Monomeric CRP regulates inflammatory responses in human intervertebral disc cells.

CRP is an acute-phase protein that is used as a biomarker to follow severity and progression in infectious and inflammatory diseases. Its pathophysiological mechanisms of action are still poorly defined. CRP in its pentameric form exhibits weak anti-inflammatory activity. The monomeric isoform (mCRP) exerts potent proinflammatory properties in chondrocytes, endothelial cells, and leucocytes. No data exist regarding mCRP effects in human intervertebral disc (IVD) cells. This work aimed to verify the pathophysiological relevance of mCRP in the aetiology and/or progression of IVD degeneration. We investigated the effects of mCRP and the signalling pathways that are involved in cultured human primary annulus fibrosus (AF) cells and in the human nucleus pulposus (NP) immortalized cell line HNPSV-1. We determined messenger RNA (mRNA) and protein levels of relevant factors involved in inflammatory responses, by quantitative real-time polymerase chain reaction (RT-qPCR) and western blot. We also studied the presence of mCRP in human AF and NP tissues by immunohistochemistry. We demonstrated that mCRP increases nitric oxide synthase 2 (NOS2), cyclooxygenase 2 (COX2), matrix metalloproteinase 13 (MMP13), vascular cell adhesion molecule 1 (VCAM1), interleukin (IL)-6, IL-8, and Lipocalin 2 (LCN2) expression in human AF and NP cells. We also showed that nuclear factor-κß (NF-κß), extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphoinositide 3-kinase (PI3K) are at play in the intracellular signalling of mCRP. Finally, we demonstrated the presence of mCRP in human AF and NP tissues. Our results indicate, for the first time, that mCRP can be localized in IVD tissues, where it triggers a proinflammatory and catabolic state in degenerative and healthy IVD cells, and that NF-κß signalling may be implicated in the mediation of this mCRP-induced state.

Histological and histomorphometric study of human palatal mucosa: Implications for connective tissue graft harvesting.

AIMS: To analyse the histological structure and histomorphometric characteristics of human hard palatal mucosa in order to determine the donor site of choice for connective tissue grafts from a histological point of view. MATERIALS AND METHODS: Palatal mucosa samples from six cadaver heads were harvested at four sites: incisal, premolar, molar and tuberosity. Histological and immunohistochemical techniques were performed, as was histomorphometric analysis. RESULTS: In the current study, we found that the density and size of cells were higher in the superficial papillary layer, whereas the thickness of the collagen bundles increased in the reticular layer. Excluding the epithelium, the mean percentage of lamina propria (LP) and submucosa (SM) was 37% and 63%, respectively (p < .001). LP thickness showed similar values in the incisal, premolar and molar regions, and a significantly greater thickness in tuberosity (p < .001). The thickness of SM increased from incisal to premolar and molar, disappearing in the tuberosity (p < .001). CONCLUSIONS: As dense connective tissue of LP is the tissue of choice for connective tissue grafts, the best donor site from a histological point of view is tuberosity because it is composed only of a thick LP without the presence of a loose submucosal layer.

The lipidomic and inflammatory profiles of visceral and subcutaneous adipose tissues are distinctly regulated by the SGLT2 inhibitor empagliflozin in Zucker diabetic fatty rats.

The pharmacological inhibition of sodium-glucose cotransporter 2 (SGLT2) has emerged as a treatment for patients with type 2 diabetes mellitus (T2DM), cardiovascular disease and/or other metabolic disturbances, although some of the mechanisms implicated in their beneficial effects are unknown. The SGLT2 inhibitor (SGLT2i) empagliflozin has been suggested as a regulator of adiposity, energy metabolism, and systemic inflammation in adipose tissue. The aim of our study was to evaluate the impact of a 6-week-empagliflozin treatment on the lipidome of visceral (VAT) and subcutaneous adipose tissue (SAT) from diabetic obese Zucker Diabetic Fatty (ZDF) rats using an untargeted metabolomics approach. We found that empagliflozin increases the content of diglycerides and oxidized fatty acids (FA) in VAT, while in SAT, it decreases the levels of several lysophospholipids and increases 2 phosphatidylcholines. Empagliflozin also reduces the expression of the cytokines interleukin-1 beta (IL-1ß), IL-6, tumor necrosis factor-alpha (TNFα), monocyte-chemotactic protein-1 (MCP-1) and IL-10, and of Cd86 and Cd163 M1 and M2 macrophage markers in VAT, with no changes in SAT, except for a decrease in IL-1ß. Empagliflozin treatment also shows an effect on lipolysis increasing the expression of hormone-sensitive lipase (HSL) in SAT and VAT and of adipose triglyceride lipase (ATGL) in VAT, together with a decrease in the adipose content of the FA transporter cluster of differentiation 36 (CD36). In conclusion, our data highlighted differences in the VAT and SAT lipidomes, inflammatory profiles and lipolytic function, which suggest a distinct metabolism of these two white adipose tissue depots after the empagliflozin treatment.

