Impact of TRP Channels on Extracellular Matrix Remodeling: Focus on TRPV4 and Collagen.

Disturbed remodeling of the extracellular matrix (ECM) is frequently observed in several high-prevalence pathologies that include fibrotic diseases of organs such as the heart, lung, periodontium, liver, and the stiffening of the ECM surrounding invasive cancers. In many of these lesions, matrix remodeling mediated by fibroblasts is dysregulated, in part by alterations to the regulatory and effector systems that synthesize and degrade collagen, and by alterations to the functions of the integrin-based adhesions that normally mediate mechanical remodeling of collagen fibrils. Cell-matrix adhesions containing collagen-binding integrins are enriched with regulatory and effector systems that initiate localized remodeling of pericellular collagen fibrils to maintain ECM homeostasis. A large cadre of regulatory molecules is enriched in cell-matrix adhesions that affect ECM remodeling through synthesis, degradation, and contraction of collagen fibrils. One of these regulatory molecules is Transient Receptor Potential Vanilloid-type 4 (TRPV4), a mechanically sensitive, Ca2+-permeable plasma membrane channel that regulates collagen remodeling. The gating of Ca2+ across the plasma membrane by TRPV4 and the consequent generation of intracellular Ca2+ signals affect several processes that determine the structural and mechanical properties of collagen-rich ECM. These processes include the synthesis of new collagen fibrils, tractional remodeling by contractile forces, and collagenolysis. While the specific mechanisms by which TRPV4 contributes to matrix remodeling are not well-defined, it is known that TRPV4 is activated by mechanical forces transmitted through collagen adhesion receptors. Here, we consider how TRPV4 expression and function contribute to physiological and pathological collagen remodeling and are associated with collagen adhesions. Over the long-term, an improved understanding of how TRPV4 regulates collagen remodeling could pave the way for new approaches to manage fibrotic lesions.

L-PRF Secretome from Both Smokers/Nonsmokers Stimulates Angiogenesis and Osteoblast Differentiation In Vitro.

Leukocyte and Platelet-Rich Fibrin (L-PRF) is part of the second generation of platelet-concentrates. L-PRF derived from nonsmokers has been used in surgical procedures, with its beneficial effects in wound healing being proven to stimulate biological activities such as cell proliferation, angiogenesis, and differentiation. Cigarette smoking exerts detrimental effects on tissue healing and is associated with post-surgical complications; however, evidence about the biological effects of L-PRF derived from smokers is limited. This study evaluated the impact of L-PRF secretome (LPRFS) derived from smokers and nonsmokers on angiogenesis and osteoblast differentiation. LPRFS was obtained by submerging L-PRF membranes derived from smokers or nonsmokers in culture media and was used to treat endothelial cells (HUVEC) or SaOs-2 cells. Angiogenesis was evaluated by tubule formation assay, while osteoblast differentiation was observed by alkaline phosphatase and osterix protein levels, as well as in vitro mineralization. LPRFS treatments increased angiogenesis, alkaline phosphatase, and osterix levels. Treatment with 50% of LPRFS derived from smokers and nonsmokers in the presence of osteogenic factors stimulates in vitro mineralization significantly. Nevertheless, differences between LPRFS derived from smokers and nonsmokers were not found. Both LPRFS stimulated angiogenesis and osteoblast differentiation in vitro; however, clinical studies are required to determine the beneficial effect of LPRFS in smokers.

FOXO1 regulates wound-healing responses in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Forkhead box-O 1 (FOXO1) is a transcription factor actively involved in oral wound healing at the epithelial barrier. However, less is known regarding the role of FOXO1 during the tissue repair response in the connective tissue compartment. This study explored the involvement of FOXO1 in the modulation of fibroblast activity related to wound healing. METHODS: Primary cultures of human gingival fibroblasts were obtained from four healthy young donors. Myofibroblastic differentiation, collagen gel contraction, cell migration, cell spreading, and integrin activation were evaluated in the presence or absence of a FOXO1 inhibitor (AS1842856). Variations in mRNA and proteins of interest were evaluated through qRT-PCR and western blot, respectively. Distribution of actin, α-smooth muscle actin, and ß1 integrin was evaluated using immunofluorescence. FOXO1 and TGF-ß1 expression in gingival wound healing was assessed by immunohistochemistry in gingival wounds performed in C57BL/6 mice. Images were analyzed using ImageJ/Fiji. ANOVA or Kruskal-Wallis test followed by Tukey's or Dunn's post-hoc test was performed. All data are expressed as mean ± SD. p < .05 was considered statistically significant. RESULTS: FOXO1 inhibition caused a decrease in the expression of the myofibroblastic marker α-SMA along with a reduction in fibronectin, type I collagen, TGF-ß1, and ß1 integrin mRNA level. The FOXO1 inhibitor also caused decreases in cell migration, cell spreading, collagen gel contraction, and ß1 integrin activation. FOXO1 and TGF-ß1 were prominently expressed in gingival wounds in fibroblastic cells located at the wound bed. CONCLUSION: The present study indicates that FOXO1 plays an important role in the modulation of several wound-healing functions in gingival fibroblast. Moreover, our findings reveal an important regulatory role for FOXO1 on the differentiation of gingival myofibroblasts, the regulation of cell migration, and collagen contraction, all these functions being critical during tissue repair and fibrosis.

