Availability, attitudes and costs of selected dental services in a low-income country

ABSTRACT The aim of this study was to determine the availability of selected dental services, attitudes towards such services and economic factors associated with their utilization in a low-income country. Data utilized for this study was collected in two phases. Phase I was a survey of all dental facilities in four selected regions in Tanzania. The survey was administered to officers-in-charge of dental facilities and other dental practitioners working within these facilities. Phase II was a series of cross-sectional studies conducted among outpatients attending four selected regional hospitals in Tanzania. Data was collected using self-administered questionnaires. Dental materials and equipment were inconsistently available and only 3/28 studied dental facilities had all the seven investigated equipment fully functional and in use. The self-rated competency among dental practitioners ranged from 6.4 to 9.8 for all six procedures questioned, but equal scores between cadres were only for tooth extractions (9.7 vs. 9.1 respectively). Competencies for placing restorations were all rated lower by other cadres (p varied from <. 05 to < .001). Most of the patients had a negative overall attitude (-1.99) towards tooth filling treatment although those with previous tooth filling experience had positive attitudes (1.00). The mean willingness-to-pay (WTP) values for tooth filling (7,398 and 7,726 Tshs for anterior and posterior teeth, respectively) were higher (p < 0.001) than for tooth extraction irrespective of tooth type (5,448 and 6,188 Tshs, respectively). Being 45+ years increased the likelihood to offer lower WTP values. More than half (55%) of the patients would experience significant financial impacts as a result of their utilization of dental services. The findings clearly suggest that there are a multitude of factors that influence the observed dental treatment patterns being predominated by tooth extractions in Tanzania. Keywords: Willingness-to-pay, costs, dental services, attitudes, TanzaniaTIIVISTELMÄ Valikoitujen hammashoitopalvelujen saatavuus, niihin liittyvät asenteet ja käytön kustannukset vähävaraisessa maassa Tutkimus selvitti valikoitujen hammashoitopalveluiden saatavuutta sekä niihin liittyviä asenteita ja käyttöön liittyviä taloudellisia tekijöitä vähävaraisessa maassa. Ensin tehtiin kyselytutkimus kaikissa hammashoitopalveluja tarjoavissa yksiköissä neljällä valitulla tansanialaisella hallintoalueella näiden yksiköiden johtajien ja hammashoitoa antavien työntekijöiden keskuudessa. Toiseksi toteutettiin sarja poikkileikkaustutkimuksia kyselyin näiden neljän hallintoalueen avohoitopotilaiden keskuudessa. Kyselyissä selvitettiin valikoitujen hammashoitopalvelujen kustannuksia, hammashoitomateriaalien ja -laitteiden saatavuutta, hammashoidon työntekijöiden kokemia valmiuksia, potilaiden asenteita ja maksuhalukkuutta paikkaushoitoon sekä potilaille hammashoidosta aiheutuviin kustannuksia. Materiaaleja ja laitteita oli vaihtelevasti saatavilla. Vain kolmessa tutkitusta 28 hoitolasta kaikki seitsemän tutkimuksessa tarkasteltua laitetta olivat käytössä ja toimintakunnossa. Hammaslääkärien kokemat valmiudet toimenpiteisiin vaihtelivat (6,4–9,8) ja olivat saman tasoisia muiden ammattiryhmien kanssa vain poistoissa (9,7 vs. 9,1). Paikkausvalmiudet olivat alempia muilla ammattiryhmillä (p <0,05–<0,001). Useimmilla potilailla oli negatiivinen kokonaisasenne (-1,99) paikkaushoitoon. Heillä, joilla oli aiempaa kokemusta paikkaushoidosta, oli positiivinen asenne (1,00). Paikkaushoidon maksuhalukkuuden keskiarvot olivat huomattavasti korkeammat (7398 Tansanian shillinkiä (Tshs) etualueella ja 7726 Tshs taka-alueella) kuin hammaspoistojen maksuhalukkuuden (5448 Tshs etualueella ja 6188 Tshs taka-alueella, p<0,001). Hammashoitopalvelujen käyttö aiheutti yli puolelle (55 %) tutkituista potilaista merkittäviä taloudellisia vaikutuksia. Tulokset osoittavat selvästi, että Tansaniassa monet tekijät vaikuttavat hammashoitopalvelujen tuotantoon, jota hallitsevat hampaiden poistot. Avainsanat: Maksuhalukkuus, kustannukset, hammashoitopalvelut, asenteet, Tansani

