductive tissues.

Candida albicans biofilms: comparative analysis of room-temperature and cryofixation for scanning electron microscopy.

Biofilms are frequently related to invasive fungal infections and are reported to be more resistant to antifungal drugs than planktonic cells. The structural complexity of the biofilm as well as the presence of a polymeric extracellular matrix (ECM) is thought to be associated with this resistant behavior. Scanning electron microscopy (SEM) after room temperature glutaraldehyde-based fixation, have been used to study fungal biofilm structure and drug susceptibility but they usually fail to preserve the ECM and, therefore, are not an optimised methodology to understand the complexity of the fungal biofilm. Thus, in this work, we propose a comparative analysis of room-temperature and cryofixation/freeze substitution of Candida albicans biofilms for SEM observation. Our experiments showed that room-temperature fixative protocols using glutaraldehyde and osmium tetroxide prior to alcohol dehydration led to a complete extraction of the polymeric ECM of biofilms. ECM from fixative and alcohol solutions were recovered after all processing steps and these structures were characterised by biochemistry assays, transmission electron microscopy and mass spectrometry. Cryofixation techniques followed by freeze-substitution lead to a great preservation of both ECM structure and C. albicans biofilm cells, allowing the visualisation of a more reliable biofilm structure. These findings reinforce that cryofixation should be the indicated method for SEM sample preparation to study fungal biofilms as it allows the visualisation of the EMC and the exploration of the biofilm structure to its fullest, as its structural/functional role in interaction with host cells, other pathogens and for drug resistance assays.

Cannabinoids as Modulators of Cell Death: Clinical Applications and Future Directions.

Endocannabinoids are bioactive lipids that modulate various physiological processes through G-protein-coupled receptors (CB1 and CB2) and other putative targets. By sharing the activation of the same receptors, some phytocannabinoids and a multitude of synthetic cannabinoids mimic the effects of endocannabinoids. In recent years, a growing interest has been dedicated to the study of cannabinoids properties for their analgesic, antioxidant, anti-inflammatory and neuroprotective effects. In addition to these well-recognized effects, various studies suggest that cannabinoids may affect cell survival, cell proliferation or cell death. These observations indicate that cannabinoids may play an important role in the regulation of cellular homeostasis and, thus, may contribute to tissue remodelling and cancer treatment. For a long time, the study of cannabinoid receptor signalling has been focused on the classical adenylyl cyclase/cyclic AMP/protein kinase A (PKA) pathway. However, this pathway does not totally explain the wide array of biological responses to cannabinoids. In addition, the diversity of receptors and signalling pathways that endocannabinoids modulate offers an interesting opportunity for the development of specific molecules to disturb selectively the endogenous system. Moreover, emerging evidences suggest that cannabinoids ability to limit cell proliferation and to induce tumour-selective cell death may offer a novel strategy in cancer treatment. This review describes the main properties of cannabinoids in cell death and attempts to clarify the different pathways triggered by these compounds that may help to understand the complexity of respective molecular mechanisms and explore the potential clinical benefit of cannabinoids use in cancer therapies.