Manifestaciones periodontales de la Granulomatosis de Wegener: Reporte de un caso clínico

La granulomatosis de Wegener o granulomatosis con Poliangitis (GPA) es una enfermedad caracterizada por inflamación y necrosis de las paredes de los vasos sanguíneos. Es de etiología desconocida, baja prevalencia y alta agresividad. Esta enfermedad puede comprometer los tejidos bucales causando agrandamiento e inflamación del tejido gingival. Se reporta el caso de un paciente de género masculino que manifiesta aumento de volumen de la encía e inflamación asociado al diagnóstico de granulomatosis de Wegener. La lesión fue eliminada quirúrgicamente y el diagnóstico se logró al combinar los hallazgos serológicos del test ANCA, manifestaciones periodontales y análisis histopatológico. El paciente fue tratado con metotrexato y corticoesteroides y no presenta recidiva de la lesión luego de 2 años de control. En este artículo se analizan las manifestaciones periodontales asociadas a la GPA resaltando la importancia de un adecuado diagnóstico de lesiones periodontales caracterizadas por agradamiento gingival e inflamación.

Wegener's granulomatosis or granulomatosis with polyangiitis (GPA) is a disease characterized by inflammation and necrosis of the blood vessel walls. It is of unknown etiology, low prevalence and high degree of aggressiveness. This disease can compromise the oral tissues, causing enlargement and inflammation of the gingival tissues. The case of a male patient who presented rapidly growing gingival tissue enlargement and inflammatory characteristics associated with the diagnosis of Wegener's granulomatosis is reported. The lesion was removed surgically and the diagnosis was achieved by combining the serological findings of the ANCA test, periodontal manifestations and histopathological analysis of the lesion. The patient was treated with methotrexate and corticosteroids and the lesion did not reappear after 2 years of control. In this article, the periodontal manifestations associated with GPA are analyzed, highlighting the importance of an adequate diagnosis of periodontal lesions characterized by gingival enlargement and inflammation.

Smoking habits do not affect biological responses induced by leucocyte and platelet-rich fibrin in periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Leucocyte- and platelet-rich fibrin has been developed to stimulate wound healing response. However, it is currently unknown whether smoking affects the biological responses elicited by leucocyte- and platelet-rich fibrin on periodontal ligament-derived mesenchymal stromal cells. This study analyzes the kinetics of biomolecule release from leucocyte- and platelet-rich fibrin derived from smokers and nonsmokers and their effect on periodontal ligament cell proliferation and migration as essential biological activities during wound healing. METHODS: Biomolecules present in leucocyte- and platelet-rich fibrin exudates and conditioned media collected from smokers and nonsmokers were analyzed by Luminex arrays. Periodontal ligament-derived mesenchymal stromal cell obtained from one nonsmoker were treated with leucocyte- and platelet-rich fibrin exudates or leucocyte- and platelet-rich fibrin conditioned media derived from both smokers and nonsmokers. The parameters evaluated included cell proliferation, determined by Ki67 immunostaining and migration assessed using transwell assays. Also, cells were treated with nicotine in the presence of fetal bovine serum 10% or leucocyte- and platelet-rich fibrin conditioned media. RESULTS: A similar biomolecular profile was detected in leucocyte- and platelet-rich fibrin exudates and leucocyte- and platelet-rich fibrin conditioned media from smokers and nonsmokers, stimulating (periodontal ligament-derived mesenchymal stromal cell) proliferation, and migration to a comparable degree. Nicotine reduced cell proliferation and migration of periodontal cells; however, this effect was recovered in the presence of leucocyte- and platelet-rich fibrin conditioned media. CONCLUSION: Leucocyte- and platelet-rich fibrin derived from smokers could be an autologous source of biomolecules to stimulate cell biological activities involved in wound healing in smokers who have difficulties in ceasing this habit. Clinical trials are required to evaluate the impact of leucocyte- and platelet-rich fibrin on healing responses in smokers.