Study of the tissue inhibitors of metalloproteinases in atherosclerosis

Introduction: Atherosclerosis is a disease of the arterial wall with high levels ofmorbidity and mortality. Modern therapeutic interventions aim towards both theattenuation of the lumen stenosis as well as the stabilization of the atheromatousplaque and the avoidance of its rupture, which is the cause of the majority ofcardiovascular events. The unstable plaque can be identified by its thin fibrous cap, alesser amount of extracellular connective proteins (collagen and elastin) and extensiveinflammation. The cleavage of collagen and elastin weakens the atheromatous plaque,and is mediated by specific proteolytic molecules, the matrix metalloproteinases(MMPs), which are mostly produced by inflammatory cells (macrophages, foam cells)and the Smooth Muscle Cells within the atheroma. Accumulating evidence pointtowards the fact that the MMPs and their tissue inhibitors (TIMPs) are involved in adelicate balance, which in great part defines the clinical outcome of the disease.Statins, that are proven to exert atheroprotective role through their effect ondecreasing cholesterol blood levels, are thought to have additional atheroprotectivemechanisms through the modification of signaling pathways that control theinflammatory process and influence the MMP and TIMP expression and function.Systematic, long term exercise is known to positively contribute in the attenuation ofcardiovascular disease. The modification of the traditional cardiovascular risk factorsthrough exercise, can only in part explain its’ atherosclerosis ameliorating results,leading scientists to search for additional atheroprotective actions of exercise. Amongothers, the modification of MMP and TIMP expression and function are considered.Aim: Subject of the present study was to investigate the effects of two antiatherosclerotictherapeutic strategies, that of atorvastatin treatment and of systematicexercise, as well as their combined effects on the MMP and TIMP function, inconjunction with the atheromatous plaques’ size and stability.Experimental design: Prospective, randomized, controlled, interventional study.Setting: Center of Experimental Surgery, Foundation of Biomedical Research,Academy of Athens.Subjects: Male ApoE-/- C57BL/6J knockout mice.Methods: At the age of 8 weeks, mice were randomized in the following groups: a)Control group (CO, n=10), b) Atorvastatin treatment group (AT, n=10), c) Exercisetreatment group (EX, n=10), d) Combined atorvastatin and exercise treatment group (AT+EX, n=10). All groups were fed atherogenic, “western type” high-fat diet for thewhole study duration (24 weeks). At the beginning of the 16th week the all groupsbesides CO began receiving the predefined therapeutic intervention. Atorvastatin(10mg/kg) was administered in group AT daily for the rest of the study duration(8weeks). Group EX underwent an exercise program on a motorized treadmill(8weeks, 5days/week, 40min/session, 12m/min, 5o inclination). Group AT+EXreceived atorvastatin treatment similar to group AT, and concurrently underwent thesame exercise program as group EX. At the end of the study all mice were sacrificed.The aortic arch was removed and was further processed for paraffin embedding andoptical microscopy sectioning, while the thoracic aorta was deep frozen for lateranalysis. The area of the lumen cross-section and that of the atheromatous plaqueswas measured histochemically, and the lumen stenosis factor was calculated.Additionally, the positive stain ratio of the plaques for collagen, elastin, macrophages,smooth muscle cells, ΜΜΡ-2, ΜΜΡ-3, ΜΜΡ-8, ΜΜΡ-9, ΤΙΜΡ-1, ΤΙΜΡ-2 andTIMP-3 was measured, by means of histochemical and immunohistochemical methods. Blood