In vitro antiplasmodial evaluation of ethanolic and n-hexane extracts of Parinari curatellifolia stem bark

Parinari curatellifolia and other Parinari species are used traditionally in many parts of Africa as a remedy for malaria among other diseases. To ascertain this folkloric claim, the antiplasmodial potential of ethanol extract of Parinari curatellifolia stem bark (EEPCSB) and n-hexane extract of Parinari curatellifolia stem bark (HEPCSB) on Plasmodium falciparum was studied. Parasites were grown in a 96-well plate containing Roswell Park Memorial Institute-1640. The wells were grouped into: control (untreated), artemether-treated, EEPCSB-treated and HEPCSBtreated groups. Treatments were administered to the tune of 10, 20, 40 and 80 μg/ml. Parasitemia was observed by microscopy after 24, 48 and 72h of incubation. EEPCSB and HEPCSB elicited dose and duration-dependent reduction (p<0.05) in parasitemia when compared with the untreated group. The recorded percentage parasite inhibition by the extracts was lower (p<0.05) when compared with artemether. There was no difference (p>0.05) in plasmodium lactate dehydrogenase activity of EEPCSB-treated and artemether-treated groups. Findings from this study show that extracts of P. curatellifolia stem bark, especially EEPCSB, demonstrated excellent inhibitory activities against P. falciparum and can be a good source of compounds for the development of novel antimalarial drugs. Keywords: Parinari curatellifolia; Extracts; Plasmodium falciparum; Parasitemia; Antiplasmodial

Naringenin and ellagic acid reduce tetrazolium salt in the absence of cells

Background: Plant derived compounds, naringenin and ellagic acid might interfere in MTT (3-(4, 5- Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide) assay in the form of reducing the tetrazolium salt in the absence of cells. Objective: Effect of naringenin and ellagic acid in MTT assay. Methods: MTT assay has been performed at different conditions like, varying the concentration of phytocompounds, incubation time, and changing the media. Results: Ellagic acid at the concentration of 80 μg/ml showed maximum absorbance of 0.579 in the presence of RPMI media (Roswell Park Memorial Institute Medium) that has 10 % newborn calf serum. Naringenin showed higher absorbance of 2.74 at 80 μg/ml in RPMI media that has 10 % newborn calf serum. Naringenin and Ellagic acid had better ability to reduce the MTT in the presence of RPMI media than anhydrous ethanol. Conclusion: Naringenin reduced the MTT better than ellagic acid in RPMI-1640 medium. These results show the need to be cautious of choosing MTT assay to measure the metabolic activity of cells treated with phytocompounds.

Abundance interaction in Candida albicans and Candida glabrata mixed biofilms under diverse conditions

Candida albicans and Candida glabrata are frequently coisolated from the oral cavity in immunosuppressive or immunocompromised individuals. Their relationship is usually defined as competition as C. glabrata can inhibit growth of C. albicans in cohabitation. In this study, eight C. albicans isolates as well as two C. glabrata strains were used to investigate the effects of culture medium (Roswell Park Memorial Institute [RPMI]-1640, YPD, YND), incubation time (24 h, 48 h, 72 h, 96 h), initial inoculum (C. glabrata: C. albicans = 2:1, 1:1, 1:2), and medium state (static and dynamic states) on viable cell enumeration and relative abundance in both Candida SB and MB. The results showed that in most cases, C. glabrata and C. albicans SB and MB flourished in RPMI-1640 at 24 h under dynamic state compared with other conditions. Except YPD medium, there were high proportions of preponderance of C. albicans over C. glabrata in MB compared with SB. High initial inoculum promoted corresponding Candida number in both SB and MB and its abundance in MB relative to SB. This study revealed an impact of several environmental conditions on the formation of C. albicans and C. glabrata SB and MB and their abundance in MB in comparison with SB, deepening our understanding of both Candida interaction and their resistance mechanism in MB. Lay Summary This study described the effects of diverse experimental conditions on the numbers of Candida albicans and Candida glabrata single biofilms and mixed biofilms and their abundance.