Cytokine profiles and the dynamic of gingivitis development in humans.

AIM: To investigate the relationship between cytokine profiles and "fast" and "slow" patterns of gingival inflammation development. MATERIALS AND METHODS: Forty-two adults participated in an experimental gingivitis study, comprising a 2-week hygiene phase (clinical examination and professional cleaning); a 3-week induction phase (absence of oral hygiene); and a 2-week resolution phase (re-establishment of oral hygiene). Plaque and gingival inflammation scores were assessed. Interferon-gamma (IFN-γ), interleukin (IL)-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and tumour necrosis factor-alpha (TNF-α) from gingival crevicular fluid were collected and measured by multiplex ELISA. Group-based-trajectory-modelling (GBTM) was used to model cytokine profiles over the induction phase. The effect of gingival inflammation on cytokine levels over time was estimated with mixed-effects modelling. RESULTS: GBTM analysis revealed two cytokine profiles, "non-organized response" (IL-4, IL-6, IL-8, IL-12, and IL-13) and "organized response" (IL-2, IL-10, and TNF-α). Among the "slow" responders, neither cytokine profile was associated with gingivitis. In contrast, a "fast" response was associated with a higher "non-organized response" factor (coef. 0.14) and a lower "organized response" factor (coef. -0.03). CONCLUSION: A "fast" gingivitis development was associated with a higher "non-organized response" and a lower "organized response", which may elucidate the role of individual variability in gingivitis susceptibility.

Matrix Stiffness Modulates Metabolic Interaction between Human Stromal and Breast Cancer Cells to Stimulate Epithelial Motility.

Breast tumors belong to the type of desmoplastic lesion in which a stiffer tissue structure is a determinant of breast cancer progression and constitutes a risk factor for breast cancer development. It has been proposed that cancer-associated stromal cells (responsible for this fibrotic phenomenon) are able to metabolize glucose via lactate production, which supports the catabolic metabolism of cancer cells. The aim of this work was to investigate the possible functional link between these two processes. To measure the effect of matrix rigidity on metabolic determinations, we used compliant elastic polyacrylamide gels as a substrate material, to which matrix molecules were covalently linked. We evaluated metabolite transport in stromal cells using two different FRET (Fluorescence Resonance Energy Transfer) nanosensors specific for glucose and lactate. Cell migration/invasion was evaluated using Transwell devices. We show that increased stiffness stimulates lactate production and glucose uptake by mammary fibroblasts. This response was correlated with the expression of stromal glucose transporter Glut1 and monocarboxylate transporters MCT4. Moreover, mammary stromal cells cultured on stiff matrices generated soluble factors that stimulated epithelial breast migration in a stiffness-dependent manner. Using a normal breast stromal cell line, we found that a stiffer extracellular matrix favors the acquisition mechanistical properties that promote metabolic reprograming and also constitute a stimulus for epithelial motility. This new knowledge will help us to better understand the complex relationship between fibrosis, metabolic reprogramming, and cancer malignancy.

The Dynamic Interaction between Extracellular Matrix Remodeling and Breast Tumor Progression.

Desmoplastic tumors correspond to a unique tissue structure characterized by the abnormal deposition of extracellular matrix. Breast tumors are a typical example of this type of lesion, a property that allows its palpation and early detection. Fibrillar type I collagen is a major component of tumor desmoplasia and its accumulation is causally linked to tumor cell survival and metastasis. For many years, the desmoplastic phenomenon was considered to be a reaction and response of the host tissue against tumor cells and, accordingly, designated as "desmoplastic reaction". This notion has been challenged in the last decades when desmoplastic tissue was detected in breast tissue in the absence of tumor. This finding suggests that desmoplasia is a preexisting condition that stimulates the development of a malignant phenotype. With this perspective, in the present review, we analyze the role of extracellular matrix remodeling in the development of the desmoplastic response. Importantly, during the discussion, we also analyze the impact of obesity and cell metabolism as critical drivers of tissue remodeling during the development of desmoplasia. New knowledge derived from the dynamic remodeling of the extracellular matrix may lead to novel targets of interest for early diagnosis or therapy in the context of breast tumors.