samples were acquired from all mice at the beginning of the study,after the atherosclerosis establishment (16th week) and at the study’s end. Blood levelsof total cholesterol, glucose, and triglycerides were determined as well as theconcentrations of ΜΜΡ-2, ΜΜΡ-3, ΜΜΡ-8, MMP-9, ΤΙΜΡ-1, TIMP-2 and TIMP-3.The thoracic aorta sections were zymographically analyzed for the determination ofMMP-2, MMP-9, TIMP-1 and TIMP-2 activity.Results: Both therapeutic interventions attenuated atherosclerosis development. Thiswas evident in the lower extent of the atheromatic lesions in the ΑΤ group, and in thelower degree of lumen stenosis in all intervention groups. Furthermore, thetherapeutic modalities acted in synergy, achieving significantly better results in thecombined intervention group. All interventions failed to differentiate the experimentalgroups regarding weight loss and blood glucose, while only the atorvastatinadministration modestly reduced blood lipids. Atheromatous plaques showed a morestable phenotype in the intervention groups, containing more collagen and elastin,with elastin being further increased in the AT+EX group. These changes wereaccompanied by a favorable modification of MMP-3, MMP-8 and MMP-9. MMP-8function was closely associated with collagen levels, while the reduction in MMP-9prevalence and function was loosely related to the elevated levels of elastin.Therepautic interventions did not affect the smooth muscle cell content of the lesions, but did reduce the presence of macrophages in the athromatous plaques, but thecombined intervention did not confer additional changes. Exercise treatment increasedTIMP-1 levels, and subsequently reduced the net proteolytic activity within theplaque. On the contrary, TIMP-2 was decreased in all intervention groups and TIMP-3 appeared unaffected. While the blood levels of the above molecules increasedduring atherosclerosis development in comparison with baseline measurements (withthe exception of TIMP-3), they did not change after the therapeutic interventions inaccordance to their intra-plaque levels. Active MMP-2 was reduced after atorvastatinadministration, increased after exercise treatment and exhibited unchanged levels afterthe combined interventions. Its proenzyme expression though, was reduced after allinterventions.Conclusions: The two therapeutic strategies act with synergy regarding the extent ofthe atheromatic lesions and lumen stenosis. Moreover, they stabilize the plaque byincreasing its content in elastin and collagen, which is achieved mainly by theirinfluence on MMP and TIMP levels. The increased elastin levels are attributed ingreat part in the modification of MMP-2/TIMP-2 balance and in the MMP-9 levels reduction that is achieved through the lower extent of macrophage prevalence insidethe plaque. MMP-8 concentration in the lesion area is correlated with the pattern ofcollagen preservation. The effects of both atorvastatin and exercise on the MMP andTIMP levels are mainly associated with the macrophage amount and type, and not inchanges regarding smooth muscle cells. The increased MMP-2, MMP-3, MMP-8 andMMP-9, as well as TIMP-1 and TIMP-2 (but not TIMP-3) blood concentration is amarker of atherosclerosis. On the other hand, blood levels of MMP-2, MMP-3 andMMP-8 are not correlated with their concentration within the atheromatous plaqueafter the therapeutic interventions, and therefore cannot serve as markers of thedisease development or amelioration.