Immunohistological study of the density and distribution of human penile neural tissue: gradient hypothesis.

Immunohistological patterns of density and distribution of neural tissue in the human penis, including the prepuce, are not fully characterized, and effects of circumcision (partial or total removal of the penile prepuce) on penile sexual sensation are controversial. This study analyzed extra- and intracavernosal innervation patterns on the main penile axes using formalin-fixed, paraffin-embedded human adult and fetal penile tissues, single- and double-staining immunohistochemistry and a variety of neural and non-neural markers, with a special emphasis on the prepuce and potential sexual effects of circumcision. Immunohistochemical profiles of neural structures were determined and the most detailed immunohistological characterizations to date of preputial nerve supply are provided. The penile prepuce has a highly organized, dense, afferent innervation pattern that is manifest early in fetal development. Autonomically, it receives noradrenergic sympathetic and nitrergic parasympathetic innervation. Cholinergic nerves are also present. We observed cutaneous and subcutaneous neural density distribution biases across our specimens towards the ventral prepuce, including a region corresponding in the adult anatomical position (penis erect) to the distal third of the ventral penile aspect. We also describe a concept of innervation gradients across the longitudinal and transverse penile axes. Results are discussed in relation to the specialized literature. An argument is made that neuroanatomic substrates underlying unusual permanent penile sensory disturbances post-circumcision are related to heightened neural levels in the distal third of the ventral penile aspect, which could potentially be compromised by deep incisions during circumcision.

The inner nuclear membrane protein Lem2 coordinates RNA degradation at the nuclear periphery.

Transcriptionally silent chromatin often localizes to the nuclear periphery. However, whether the nuclear envelope (NE) is a site for post-transcriptional gene repression is not well understood. Here we demonstrate that Schizosaccharomyces pombe Lem2, an NE protein, regulates nuclear-exosome-mediated RNA degradation. Lem2 deletion causes accumulation of RNA precursors and meiotic transcripts and de-localization of an engineered exosome substrate from the nuclear periphery. Lem2 does not directly bind RNA but instead interacts with the exosome-targeting MTREC complex and its human homolog PAXT to promote RNA recruitment. This pathway acts largely independently of nuclear bodies where exosome factors assemble. Nutrient availability modulates Lem2 regulation of meiotic transcripts, implying that this pathway is environmentally responsive. Our work reveals that multiple spatially distinct degradation pathways exist. Among these, Lem2 coordinates RNA surveillance of meiotic transcripts and non-coding RNAs by recruiting exosome co-factors to the nuclear periphery.

In Vivo Effects of Conditioned Medium from Human Uterine Cervical Stem Cells in an Ovarian Cancer Xenograft Mouse Model.

BACKGROUND/AIM: Ovarian cancer is the most lethal of all gynecological cancers, despite advances in surgical techniques and medical treatments. During the last years, therapies based on mesenchymal stem cells and particularly their secretome (conditioned medium, CM) have emerged as promising treatments for various types of tumors. MATERIALS AND METHODS: In the present study, we evaluated the in vivo antitumor effect of human uterine cervical stem cell conditioned medium (hUCESC-CM) after intraperitoneal administration in an ovarian cancer mouse model. RESULTS: We found that intraperitoneal injection of hUCESC-CM in immunodeficient mice, injected fifty days previously with the human ovarian adenocarcinoma SKOV-3 cell line, significantly reduced abdominal tumor growth, and significantly increased overall survival, compared to control mice. CONCLUSION: hUCESC-CM could be an alternative approach to intraperitoneal treatment of ovarian cancer, either administered alone and/or with conventional chemotherapy.