Preventing first births among adolescents in Mexico City's public abortion programme.

INTRODUCTION: We examined parity and age among women seeking an abortion in Mexico City's public first-trimester abortion programme, Interrupcion Legal de Embarazo (ILE). We hypothesised that younger women, especially students, used abortion to prevent first births while older women used abortion to limit births. METHODS: We used clinical data from a sample of 47 462 women who had an abortion between 2007 and 2016 and classified them as nulliparous or parous according to previous births prior to the abortion. We used logistic regression to identify sociodemographic and clinical factors associated with using abortion to prevent a first birth (nulliparous) versus limiting births (parous) and calculated absolute multivariable predicted probabilities. RESULTS: Overall, 41% of abortions were in nulliparous women seeking to prevent a first birth, and 59% were in women who already had one or more children. The adjusted probability of using abortion to prevent a first birth was 80.4% (95% CI 78.3 to 82.4) for women aged 12-17 years and 54.3% (95% CI 51.6 to 57.0) for women aged 18-24 years. Adolescents (aged 12-17 years) who were employed or students had nearly 90% adjusted probability of using abortion to prevent a first birth (employed 87.8%, 95% CI 82.9 to 92.8; students 88.5%, 95% CI 82.9 to 94.1). At all ages, employed women and students had higher probabilities of using abortion to prevent a first birth compared with unemployed women and women who work in the home. CONCLUSION: Legal first-trimester abortion services in Mexico can help prevent first births in adolescents, especially students.

Congo red test for identification of preeclampsia: Results of a prospective diagnostic case-control study in Bangladesh and Mexico.

BACKGROUND: Misfolded proteins in the urine of women with preeclampsia bind to Congo Red dye (urine congophilia). We evaluated a beta prototype of a point-of-care test for the identification of urine congophilia in preeclamptic women. METHODS: Prospective diagnostic case-control study conducted in 409 pregnant women (n = 204 preeclampsia; n = 205 uncomplicated pregnancies) presenting for delivery in two tertiary level hospitals located in Bangladesh and Mexico. The GV-005, a beta prototype of a point-of-care test for detecting congophilia, was performed on fresh and refrigerated urine samples. The primary outcome was the prevalence of urine congophilia in each of the two groups. Secondary outcome was the likelihood of the GV-005 (index test) to confirm and rule-out preeclampsia based on an adjudicated diagnosis (reference standard). FINDINGS: The GV-005 was positive in 85% of clinical cases (83/98) and negative in 81% of clinical controls (79/98) in the Bangladesh cohort. In the Mexico cohort, the GV-005 test was positive in 48% of clinical cases (51/106) and negative in 77% of clinical controls (82/107). Adjudication confirmed preeclampsia in 92% of Bangladesh clinical cases (90/98) and 61% of Mexico clinical cases (65/106). The odds ratio of a urine congophilia in adjudicated cases versus controls in the Bangladesh cohort was 34.5 (14.7 - 81.1) (p<0.001) compared to 4.2 (2.1 - 8.4; p<0.001) in the Mexico cohort. INTERPRETATION: The GV-005, a beta prototype of a point-of-care test for detection of urine congophilia, is a promising tool for rapid identification of preeclampsia. FUNDING: Saving Lives at Birth.

Contraceptive Receipt Among First-Trimester Abortion Clients and Postpartum Women in Urban Mexico.

CONTEXT: In Mexico, first-trimester abortion is legal in Mexico City and is available in the public and private sectors. Understanding subsequent contraceptive uptake and method mix among first-trimester abortion clients relative to that of women who deliver a live birth at a health facility could help identify where improvements in care following an obstetric event can be made across the health system. METHODS: This article uses a retrospective cohort study to compare uptake of contraception prior to discharge between abortion clients in Mexico City's public abortion program and postpartum women from urban settings. The two data sources were clinical records of 45,233 abortion clients in Mexico City and information from a population-based survey of 1,289 urban women on their immediate postpartum contraceptive adoption. The primary outcome investigated was receipt of any reversible modern contraceptive method; secondary outcomes were level of method effectiveness and method type. Logistic regression and calculated multivariable probabilities were used to control for the effects of sociodemographic factors across the two data sources. RESULTS: The adjusted probability of uptake of any reversible modern method of contraception was higher among abortion clients than among postpartum women (67% vs. 48%). However, among all women who had received a contraceptive method, abortion clients had a lower adjusted probability of having received a long-acting reversible contraceptive than did postpartum women (49% vs. 82%) and a higher probability of having received a moderately effective method (38% vs. 13%). The adjusted probability of implant uptake was higher among abortion clients than among postpartum women (9% vs. 3%), while the adjusted probability of IUD uptake was lower (38% vs. 78%). CONCLUSIONS: Women receiving abortions in Mexico City's public abortion program were more likely than urban postpartum women to receive a reversible modern contraceptive method before leaving the facility. Women should be offered the full range of contraceptive methods after any obstetric event, to help them prevent unintended pregnancy and avoid short interpregnancy intervals.