Εισαγωγή: Η αθηρωμάτωση αποτελεί μια πάθηση του αρτηριακού τοιχώματος με εξαιρετικά μεγάλη νοσηρότητα και θνητότητα. Στόχος των παρεμβάσεων αντιμετώπισής της αποτελεί πλέον εκτός του μετριασμού του στενωτικού της χαρακτήρα, η σταθεροποίηση των αθηρωματικών πλακών, που με τη ρήξη τους μπορούν να προκαλέσουν πληθώρα καρδιαγγειακών συμβαμάτων. Η ασταθήςαθηρωματική πλάκα χαρακτηρίζεται μεταξύ άλλων από λεπτή ινώδη κάψα,μειωμένες συγκεντρώσεις των εξωκυττάριων συνδετικών πρωτεϊνών (κολλαγόνου και ελαστίνης), κι εκτεταμένη φλεγμονώδη αντίδραση. Η λύση του κολλαγόνου και της ελαστίνης αποδυναμώνει την αθηρωματική πλάκα, και καταλύεται από συγκεκριμένα πρωτεολυτικά ένζυμα, τις μεταλλοπρωτεϊνάσες (MMPs), οι οποίες παράγονται κυρίως από τα φλεγμονώδη κύτταρα (μακροφάγα, αφρώδη κύτταρα) και τα λεία μυϊκά κύτταρα της αθηρωματικής περιοχής. Υπάρχουν ισχυρές ενδείξεις ότι οι MMPsκαι οι αναστολείς τους (TIMPs) βρίσκονται σε μια λεπτή ισορροπία που καθορίζει σε μεγάλο βαθμό την κλινική έκβαση της νόσου. Οι στατίνες, που αποδεδειγμένα ασκούν αθηροπροστατευτικό ρόλο μέσω της μείωσης της χοληστερόλης στο αίμα, πιστεύεται τελευταία ότι ασκούν επιπλέον προστασία μέσω της ρύθμισης μονοπατιών σήμανσης που ελέγχουν τη φλεγμονώδη αντίδραση και επηρεάζουν την έκφραση και λειτουργία των MMPs και των αναστολέων τους. Η συστηματική, μακροχρόνια άσκηση είναι γνωστό ότι συμβάλλει στη μείωση της καρδιαγγειακής νοσηρότητας και θνητότητας.Η τροποποίηση των κλασσικών παραγόντων καρδιαγγειακού κινδύνου μπορεί μόνο εν μέρει να εξηγήσει τα ευεργετικά αποτελέσματα της άσκησης οδηγώντας τους ερευνητές να αναζητήσουν επιπλέον αντιαθηρογόνες δράσεις της, μεταξύ άλλων και της τροποποίησης της έκφρασης και λειτουργίας των MMPs και των TIMPs.Σκοπός: Αντικείμενο της παρούσας διδακτορικής διατριβής ήταν η διερεύνηση της επίδρασης δύο θεραπευτικών στρατηγικών έναντι της αθηρωμάτωσης, της χορήγησης ατορβαστατίνης και της συστηματικής άσκησης, αλλά και του συνδυασμού τους, στη λειτουργία των MMPs και των αναστολέων τους και η συσχέτιση των επιδράσεων αυτών με την εξέλιξη της αθηρωματικής νόσου, όπως αυτή προσδιορίζεται από το μέγεθος και την σταθερότητα των αθηρωματικών αλλοιώσεων.Πειραματικός σχεδιασμός: Προοπτική, τυχαιοποιημένη, ελεγχόμενη μελέτη παρέμβασης. Τοποθεσία: Κέντρο Πειραματικής Χειρουργικής, Ίδρυμα Ιατροβιολογικών Ερευνών Ακαδημίας Αθηνών. Πειραματόζωα: Αρσενικοί μύες με ομόζυγη έλλειψη της απολιποπρωτεϊνης Ε(ApoE-/- C57BL/6J knockout mice).Μέθοδος: Την 8η εβδομάδα ηλικίας τους οι μύες τυχαιοποιήθηκαν στις παρακάτω ομάδες: α) Ομάδα ελέγχου αθηρωμάτωσης (CO, n=10), β) Ομάδα χορήγησης ατορβαστατίνης. (ΑΤ, n=10) γ) Ομάδα άσκησης (EX, n=10). δ) Ομάδα συνδυασμένης παρέμβασης ατορβαστατίνης και άσκησης (AT+EX, n=10). Σε όλες τις ομάδες χορηγήθηκε ειδική αθηρογόνος τροφή, με υψηλή περιεκτικότητα σε λιπαρά, για 24 εβδομάδες. Την 16η πειραματική εβδομάδα, ξεκίνησαν οι θεραπευτικές παρεμβάσεις στις ομάδες πλην τηs CO. Στους μύες της ομάδας AT χορηγήθηκε ατορβαστατίνη10mg/Kgr βάρους/ημέρα μέχρι το τέλος της μελέτης. Οι μύες της ομάδας EXυποβλήθηκαν σε πρόγραμμα εκγύμνασης σε ειδικό κυλιόμενο τάπητα μέχρι το πέρας της μελέτης, και οι μύες της ομάδας AT+EX έλαβαν ατορβαστατίνη ομοίως με τους μύες της ομάδας AT και ταυτόχρονα ακολούθησαν πρόγραμμα άσκησης ομοίως με τους μύες της ομάδας EX. Με το τέλος της μελέτης ακολούθησε ευθανασία των μυών. Έγινε λήψη του αορτικού τόξου των μυών προς έγκλειση σε παραφίνη και τομές μικροσκοπίας, και συντήρηση σε βαθιά κατάψυξη της θωρακικής αορτής. Με ιστολογική ανάλυση προσδιορίστηκε το εμβαδό του αγγειακού αυλού και των αθηρωματικών πλακών, και ο βαθμός στένωσης του αυλού κατά μήκος τους αορτικού τόξου. Επιπλέον, με ιστοχημικές και ανοσοϊστοχημικές μεθόδους προσδιορίστηκε το ποσοστό της επιφάνειας της αθηρωματικής πλάκας με θετική χρώση για κολλαγόνο,ελαστίνη, μακροφάγα, λεία μυϊκά κύτταρα, ΜΜΡ-2, ΜΜΡ-3, ΜΜΡ-8, ΜΜΡ-9,ΤΙΜΡ-1, ΤΙΜΡ-2 και TIMP-3. Αιμοληψίες έγιναν σε όλους τους μύες κατά την έναρξη της μελέτης, μετά την ανάπτυξη αθηρωμάτωσης (16η εβδομάδα) και κατά τη λήξη της μελέτης και προσδιορίστηκαν τα επίπεδα της γλυκόζης, της ολικής χοληστερόλης, των τριγλυκεριδίων, της ΜΜΡ-2, της ΜΜΡ-3, της ΜΜΡ-8, της MMP-9, του ΤΙΜΡ-1, του TIMP-2 και του TIMP-3 στο αίμα. Τα τμήματα της θωρακικής αορτής αναλύθηκαν με ζυμογραφία για προσδιορισμό της ενεργού/ανασταλτικής δράσης της MMP-2, της MMP-9, του TIMP-1 και του TIMP-2.