WISP-2 modulates the induction of inflammatory mediators and cartilage catabolism in chondrocytes.

Wnt-1 inducible signaling pathway protein 2 (WISP-2/CCN5) is a recently identified adipokine that has been described as an important mediator of canonical Wnt activation in adipogenic precursor cells. In osteoarthritis (OA), the most common form of arthritis, chondrocytes exhibit aberrant and increased production of pro-inflammatory mediators and matrix degrading enzymes such as IL-1ß and MMP-13. Although recent evidence suggests a role for Wnt signaling in OA physiopathology, little is known about the involvement of WISP-2 in cartilage degradation. In the present study, we determined the expression of WISP-2 in healthy and OA human chondrocytes. WISP-2 expression is modulated along chondrocyte differentiation and downregulated at the onset of hypertrophy by inflammatory mediators. We also investigated the effect of WISP-2 on cartilage catabolism and performed WISP-2 loss-of-function experiments using RNA interference technology in human T/C-28a2 immortalized chondrocytes. We demonstrated that recombinant human WISP-2 protein reduced IL-1ß-mediated chondrocyte catabolism, that IL-1ß and WNT/b-catenin signaling pathways are involved in rhWISP-2 protein and IL-1ß effects in human chondrocytes, and that WISP-2 has a regulatory role in attenuating the catabolic effects of IL-1ß in chondrocytes. Gene silencing of WISP-2 increased the induction of the catabolic markers MMP-13 and ADAMTS-5 and the inflammatory mediators IL-6 and IL-8 triggered by IL-1ß in human primary OA chondrocytes in a Wnt/ß-catenin dependent manner. In conclusion, here we have shown for the first time that WISP-2 may have relevant roles in modulating the turnover of extracellular matrix in the cartilage and that its downregulation may detrimentally alter the inflammatory environment in OA cartilage. We also proved the participation of Wnt/ß-catenin signaling pathway in these processes. Thus, targeting WISP-2 might represent a potential therapeutical approach for degenerative and/or inflammatory diseases of musculoskeletal system, such as osteoarthritis.

Activation of Hypothalamic AMP-Activated Protein Kinase Ameliorates Metabolic Complications of Experimental Arthritis.

OBJECTIVE: To investigate whether thermogenesis and the hypothalamus may be involved in the physiopathology of experimental arthritis (EA). METHODS: EA was induced in male Lewis rats by intradermal injection of Freund's complete adjuvant (CFA). Food intake, body weight, plasma cytokines, thermographic analysis, gene and protein expression of thermogenic markers in brown adipose tissue (BAT) and white adipose tissue (WAT), and hypothalamic AMP-activated protein kinase (AMPK) were analyzed. Virogenetic activation of hypothalamic AMPK was performed. RESULTS: We first demonstrated that EA was associated with increased BAT thermogenesis and browning of subcutaneous WAT leading to elevated energy expenditure. Moreover, rats experiencing EA showed inhibition of hypothalamic AMPK, a canonical energy sensor modulating energy homeostasis at the central level. Notably, specific genetic activation of AMPK in the ventromedial nucleus of the hypothalamus (a key site modulating energy metabolism) reversed the effect of EA on energy balance, brown fat, and browning, as well as promoting amelioration of synovial inflammation in experimental arthritis. CONCLUSION: Overall, these data indicate that EA promotes a central catabolic state that can be targeted and reversed by the activation of hypothalamic AMPK. This might provide new therapeutic alternatives to treat rheumatoid arthritis (RA)-associated metabolic comorbidities, improving the overall prognosis in patients with RA.

Nucleolar release of rDNA repeats for repair involves SUMO-mediated untethering by the Cdc48/p97 segregase.