RESUMEN Contexto: En México, el aborto de primer trimestre es legal en la Ciudad de México y está disponible en los sectores público y privado. Comprender la forma en que las clientas de aborto de primer trimestre adoptan el uso de anticonceptivos y la combinación de métodos subsiguientes en comparación a como lo hacen las mujeres que dan a luz a un nacido vivo en una institución de salud, podría ayudar a identificar dónde, en el sistema de salud, se pueden realizar mejoras en la atención después de un evento obstétrico. Métodos: Este artículo utiliza un estudio de cohorte retrospectivo para comparar la adopción de anticonceptivos por parte de clientas de servicios de aborto que participan en el Programa de Interrupción Legal del Embarazo en el sector público de la Ciudad de México y las mujeres posparto de entornos urbanos, previo a ser dadas de alta de la institución de salud. Las dos fuentes de datos fueron los registros clínicos de 45,233 clientas de servicios de aborto en la Ciudad de México y la información de una encuesta poblacional aplicada a 1,289 mujeres urbanas sobre su adopción inmediata de anticonceptivos posparto. El resultado primario investigado fue la recepción de cualquier método anticonceptivo moderno reversible; los resultados secundarios fueron el nivel de efectividad del método y el tipo de método. Se utilizó regresión logística y probabilidades multivariadas calculadas para controlar los efectos de los factores sociodemográficos en las dos fuentes de datos. Resultados: La probabilidad ajustada de la adopción de cualquier método anticonceptivo moderno reversible fue mayor entre las usuarias de aborto que entre las mujeres en período posparto (67% vs. 48%). Sin embargo, en el total de mujeres que habían recibido un método anticonceptivo, las clientas de servicios de aborto tuvieron una probabilidad ajustada menor de haber recibido un anticonceptivo reversible de acción prolongada que las mujeres posparto (49% frente a 82%) y una probabilidad más alta de haber recibido un método anticonceptivo moderadamente eficaz (38% vs. 13%). La probabilidad ajustada de adopción del implante fue mayor entre las usuarias de aborto que entre las mujeres en período posparto (9% vs. 3%), mientras que la probabilidad ajustada de adopción del DIU fue menor (38% vs. 78%). Conclusiones: Las mujeres que se recibieron servicios de aborto en el Programa de Interrupción Legal del Embarazo en el sector público de la Ciudad de México tuvieron más probabilidades que las mujeres urbanas en período posparto de recibir un método anticonceptivo moderno reversible antes de ser dadas de alta de la institución de salud. A las mujeres se les debe ofrecer la gama completa de métodos anticonceptivos después de cualquier evento obstétrico, para ayudarlas a prevenir embarazos no deseados y evitar intervalos cortos entre embarazos.