The prevalence and clinical significance of microcolonies when tested according to contemporary interpretive breakpoints for fluconazole against Candida species using E-test

Changes in the interpretive-breakpoints for antifungals against various Candida species have raised the need to examine the significance of the phenomenon of the growth of microcolonies in agar diffusion inhibition zones, which has generally been considered negligible. The objective was to determine the incidence of cases in which microcolonies demonstrate fluconazole resistance according to current interpretive-breakpoints and whether their growth is associated with therapeutic failure. The fluconazole minimum inhibitory concentrations (MICs) of 100 blood culture isolates of Candida were performed by E-test on Roswell Park Memorial Institute (RPMI) agar and examined for the appearance of microcolonies. Fluconazole MICs of microcolonies were then determined over three generations. The significance of the phenomenon of microcolonies was determined according to clinical data retrieved from electronic files. Microcolonies were a common phenomenon among Candida isolates following incubation on RPMI agar, with a higher frequency among C. albicans isolates as compared to non-albicans Candida across generations (57–93% vs 31–93%, respectively) and a similar fluconazole susceptibility rate over three generations. The rate of microcolonies was similar in both patients with successful and unsuccessful outcome (41% vs 42%, respectively). Microcolonies are a common phenomenon. No increase in MIC was demonstrated throughout three generations of microcolony inoculation on RPMI, and no difference in clinical outcome was observed.

Long live the worms: methods for maintaining and assessing the viability of intestinal stages of Parascaris spp. in vitro

In vitro maintenance of helminth parasites enables a variety of molecular, pharmaceutical and immunological analyses. Currently, the nutritional and environmental in vitro requirements of the equine ascarid parasite, Parascaris spp., have not been determined. Additionally, an objective method for assessing viability of Parascaris spp. intestinal stages does not exist. The purpose of this study was to ascertain the in vitro requirements of intestinal stages of Parascaris spp., and to develop a viability assessment method. A total of 1045 worms were maintained in a total of 212 cultures. Worms obtained from naturally infected foals at necropsy were immediately placed in culture flasks containing 200 mL of culture media. A variety of media types, nutrient supplementation and environmental conditions were examined. A motility-based scoring system was used to assess worm viability. Worms maintained in Roswell Park Memorial Institute-1640 had significantly better viability than any other media (P < 0.0001) and all media types supplemented with any of the nutrients examined (P < 0.0001). The use of a platform rocker also significantly improved viability (P = 0.0305). This is the first study to examine the requirements for maintaining Parascaris spp. intestinal stages in vitro and to evaluate their viability based on movement using an objective scoring system.

In vitro culture of L3 larvae of nematodes obtained from the African giant snail Lissachatina fulica (Mollusca: Gastropoda) in Santa Fe de Antioquia

Introducción. Más de 170 municipios colombianos están invadidos por Lissachatina fulica, caracol africano que puede portar larvas de nematodos de interés en salud humana y veterinaria. Los parásitos entran al caracol huésped intermediario en el estadio de larva L1, y allí cambian a L2 y L3, formas estas capaces de infectar a vertebrados.Objetivo. Estandarizar el cultivo in vitro de las L3 portadas por especímenes de L. fulica recolectados en Santa Fe de Antioquia.Materiales y métodos. Entre julio y noviembre de 2014 se recolectaron 10 caracoles, se sacrificaron y se digirieron con ácido clorhídrico al 0,7 %. Las larvas se recuperaron mediante la técnica de Baermann; se cultivaron 36 días en los medios Schneider, mínimo esencial de Eagle modificado por Dulbecco (Dulbecco’s Modified Eagles Minimal Essential Medium, DMEM), y Roswell Park Memorial Institute (RPMI), con suero fetal bovino (SFB) al 20 % y sin este, y agua destilada con SFB al 20 %. Los medios de cultivo se cambiaron cada 36 horas. Las larvas se midieron con el microscopio utilizando reglilla ocular, y se evaluaron la supervivencia, la longitud y el ancho. Se calcularon datos estadísticos de resumen y se hicieron gráficos de cajas y bigotes, así como la prueba t de Student. El nivel de significación (p) se estableció como menor de 0,05.Resultados. El 50 % de las larvas sobrevivió, 85 % en DMEM con SFB al 20 %, el 70 % con RPMI más SFB al 20 %, el 60 % en RPMI, el 50 % en Schneider más SFB al 20 %, el 45 % en Schneider y el 40 % en DMEM. El control sobrevivió diez días. Hubo diferencias significativas entre la longitud inicial promedio de las larvas y la longitud final promedio en los medios con suplementos: inicial, 645,83 μm; final en DMEM más SFB al 20 %, 732,65 μm (p<0,001); en RPMI más SFB al 20 %, 718,79 μm (p<0,001), y en Schneider más SFB al 20 %, 696,12 μm (p<0,01). No hubo diferencias significativas entre la anchura inicial promedio, de 24,99 μm, y la final.Conclusiones. El mejor medio para cultivar las L3 de L. fulica fue el DMEM más SFB al 20 %. En la evaluación del crecimiento larval, la longitud fue más informativa que la anchura. Las larvas estudiadas no correspondieron a Angiostrongylus cantonensis, A. costaricensis ni Aelurostrongylus abstrusus.