Αποτελέσματα: Και οι δύο θεραπευτικές παρεμβάσεις επιβράδυναν την εξέλιξη της αθηρωματικής πλάκας. Αυτό εκφράστηκε ως μειωμένη έκταση του αθηρώματος στις ομάδες που έλαβαν ατορβαστατίνη, και ως μειωμένος βαθμός στένωσης σε όλες τις ομάδες παρέμβασης. Επιπλέον, οι δύο θεραπευτικές παρεμβάσεις εμφάνισαν συνέργεια επιτυγχάνοντας σημαντικά καλύτερα αποτελέσματα στην ομάδα συνδυασμένης παρέμβασης. Όλες οι παρεμβάσεις απέτυχαν να διαφοροποιήσουν τις ομάδες σε σχέση με την απώλεια βάρους και τη γλυκόζη αίματος, ενώ μόνο η χορήγηση ατορβαστατίνης πέτυχε μέτριου βαθμού μείωση των λιπιδίων του αίματος.Οι αθηρωματικές πλάκες εμφάνισαν σταθερότερο φαινότυπο στις ομάδες παρέμβασης, με περισσότερο κολλαγόνο κιι ελαστίνη, και με την ελαστίνη να είναι περεταίρω αυξημένη στην ομάδα συνδυασμού παρεμβάσεων. Οι αλλαγές αυτές συνοδεύτηκαν από ευνοϊκή ρύθμιση των MMP-3, MMP-8 και MMP-9. Η λειτουργικότητα της MMP-8 φάνηκε να είναι σε στενή συνάρτηση με τα επίπεδα κολλαγόνου, ενώ η μείωση της MMP-9 σχετίζεται εν μέρει με τα αυξημένα επίπεδα ελαστίνης. Οι θεραπευτικές παρεμβάσεις δε μετέβαλλαν σημαντικά τη σύσταση της αθηρωματικής πλάκας σε λεία μυϊκά κύτταρα, μείωσαν όμως το περιεχόμενό της σε μακροφάγα, χωρίς ο συνδυασμός τους να επιφέρει επιπλέον οφέλη. Η παρέμβαση της άσκησης αύξησε τα επίπεδα του TIMP-1, μεταβάλλοντας την καθαρή πρωτεολυτική δραστηριότητα εντός της πλάκας. Αντίθετα, ο TIMP-2 μειώθηκε σε όλες τις ομάδες θεραπευτικής παρέμβασης, ενώ ο TIMP-3 δεν παρουσίασε μεταβολή. Τα επίπεδα των μορίων αυτών στο αίμα, αν και αυξήθηκαν σε συνάρτηση με την ανάπτυξη αθηρωμάτωσης (με εξαίρεση τον TIMP-3), δεν μετέβαλαν τις συγκεντρώσεις τους στο αίμα μετά από τις θεραπευτικές παρεμβάσεις αντίστοιχα με τα επίπεδά τους στην πλάκα. Η ενεργός μορφή της MMP-2 μειώθηκε μετά από χορήγηση ατορβαστατίνης,αυξήθηκε με την άσκηση και παρέμεινε σε σταθερά επίπεδα κατά τον θεραπευτικό συνδυασμό. Τα προένζυμα όμως της MMP-2 μειώθηκαν μετά από όλες τις παρεμβάσεις. Συμπεράσματα: Οι δύο θεραπευτικές παρεμβάσεις παρουσιάζουν συνέργεια όσον αφορά στην έκταση των αθηρωματικών αλλοιώσεων και τη στένωση, με το συνδυασμό των δύο στρατηγικών να δίνει εμφανώς καλύτερα αποτελέσματα. Επίσης σταθεροποιούν το φαινότυπο της πλάκας, αυξάνοντας την περιεκτικότητα σε ελαστίνη και κολλαγόνο, δράσεις προς σταθεροποίηση της αθηρωματικής πλάκας που είναι σε μεγάλο βαθμό συνάρτηση της επίδρασής τους στα επίπεδα MMPs καιTIMPs. Ο μηχανισμός της αύξησης της ελαστίνης σχετίζεται με την ελαττωμένη τηςλύση, μέσω της ισορροπίας MMP-2/TIMP-2 και μέσω της μείωσης των επιπέδωνMMP-9 που προκύπτουν από τη μεταβολή της σύστασης της αθηρωματικής πλάκας σε μακροφάγα κύτταρα. Η συγκέντρωση της MMP-8 στην αθηρωματική πλάκα παρουσιάζει ισχυρή συσχέτιση με το μοτίβο συντήρησης του κολλαγόνου. Οι επιδράσεις της ατορβαστατίνης και της άσκησης στα επίπεδα των MMPs και τωνTIMPs σχετίζονται πρωτίστως με μεταβολές στο πλήθος και τον φαινότυπο των μακροφάγων, και όχι σε μεταβολές στα λεία μυϊκά κύτταρα. Η αύξηση των συγκεντρώσεων της MMP-2, MMP-3, MMP-8 και MMP-9, όπως επίσης και τωνTIMP-1 και TIMP-2 (αλλά όχι του TIMP-3) στο αίμα αποτελούν δείκτες αθηρωμάτωσης. Οι συγκεντρώσεις όμως των δεικτών των MMP-2, MMP-3 καιMMP-8 στο αίμα δεν σχετίζονται άμεσα με τις συγκεντρώσεις τους στην αθηρωματική πλάκα έπειτα από τις θεραπευτικές παρεμβάσεις και άρα δεν μπορούν να αποτελούν δείκτες της εξέλιξης της νόσου