RÉSUMÉ Contexte: Au Mexique, l'avortement au premier trimestre de la grossesse est légal dans la ville de Mexico et peut être obtenu dans le secteur public et privé. Comprendre l'adoption ultérieure de la contraception par les patientes de l'avortement au premier trimestre et leur éventail de méthodes, par rapport aux femmes qui accouchent d'un enfant vivant en structure sanitaire pourrait aider à identifier les possibilités d'amélioration des soins après un événement obstétrical dans l'ensemble du système de santé. Méthodes: Sur la base d'une étude de cohorte rétrospective, cet article compare l'adoption de la contraception avant la sortie de la structure de soins, entre les patientes ayant subi un avortement dans le cadre du programme public d'avortement de Mexico et les femmes post-partum en milieu urbain. Les deux sources de données considérées sont les dossiers cliniques de 45 233 patientes de l'avortement à Mexico et l'information obtenue d'une enquête en population relative à 1 289 femmes urbaines concernant leur adoption immédiate de la contraception après l'accouchement. Le résultat principal examiné était l'obtention d'une méthode contraceptive moderne réversible quelconque; les résultats secondaires étaient le niveau d'efficacité de la méthode et le type de méthode. Pour les deux sources de données, les effets de facteurs sociodémographiques ont été contrôlés par régression logistique et probabilités multivariables calculées. Résultats: La probabilité corrigée d'adoption d'une méthode de contraception moderne réversible quelconque s'est avérée supérieure parmi les patientes de l'avortement (67% contre 48% chez les femmes post-partum). Cependant, sur la totalité des femmes ayant reçu une méthode contraceptive, les patientes de l'avortement présentaient une moindre probabilité corrigée d'avoir obtenu une méthode réversible à longue durée d'action (49% contre 82% des femmes post-partum) et une plus forte probabilité d'avoir obtenu une méthode modérément efficace (38% contre 13%). La probabilité corrigée d'adoption de l'implant s'est révélée supérieure parmi les clientes de l'avortement (9% contre 3% chez les femmes post-partum), tandis que la probabilité corrigée d'adoption du DIU était plus faible (38% contre 78%). Conclusions: Les femmes qui obtiennent un avortement dans le cadre du programme public d'avortement de Mexico étaient plus susceptibles que leurs homologues post-partum urbaines de recevoir une méthode contraceptive moderne réversible avant de quitter la structure. La gamme complète de méthodes contraceptives doit être proposée aux femmes après tout événement obstétrical, pour les aider à éviter les grossesses non planifiées et les intervalles de grossesse courts.

Anti-herpetic Activity of Macrocystis pyrifera and Durvillaea antarctica Algae Extracts Against HSV-1 and HSV-2.

Herpes simplex viruses (HSVs) type 1 (HSV-1) and type 2 (HSV-2) are highly prevalent in the human population, and the infections they produce are lifelong with frequent reactivations throughout life. Both viruses produce uncomfortable and sometimes painful lesions in the orofacial and genital areas, as well as herpetic gingivostomatitis, among other clinical manifestations. At present, the most common treatments against HSVs consist of nucleoside analogs that target the viral polymerases. However, such drugs are poorly effective for treating skin lesions, as they only reduce in 1-2 days the duration of the herpetic lesions. Additionally, viral isolates resistant to these drugs can emerge in immunosuppressed individuals, and second-line drugs for such variants are frequently accompanied by adverse effects requiring medical supervision. Thus, novel or improved therapeutic drugs for treating HSV lesions are needed. Here, we assessed the potential antiviral activity of aqueous extracts obtained from two brown macroalgae, namely Macrocystis pyrifera and Durvillaea antarctica against HSVs. Both extracts showed antiviral activity against acyclovir-sensitive and acyclovir-resistant HSV-1 and HSV-2. Our analyses show that there is a significant antiviral activity associated with proteins in the extract, although other compounds also seem to contribute to inhibiting the replication cycle of these viruses. Evaluation of the algae extracts as topical formulations in an animal model of HSV-1 skin infection significantly reduced the severity of the disease more than acyclovir, as well as the duration of the herpetic lesions, when compared to mock-treated animals, with the D. antarctica extract performing best. Taken together, these findings suggest that these algae extracts may be potential phytotherapeutics against HSVs and may be useful for the treatment and reduction of common herpetic manifestations in humans.

A repeat dose of misoprostol 800 mcg following mifepristone for outpatient medical abortion at 64-70 and 71-77 days of gestation: A retrospective chart review.

OBJECTIVE: To compare the effectiveness of outpatient medical abortion with mifepristone 200 mg and two misoprostol 800 mcg doses at 64-70 and 71-77 days of gestation. STUDY DESIGN: We conducted a retrospective chart review of medical abortion outcomes among clients with 64-77 day gestations at a Mexico City public clinic between February 2014 and November 2016 who took mifepristone 200 mg followed 24-48 h later by two doses of misoprostol 800 mcg four hours apart (first dose buccally, second dose sublingually). The primary outcome was successful medical abortion, defined as pregnancy expulsion without surgical intervention. We also assessed additional management and visits to other facilities. We compared outcomes by gestational age (64-70 vs 71-77 days). RESULTS: Of 602 charts reviewed, we analyzed 232 and 218 in the respective groups for effectiveness; nearly 25% of clients were lost to follow up. Treatment success occurred in 231 (99.6%, 95% CI 97.6-100%) clients at 64-70 days and 213 (97.7%, 95% CI 94.7-99.3%) clients at 71-77 days (p = 0.11). Ongoing pregnancy occurred in 1 (0.4%, 95% CI 0-2.4%) and 3 (1.4%, 95% CI 0.3-4.0%) clients, respectively (p = 0.36). Two charts from the 71-77 days group documented visits to other facilities: one bleeding concern prior to scheduled follow up and a hemorrhage during an aspiration intervention. CONCLUSIONS: Regimen effectiveness was high at 64-70 and 71-77 days among clients who attended follow up. However, with 25% attrition, it is difficult to draw definitive conclusions about effectiveness and associated safety. IMPLICATIONS: Mifepristone 200 mg followed by two doses of misoprostol 800 mcg four hours apart is a promising medical abortion regimen to improve efficacy in pregnancies from 64-77 days of gestation as compared to regimens with an initial single misoprostol dose. Prospective research is recommended to achieve more robust efficacy estimates.