Ribosomal RNA genes (rDNA) are highly unstable and susceptible to rearrangement due to their repetitive nature and active transcriptional status. Sequestration of rDNA in the nucleolus suppresses uncontrolled recombination. However, broken repeats must be first released to the nucleoplasm to allow repair by homologous recombination. Nucleolar release of broken rDNA repeats is conserved from yeast to humans, but the underlying molecular mechanisms are currently unknown. Here we show that DNA damage induces phosphorylation of the CLIP-cohibin complex, releasing membrane-tethered rDNA from the nucleolus in Saccharomyces cerevisiae. Downstream of phosphorylation, SUMOylation of CLIP-cohibin is recognized by Ufd1 via its SUMO-interacting motif, which targets the complex for disassembly through the Cdc48/p97 chaperone. Consistent with a conserved mechanism, UFD1L depletion in human cells impairs rDNA release. The dynamic and regulated assembly and disassembly of the rDNA-tethering complex is therefore a key determinant of nucleolar rDNA release and genome integrity.

Is there a correlation between HER2 gene amplification level and response to neoadjuvant treatment with trastuzumab and chemotherapy in HER2-positive breast cancer?

There are contradictory data regarding the correlation between HER2 amplification level determined by in situ hybridization and evolution after treatment with anti-HER2 therapies. The aim of this study was to correlate quantitative results of FISH (ratio HER2/CEP17 and number of HER2 signals/nucleus) with pathological response achieved after neoadjuvant treatment with trastuzumab and chemotherapy. For this purpose, we analysed 100 consecutive HER2-positive cases of breast carcinoma treated with neoadjuvant therapy. HER2 amplification determined by FISH was found in 92 of the 100 cases studied. pCR was obtained in 58% of the patients whose tumours presented amplification. In contrast, no pCR was obtained in the 8 patients with non-amplified tumours. A significant direct correlation between HER2 high amplification (HER2/CEP17 ratio > 5 or HER2 signals/nucleus > 10) and pCR was found. In conclusion, HER2 amplification levels are clinically relevant because they provide oncologists with valuable information on the possibilities of achieving pCR after neoadjuvant treatment.

ESCRT recruitment by the S. cerevisiae inner nuclear membrane protein Heh1 is regulated by Hub1-mediated alternative splicing.

Misassembled nuclear pore complexes (NPCs) are removed by sealing off the surrounding nuclear envelope (NE), which is conducted by the endosomal sorting complexes required for transport (ESCRT) machinery. Recruitment of ESCRT proteins to the NE is mediated by the interaction between the ESCRT member Chm7 and the inner nuclear membrane protein Heh1, which belongs to the conserved LEM family. Increased ESCRT recruitment results in excessive membrane scission at damage sites but its regulation remains poorly understood. Here, we show that Hub1-mediated alternative splicing of HEH1 pre-mRNA, resulting in production of its shorter form Heh1-S, is critical for the integrity of the NE in Saccharomyces cerevisiae ESCRT-III mutants lacking Hub1 or Heh1-S display severe growth defects and accumulate improperly assembled NPCs. This depends on the interaction of Chm7 with the conserved MSC domain, which is only present in the longer variant Heh1-L. Heh1 variants assemble into heterodimers, and we demonstrate that a unique splice segment in Heh1-S suppresses growth defects associated with the uncontrolled interaction between Heh1-L and Chm7. Together, our findings reveal that Hub1-mediated splicing generates Heh1-S to regulate ESCRT recruitment to the NE.This article has an associated First Person interview with the first author of the paper.

Merkel cells of human oral mucosa express the pluripotent stem cell transcription factor Sox2.