Ιn vitro μελέτη της ικανότητας διαφοροποίησης των μεγάλων μονοπυρήνων του περιφερικού αίματος προς κύτταρα που παράγουν ινσουλίνη

Εισαγωγή. Σε πρόσφατες έρευνες έχει παρατηρηθεί η ικανότητα των μονοκυττάρων του περιφερικού αίματος να διαφοροποιούνται προς κύτταρα με παρόμοια μορφολογία με τα μεσεγχυματικά κύτταρα. Σκοπός της μελέτης είναι να ερευνηθεί η ικανότητα των μονοκυττάρων για διαφοροποίηση και παραγωγή ινσουλίνης in vitro μετά από τη χορήγηση ή μη αυξητικών παραγόντων και αναλόγου του Παρόμοιου-με-Γλουκαγόνο Πεπτιδίου-1. Η επίδραση του αναλόγου του παρόμοιου-με-γλυκαγόνη πεπτιδίου-1 στη διαφοροποίηση των μονοκυττάρων μελετάται για πρώτη φορά. (2)Μέθοδοι Μονοκύτταρα του περιφερικού αίματος απομονώθηκαν από υγιείς δότες και καλλιεργήθηκαν για δεκατέσσερις ημέρες. Στις περισσότερες καλλιέργειες χρησιμοποιήθηκαν αυξητικοί παράγοντες και λιραγλουτίδη για να επαγάγουν τη διαφοροποίηση προς παγκρεατικά κύτταρα. Οι αυξητικοί παράγοντες είναι: ο αυξητικός παράγοντας των μονοκυττάρων, η ιντερλευκίνη 3, ο αυξητικός παράγοντας των ηπατοκυττάρων και ο αυξητικός παράγοντας των επιδερμικών κυττάρων. Οι υπόλοιπες καλλιέργειες έγιναν μόνο με τη χρήση θρεπτικού υλικού RPMI (Roswell Park Memorial Institute culture medium) και ανθρώπινου ορού αίματος. Τα επίπεδα ινσουλίνης μετρήθηκαν με τη μέθοδο του ενζυμικού ανοσοφθορισμού. Οι μετρήσεις έγιναν στην έναρξη των καλλιεργειών, στις επτά και στις δεκατέσσερις ημέρες. Η κυτταρική μορφολογία παρατηρήθηκε με το οπτικό και το ηλεκτρονικό μικροσκόπιο. Έγινε ιστολογική χρώση των κυττάρων με May Grunwald-Giemsa χρώση. Οι υποδοχείς της κυτταρικής μεμβράνης ανιχνεύθηκαν με κυτταρομετρία ροής στις επτά και δεκατέσσερις ημέρες καλλιέργειας. Η στατιστική ανάλυση έγινε με παραμετρικές και μη παραμετρικές δοκιμασίες, ανάλογα αν τα δείγματα ακολουθούν την κανονική κατανομή ή όχι. (3)Αποτελέσματα Τα μονοκύτταρα ήταν ικανά να παράγουν και να εκκρίνουν υψηλά επίπεδα ινσουλίνης ύστερα από επτά ημέρες σε καλλιέργεια. Περαιτέρω αύξηση της έκκρισης ινσουλίνης παρατηρήθηκε έπειτα από δεκατέσσερις ημέρες σε καλλιέργεια. Τα κύτταρα ήταν ικανά να διαφοροποιούνται και να συνθέτουν ινσουλίνη, ακόμη και όταν δεν είχαν προστεθεί αυξητικοί παράγοντες στο καλλιεργητικό υλικό. Η παραγωγή ινσουλίνης δεν διαφέρει στατιστικώς σημαντικά μεταξύ των καλλιεργειών