Galectin-8 mediates fibrogenesis induced by cyclosporine in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: During cyclosporine-induced gingival overgrowth, the homeostatic balance of gingival connective tissue is disrupted leading to fibrosis. Galectins are glycan-binding proteins that can modulate a variety of cellular processes including fibrosis in several organs. Here, we study the role of galectin-8 (Gal-8) in the response of gingival connective tissue cells to cyclosporine. METHODS: We used human gingival fibroblasts and mouse NIH3T3 cells treated with recombinant Gal-8 and/or cyclosporine for analyzing specific mRNA and protein levels through immunoblot, real-time polymerase chain reaction, ELISA and immunofluorescence, pull-down with Gal-8-Sepharose for Gal-8-to-cell surface glycoprotein interactions, short hairpin RNA for Gal-8 silencing and Student's t test and ANOVA for statistical analysis. RESULTS: Galectin-8 stimulated type I collagen and fibronectin protein levels and potentiated CTGF protein levels in TGF-ß1-stimulated human gingival fibroblasts. Gal-8 interacted with α5ß1-integrin and type II TGF-ß receptor. Gal-8 stimulated fibronectin protein and mRNA levels, and this response was dependent on FAK activity but not Smad2/3 signaling. Cyclosporine and tumor necrosis factor alpha (TNF-α) increased Gal-8 protein levels. Finally, silencing of galectin-8 in NIH3T3 cells abolished cyclosporine-induced fibronectin protein levels. CONCLUSION: Taken together, these results reveal for the first time Gal-8 as a fibrogenic stimulus exerted through ß1-integrin/FAK pathways in human gingival fibroblasts, which can be triggered by cyclosporine. Further studies should explore the involvement of Gal-8 in human gingival tissues and its role in drug-induced gingival overgrowth.

Cetylpyridinium chloride blocks herpes simplex virus replication in gingival fibroblasts.

Infections with herpes simplex viruses are lifelong and highly prevalent worldwide. Individuals with clinical symptoms elicited by HSVs may suffer from occasional or recurrent herpetic lesions in the orofacial and genital areas. Despite the existence of nucleoside analogues that interfere with HSV replication, such as acyclovir, these drugs are somewhat ineffective in treating skin lesions as topical formulations only reduce in one or few days the duration of the herpetic ulcers. Cetylpyridinium chloride (CPC) is a quaternary ammonium compound present in numerous hygiene products, such as mouthwashes, deodorants, aphtae-treating formulations and oral tablets as an anti-septic to limit bacterial growth. Some reports indicate that CPC can also modulate host signaling pathways, namely NF-κB signaling. Because HSV infection is modulated by NF-κB, we sought to assess whether CPC has antiviral effects against HSVs. Using wild-type HSV-1 and HSV-2, as well as viruses that are acyclovir-resistant or encode GFP reporter genes, we assessed the antiviral capacity of CPC in epithelial cells and human gingival fibroblasts expanded from the oral cavity and its mechanism of action. We found that a short, 10-min exposure to CPC added after HSV entry into the cells, significantly limited viral replication in both cell types by impairing viral gene expression. Interestingly, our results suggest that CPC blocks HSV replication by interfering with the translocation of NF-κB into the nucleus of HSV-infected cells. Taken together, these findings suggest that formulations containing CPC may help limit HSV replication in infected tissues and consequently reduce viral shedding.

Diabetes alters the involvement of myofibroblasts during periodontal wound healing.