Deregulation of the Kallikrein Protease Family in the Salivary Glands of the Sjogren's Syndrome ERdj5 Knockout Mouse Model

Introduction The purpose of this study was to identify differentially expressed proteins in salivary glands of the ERdj5 knockout mouse model for Sjogren's syndrome and to elucidate possible mechanisms for the morbid phenotype development. At the same time, we describe for the first time the sexual dimorphism of the murine submandibular salivary gland at the proteome level. Methods We performed Liquid Chromatography/Mass Spectrometry in salivary gland tissues from both sexes of ERdj5 knockout and 129SV wildtype mice. The resulting list of proteins was evaluated with bioinformatic analysis and selected proteins were validated by western blot and immunohistochemistry and further analyzed at the transcription level by qRT-PCR. Results We identified 88 deregulated proteins in females, and 55 in males in wildtype vs knockout comparisons. In both sexes, Kallikrein 1b22 was highly upregulated (fold change&gt;25, ANOVA p&lt;0.0001), while all other proteases of this family were either downregulated or not significantly affected by the genotype. Bioinformatic analysis revealed a possible connection with the downregulated NGF that was further validated by independent methods. Concurrently, we identified 416 proteins that were significantly different in the salivary gland proteome of wildtype female vs male mice and highlighted pathways that could be driving the strong female bias of the pathology. Conclusion Our research provides a list of novel targets and supports the involvement of an NGF-mediating proteolytic deregulation pathway as a focus point towards the better understanding of the underlying mechanism of Sjogren's syndrome.Funding agencies:  Linköping’s University (PM, NY, MT, and GS) and the State Scholarships Foundation, IKY, Greece (EA).</p

MAPK Pathways in Ocular Pathophysiology: Potential Therapeutic Drugs and Challenges

Mitogen-activated protein kinase (MAPK) pathways represent ubiquitous cellular signal transduction pathways that regulate all aspects of life and are frequently altered in disease. Once activated through phosphorylation, these MAPKs in turn phosphorylate and activate transcription factors present either in the cytoplasm or in the nucleus, leading to the expression of target genes and, as a consequence, they elicit various biological responses. The aim of this work is to provide a comprehensive review focusing on the roles of MAPK signaling pathways in ocular pathophysiology and the potential to influence these for the treatment of eye diseases. We summarize the current knowledge of identified MAPK-targeting compounds in the context of ocular diseases such as macular degeneration, cataract, glaucoma and keratopathy, but also in rare ocular diseases where the cell differentiation, proliferation or migration are defective. Potential therapeutic interventions are also discussed. Additionally, we discuss challenges in overcoming the reported eye toxicity of some MAPK inhibitors

A double-crosslinked nanocellulose-reinforced dexamethasone-loaded collagen hydrogel for corneal application and sustained anti-inflammatory activity