Merkel cells are neuroendocrine cells associated to a neural sensitive ending and localized primarily in the epidermis, although they are also found in oral mucosa. Sox2 or SRY-box2 is a key transcription factor important in the maintenance of embryonic neural crest stem cell pluripotency. Sox2 has been described in Merkel cells of skin and in Merkel cell carcinomas, but not specifically in oral Merkel cells. The aims of the present study were to analyze the density of Merkel cells in human oral mucosa and to study the expression of Sox2 in these cells. For these purposes, immunohistochemical analyses for Sox2 and CK20 (the best marker for Merkel cells) were automatically performed on sections of normal human oral mucosa. Double immunofluorescence for Sox2 and CK20 was also performed. To analyze the density of Merkel cells, CK20 positive cells were counted in each sample and the length of the epithelial apical edge was measured (cells/mm). Merkel cells, demonstrated by CK20 immunoreactivity, were found in 95% of oral mucosa specimens studied (n=21). Mean density of Merkel cells in oral mucosa was 1.71±2.34 cells/mm. Sox2 immunoreactivity was found in the nuclei of scattered cells located at the basal layer. Serial sections immunostained for Sox2 and CK20 showed that Sox2-positive cells of oral mucosa coexpressed CK20, confirming that they were Merkel cells. Immunofluorescence for Sox2 and CK20 showed colocalization of both markers, demonstrating that virtually all oral Merkel cells expressed Sox2. This transcription factor could play a role in Merkel cell maturation and maintenance.

Preclinical Evaluation of the Antimicrobial-Immunomodulatory Dual Action of Xenohormetic Molecules against Haemophilus influenzae Respiratory Infection.

Chronic obstructive pulmonary disease (COPD) is characterized by abnormal inflammation and impaired airway immunity, providing an opportunistic platform for nontypeable Haemophilus influenzae (NTHi) infection. In this context, therapies targeting not only overactive inflammation without significant adverse effects, but also infection are of interest. Increasing evidence suggests that polyphenols, plant secondary metabolites with anti-inflammatory and antimicrobial properties, may be protective. Here, a Cistus salviifolius plant extract containing quercetin, myricetin, and punicalagin was shown to reduce NTHi viability. Analysis of these polyphenols revealed that quercetin has a bactericidal effect on NTHi, does not display synergies, and that bacteria do not seem to develop resistance. Moreover, quercetin lowered NTHi airway epithelial invasion through a mechanism likely involving inhibition of Akt phosphorylation, and reduced the expression of bacterially-induced proinflammatory markers il-8, cxcl-1, il-6, pde4b, and tnfα. We further tested quercetin's effect on NTHi murine pulmonary infection, showing a moderate reduction in bacterial counts and significantly reduced expression of proinflammatory genes, compared to untreated mice. Quercetin administration during NTHi infection on a zebrafish septicemia infection model system showed a bacterial clearing effect without signs of host toxicity. In conclusion, this study highlights the therapeutic potential of the xenohormetic molecule quercetin against NTHi infection.

Association between hospital interval and survival in patients with oral cancer: A waiting time paradox.

BACKGROUND: In early diagnosis studies on symptomatic cancer, survival was the most recommended outcome. The magnitude and impact of the patient interval and primary care interval is well-known in oral cancer; however, the hospital interval and its influence on surviving this neoplasia are not well known. AIMS: To quantify the interval between the first contact with the specialist and the start of treatment for patients with oral cancer and to evaluate whether there was a link between this interval and disease survival. METHODS: We designed a hospital-based study that included 228 patients diagnosed with oral/oropharyngeal squamous cell carcinoma between 1998 and 2008 at A Coruña University Hospital (Spain) who were followed up until 2016. The data were extracted retrospectively from hospital medical charts. The study interval was defined in the context of the "pathways to treatment" model as the interval from the first specialist visit (start point) to the start of treatment (end point). We calculated the total interval (from first symptom to treatment) to evaluate the relative length of the hospital interval, and we considered the variables age, sex, location, comorbidity and tumour classification stage. Survival time was defined as the interval from the first treatment to death or censoring. RESULTS: The median hospital interval was 20 days, with an interquartile range of 15-29.1 days. The most relevant prognostic variable was the tumour stage (III-IV: Exp. ß = 2.8, p = 0.001). The hospital interval was part of the multivariate model, and its association with mortality showed a V-shaped association, where patients with short hospital intervals (3-18 days) and those with long hospital intervals (26-55 days) had significantly higher mortality than those with medium hospital intervals (19-25 days). CONCLUSION: The hospital interval represents a relevant interval for the patient's path towards treatment, has prognostic implications and is subject to a severity bias (waiting time paradox) that should be avoided.