OBJECTIVES: Myofibroblasts constitute a specific cell phenotype involved in connective tissue healing. Diabetes alters the wound healing response. However, it is not clear whether diabetes modifies the involvement of myofibroblasts in periodontal wounds. MATERIALS AND METHODS: Type I diabetes was induced in rats through streptozotocin injection, and periodontal wounds were performed. Wound healing was evaluated histologically at 2, 5, 7, and 15 days by measuring epithelial migration, neutrophil infiltration, and collagen and biofilm formation. Distribution of myofibroblasts was evaluated through immunofluorescence for α-smooth muscle actin. Data analyses were performed using the Shapiro-Wilk, ANOVA, or Kruskal-Wallis tests. RESULTS: Diabetic wounds were characterized by delayed epithelial closure, increased neutrophil infiltration, biofilm formation, and reduced collagen formation. Quantification of the myofibroblasts showed a significant reduction at 5 and 7 days in wounds of diabetic rats and an increase at 15 days when compared to wounds of non-diabetic rats. CONCLUSIONS: Diabetic wound healing was associated with decreased epithelial and connective tissue healing, increased levels of inflammation, and biofilm formation. Myofibroblast differentiation was delayed in diabetic periodontal wounds at early time points. However, myofibroblasts persisted at later time points of healing. The present study suggests that diabetes alters the involvement of myofibroblasts during periodontal wound healing.

A non-inferiority study of outpatient mifepristone-misoprostol medical abortion at 64-70 days and 71-77 days of gestation.

OBJECTIVES: This open-label non-inferiority study assessed efficacy of a common outpatient medical abortion regimen among people with pregnancies 64-70 days and 71-77 days of gestation. STUDY DESIGN: We defined non-inferiority by a 6% margin of method success. People with intrauterine pregnancies 64-77 days' gestational age by abdominal ultrasound seeking medical abortion at one of eight clinics and met eligibility criteria were offered participation. Consenting participants took mifepristone 200 mg followed 24-48 h later by misoprostol 800 mcg buccally, and returned after one week for provider evaluation and abdominal ultrasound to determine abortion status. Participants recorded medication use, pregnancy expulsion, daily bleeding and pain scores until the one-week follow up. Clinic staff interviewed participants prior to study discharge to assess acceptability. RESULTS: Seven hundred and nineteen participants were enrolled, 393 and 326 in the respective groups. Successful expulsion without surgical intervention was achieved in 92.3% of the earlier gestational age group and 86.7% of the later group (difference in proportions 5.6%, 1-sided 95% CI 9.6). Ongoing pregnancy accounted for 3.6% and 8.7% (p = 0.007) of outcomes, respectively. Participants in the 71-77 day group reported nausea and weakness more frequently. Pain, bleeding and acceptability measures between groups were similar. CONCLUSION: Although the success rate at 71-77 days of gestation was within the non-inferiority margin, we cannot rule out that it is statistically worse than in the previous gestational week. Significantly more ongoing pregnancies in the later group raise concerns about using the regimen at 71-77 days.

Uncoupled inflammatory, proliferative, and cytoskeletal responses in senescent human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Aging is characterized by a decline in tissue structure and function that may be explained by the development of cellular senescence. However, the acquisition of specific phenotypic responses in senescent gingival fibroblasts is still poorly understood. Here, we have analyzed whether proliferation of primary cultures of human gingival fibroblasts may affect different cell functions relevant to cellular senescence and tissue deterioration. METHODS: Human gingival fibroblasts from five young donors were expanded until cellular senescence was achieved. Cellular senescence was evaluated by determining modifications in cell size, cell proliferation, p16 and p21 mRNA levels, H2Ax phosphorylation, cell viability, and senescence-associated beta-galactosidase staining. Inflammation was evaluated by analyzing the secretion of cytokines and nuclear translocation of NF-κB. Collagen remodeling was evaluated using a collagen gel contraction assay. Immunofluorescence and confocal microscopy were used to determine changes in the localization of the cytoskeletal proteins. Data analysis was performed to identify changes between cultures of the same donor at early and late passages using the paired sample t test or the Wilcoxon matched-pairs signed-rank test. RESULTS: Late passage cells showed changes compatible with cellular senescence that included increased cell size, reduced cell proliferation, staining for SA-beta gal, phosphorylated H2Ax, and increased p16 and p21 mRNA levels. Late passage cells showed a decrease in collagen contraction and reduced co-localization between the cytoskeletal proteins actin and vinculin. Importantly, late passage cells neither demonstrated changes in the secretion of inflammatory cytokines nor NF-κB activation. CONCLUSION: Our results imply that cytoskeletal changes and inhibition of cell proliferation represent early modifications in the structure and function of senescent gingival fibroblasts that are not coupled with the acquisition of an inflammatory phenotype. Further studies are needed to clarify the impact of different senescence stages during aging of the periodontium.