In cases of blinding disease or trauma, hydrogels have been proposed as scaffolds for corneal regenera-tion and vehicles for ocular drug delivery. Restoration of corneal transparency, augmenting a thin cornea and postoperative drug delivery are particularly challenging in resource-limited regions where drug avail-ability and patient compliance may be suboptimal. Here, we report a bioengineered hydrogel based on porcine skin collagen as an alternative to human donor corneal tissue for applications where long-term stability of the hydrogel is required. The hydrogel is reinforced with cellulose nanofibers extracted from the Ciona intestinalis sea invertebrate followed by double chemical and photochemical crosslinking. The hydrogel is additionally loaded with dexamethasone to provide sustained anti-inflammatory activity. The reinforced double-crosslinked hydrogel after drug loading maintained high optical transparency with sig-nificantly improved mechanical characteristics compared to non-reinforced hydrogels, while supporting a gradual sustained drug release for 60 days in vitro . Dexamethasone, after exposure to crosslinking and sterilization procedures used in hydrogel production, inhibited tube formation and cell migration of TNF alpha-stimulated vascular endothelial cells. The drug-loaded hydrogels suppressed key pro-inflammatory cytokines CCL2 and CXCL5 in TNF alpha-stimulated human corneal epithelial cells. Eight weeks after intra-stromal implantation in the cornea of 12 New-Zealand white rabbits subjected to an inflammatory suture stimulus, the dexamethasone-releasing hydrogels suppressed TNF alpha, MMP-9, and leukocyte and fibrob-last cell invasion, resulting in reduced corneal haze, sustained corneal thickness and stromal morphology, and reduced overall vessel invasion. This collagen-nanocellulose double-crosslinked hydrogel can be im-planted to treat corneal stromal disease while suppressing inflammation and maintaining transparency after corneal transplantation. Statement of significance To treat blinding diseases, hydrogel scaffolds have been proposed to facilitate corneal restoration and ocular drug delivery. Here, we improve on a clinically tested collagen-based scaffold to improve me-chanical robustness and enzymatic resistance by incorporating sustainably sourced nanocellulose and dual chemical-photochemical crosslinking to reinforce the scaffold, while simultaneously achieving sus-tained release of an incorporated anti-inflammatory drug, dexamethasone. Evaluated in the context of a corneal disease model with inflammation, the drug-releasing nanocellulose-reinforced collagen scaffold maintained the cornea's transparency and resisted degradation while suppressing inflammation postop-Funding Agencies|European Commission [667400]; LinkoCare Life Sciences AB; NaturaLens AB</p

Saliva antibody-fingerprint of reactivated latent viruses after mild/asymptomatic COVID-19 is unique in patients with myalgic-encephalomyelitis/chronic fatigue syndrome

Background Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic disease considered to be triggered by viral infections in a majority of cases. Symptoms overlap largely with those of post-acute sequelae of COVID-19/long-COVID implying common pathogenetic mechanisms. SARS-CoV-2 infection is risk factor for sustained latent virus reactivation that may account for the symptoms of post-viral fatigue syndromes. The aim of this study was first to investigate whether patients with ME/CFS and healthy donors (HDs) differed in their antibody response to mild/asymptomatic SARS-CoV-2 infection. Secondly, to analyze whether COVID-19 imposes latent virus reactivation in the cohorts. Methods Anti-SARS-CoV-2 antibodies were analyzed in plasma and saliva from non-vaccinated ME/CFS (n=95) and HDs (n=110) using soluble multiplex immunoassay. Reactivation of human herpesviruses 1-6 (HSV1, HSV2, VZV, EBV, CMV, HHV6), and human endogenous retrovirus K (HERV-K) was detected by anti-viral antibody fingerprints in saliva. Results At 3-6 months after mild/asymptomatic SARS-CoV-2 infection, virus-specific antibodies in saliva were substantially induced signifying a strong reactivation of latent viruses (EBV, HHV6 and HERV-K) in both cohorts. In patients with ME/CFS, antibody responses were significantly stronger, in particular EBV-encoded nuclear antigen-1 (EBNA1) IgG were elevated in patients with ME/CFS, but not in HDs. EBV-VCA IgG was also elevated at baseline prior to SARS-infection in patients compared to HDs. Conclusion Our results denote an altered and chronically aroused anti-viral profile against latent viruses in ME/CFS. SARS-CoV-2 infection even in its mild/asymptomatic form is a potent trigger for reactivation of latent herpesviruses (EBV, HHV6) and endogenous retroviruses (HERV-K), as detected by antibody fingerprints locally in the oral mucosa (saliva samples). This has not been shown before because the antibody elevation is not detected systemically in the circulation/plasma.Funding: Swedish Research Council [211832Pj01H2/Infection-Autoimmunity-B-lymphoma]; Swedish Cancer Society; Linkoeping University;  [4.3-2019-00201 GD-2020-138]</p