MCH Regulates SIRT1/FoxO1 and Reduces POMC Neuronal Activity to Induce Hyperphagia, Adiposity, and Glucose Intolerance.

Melanin-concentrating hormone (MCH) is an important regulator of food intake, glucose metabolism, and adiposity. However, the mechanisms mediating these actions remain largely unknown. We used pharmacological and genetic approaches to show that the sirtuin 1 (SIRT1)/FoxO1 signaling pathway in the hypothalamic arcuate nucleus (ARC) mediates MCH-induced feeding, adiposity, and glucose intolerance. MCH reduces proopiomelanocortin (POMC) neuronal activity, and the SIRT1/FoxO1 pathway regulates the inhibitory effect of MCH on POMC expression. Remarkably, the metabolic actions of MCH are compromised in mice lacking SIRT1 specifically in POMC neurons. Of note, the actions of MCH are independent of agouti-related peptide (AgRP) neurons because inhibition of γ-aminobutyric acid receptor in the ARC did not prevent the orexigenic action of MCH, and the hypophagic effect of MCH silencing was maintained after chemogenetic stimulation of AgRP neurons. Central SIRT1 is required for MCH-induced weight gain through its actions on the sympathetic nervous system. The central MCH knockdown causes hypophagia and weight loss in diet-induced obese wild-type mice; however, these effects were abolished in mice overexpressing SIRT1 fed a high-fat diet. These data reveal the neuronal basis for the effects of MCH on food intake, body weight, and glucose metabolism and highlight the relevance of SIRT1/FoxO1 pathway in obesity.

POU1F1 transcription factor promotes breast cancer metastasis via recruitment and polarization of macrophages.

Cancer progression requires cells surrounding tumors be reeducated and activated to support tumor growth. Oncogenic signals from malignant cells directly influence stromal composition and activation, but the factors mediating this communication are still not well understood. We have previously shown that the transcription factor POU class 1 homeobox 1 (POU1F1), also known as Pit-1, induces profound changes on neoplastic cell-autonomous processes favoring metastasis in human breast cancer. Here we describe for the first time Pit-1-mediated paracrine actions on macrophages in the tumor microenvironment by using cell lines in vitro, zebrafish and mouse models in vivo, and samples from human breast cancer patients. Through the release of CXCL12, Pit-1 in tumor cells was found to mediate the recruitment and polarization of macrophages into tumor-associated macrophages (TAMs). In turn, TAMs collaborated with tumor cells to increase tumor growth, angiogenesis, extravasation and metastasis to lung. Our data reveal a new mechanism of cooperation between tumor cells and macrophages favoring metastasis and poor clinical outcome in human breast cancer, which suggests that Pit-1 and CXCL12 should be further studied as potential prognostic and therapeutic indicators. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Oral mucosal peeling related to dentifrices and mouthwashes: A systematic review

Background: The aim of this systematic review was to summarise the clinical information available about oral mucosal peeling (OMP) and to explore its aetiopathogenic association with dentifrices and mouthwashes. Material and Methods: PICOS outline: Population: subjects diagnosed clinically and/or pathologically. Intervention: exposition to oral hygiene products. Comparisons: patients using products at different concentrations. Out-comes: clinicopathological outcomes (primary) and oral epithelial desquamation (secondary) after use. Study de-sign: any. Exclusion criteria: reports on secondary or unpublished data, in vitro studies. Data were independently extracted by two reviewers. Results: Fifteen reports were selected from 410 identified. Descriptive studies mainly showed low bias risk, ex-perimental studies mostly an "unclear risk". Dentifrices or mouthwashes were linked to OMP, with an unknown origin in 5 subjects. Sodium lauryl-sulphate (SLS) was behind this disorder in 21 subjects, tartar-control dentifrices in 2, and flavouring agents in 1 case. Desquamation extension was linked to SLS concentration. Most cases were painless, leaving normal mucosa after desquamation. Tartar-control dentifrices caused ulcerations more frequently. Conclusions: OMP management should consider differential diagnosis with oral desquamative lesions, particularly desquamative gingivitis, with a guided clinical interview together with pathological confirmation while discouraging the use of the product responsible for OMP

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