The role of exercise training and the endocannabinoid system in atherosclerotic plaque burden and composition in Apo-E-deficient mice

Introduction: We investigated the effect of combining exercise training and treatment with an endocannabinoid receptor 1 inhibitor (Rimonabant) on atherosclerosis burden and composition. Methods: Forty-eight apolipoprotein E-deficient (ApoE-/-) mice were kept on a 16-week high-fat diet. Mice were then placed on a normal diet and were randomized to the following groups with n=12 mice for 6 more weeks: 1) Control (Co) - no intervention; 2) Exercise (Ex) - exercise training on treadmill; 3) Rimonabant (Ri) - oral administration of rimonabant (10 mg/kg/day); or 4) Rimonabant+Exercise (RiEx) - combination of Ri and Ex groups treatment. At the end, all animals were sacrificed, and blood samples, as well as aortic root specimens, were obtained for histomorphometric analysis and quantification of the serum and plaque content of matrix metalloproteinases (MMPs). Results: The mean plaque area was significantly smaller (RiEx: 43.18±1.72%, Ri: 44.66±3.1%, Ex: 49±4.10%, Co: 70.43±2.83%) in all active treatment groups relative to the Co group (p<0.01). Conversely, the relative concentrations of collagen and elastin were increased significantly across all treatment groups compared to Co (p<0.05). Immunohistochemical analysis revealed significantly reduced macrophage content within plaques after all interventions, with the most pronounced effect observed after combined treatment (RiEx: 9.4±3.92%, Ri: 15±2.45%, Ex: 19.78±2.79%, Co: 34.25±4.99%; p<0.05). Within plaques, the TIMP-1 concentration was significantly upregulated in exercise-treated groups. MMP-3 and MMP-9 concentrations were equivalently decreased in all three active treatment groups compared to controls (p<0.001). Discussion: Both exercise and rimonabant treatments induced plaque regression and promoted plaque stability. The combined treatment failed to show additive or synergistic benefits relative to either intervention alone

The Complementary Effects of Atorvastatin and Exercise Treatment on the Composition and Stability of the Atherosclerotic Plaques in ApoE Knockout Mice

<div><p>Aim</p><p>This study aimed to investigate the effects of combined atorvastatin and exercise treatment on the composition and stability of the atherosclerotic plaques in apolipoproteinE (apoE) knockout mice.</p><p>Methods</p><p>Forty male, apoE^−/− mice were fed a high-fat diet for 16 weeks. Thereafter, while maintained on high-fat diet, they were randomized into four (n = 10) groups for 8 additional weeks: Group CO: Control. Group AT: Atorvastatin treatment (10 mg/Kg/day). Group EX: Exercise-training on treadmill. Group AT+EX: Atorvastatin and simultaneous exercise training. At the study’s end, plasma cholesterol levels, lipids and triglycerides were measured, along with the circulating concentrations of matrix-metalloproteinases (MMP-2,3,8,9) and their inhibitors (TIMP-1,2,3). Plaque area and the relative concentrations of collagen, elastin, macrophages, smooth muscle cells, MMP-2,3,8,9 and TIMP-1,2,3 within plaques were determined. Lastly, MMP activity was assessed in the aortic arch.</p><p>Results</p><p>All intervention groups showed a lower degree of lumen stenosis, with atheromatous plaques containing more collagen and elastin. AT+EX group had less stenosis and more elastin compared to single intervention groups. MMP-3,-8 -9 and macrophage intra-plaque levels were reduced in all intervention groups. EX group had increased TIMP-1 levels within the lesions, while TIMP-2 was decreased in all intervention groups. The blood levels of the above molecules increased during atherosclerosis development, but they did not change after the therapeutic interventions in accordance to their intra-plaque levels.</p><p>Conclusion</p><p>The two therapeutic strategies act with synergy regarding the extent of the lesions and lumen stenosis. They stabilize the plaque, increasing its content in elastin and collagen, by influencing the MMP/TIMP equilibrium, which is mainly associated with the macrophage amount. While the increased MMP-2,-3,-8 -9, as well as TIMP-1 and TIMP-2 circulating levels are markers of atherosclerosis, they are not correlated with their corresponding concentrations within the lesions after the therapeutic interventions, and cannot serve as markers for the disease development/amelioration.</p></div

Representative images from zymograms for the identification of metalloproteinase activity and reverse zymograms for the identification of TIMP activity.

<p>A) Zymograhy, MMP-2, MMP-9 and proMMP-2 identification. B) Reverse zymography, TIMP-1 and TIMP-2 identification.</p