που καλλιεργήθηκαν παρουσία αυξητικών παραγόντων-λιραγλουτίδης και των καλλιεργειών που καλλιεργήθηκαν απουσία αυξητικών παραγόντων. Τα διαφοροποιημένα μονοκύτταρα συνδέονταν με τα γειτονικά τους με αποφυάδες. Η μορφολογία του συνόλου των κυττάρων έμοιαζε με τα μεσεγχυματικά, τα δενδριτικά και τα προγονικά κύτταρα της μυελοειδούς σειράς. Τα κύτταρα διατήρησαν τους ώριμους υποδοχείς (CD14, CD45, CD16) στη βασική μεμβράνη τους και παράλληλα ανέπτυξαν προγονικούς υποδοχείς στην κυτταρική τους μεμβράνη (CD33, CD34, CD209).(4)Συμπεράσματα Τα μονοκύτταρα μπορούν να αποκτήσουν μορφολογικά χαρακτηριστικά των πολυδύναμων κυττάρων, όταν καλλιεργηθούν κάτω από συγκεκριμένες συνθήκες in vitro. Η μελέτη μας έδειξε για πρώτη φορά ότι τα διαφοροποιημένα μονοκύτταρα μπορούν να συνθέσουν και να εκκρίνουν ινσουλίνη σε μεγάλη ποσότητα. Είναι αξιοσημείωτο ότι τα κύτταρα που καλλιεργήθηκαν χωρίς αυξητικούς παράγοντες και λιραγλουτίδη, σταθερά έκκριναν ινσουλίνη με γλυκοζο-εξαρτώμενο τρόπο. Επίσης είναι εντυπωσιακή η οργάνωση των κυττάρων με τις κυτταρικές συνδέσεις στον πυθμένα των καλλιεργητικών τρυβλίων. Αντίστοιχες εικόνες in vivo σχηματίζουν τα μεσεγχυματικά κύτταρα στον μυελό των οστών. Τα ινσουλινοπαραγωγά κύτταρα παρουσιάζουν ενδεχομένως κλινική χρησιμότητα, αφού μπορούν θεωρητικά να χρησιμοποιηθούν ως μόσχευμα σε θεραπείες αναπλήρωσης των νησιδίων του παγκρέατος.

15. KONSENTRASI PROTEIN ANTIGEN EKSKRETORI/SEKRETORI DAN SOMATIK PADA Fasciola gigantica DAN Eurytrema pancreaticum The Protein Concentration of Excretory/Secretory and Somatic Antigen from Fasciola gigantica and Eurytrema pancreaticum

The study was aimed at finding out the difference of protein content between excretory/secretory and somatic antigen of Fasciola gigantica as well as between excretory/secretory of Fasciola gigantica and Eurytrema pancreaticum. As many as 32 Fasciola gigantica and 30 Eurytrema pancreaticum were placed into 80 ml Roswell Park Memorial Institute (RPMI) 1640 and incubated for 6 hours. Excretory/secretory as well as somatic antigen was isolated and the concentration of protein was determined, using Lowry method. The result revealed the different of protein concentration. The concentrations of excretory/secretory antigen protein in Fasciola gigantica and Eurytrema pancreaticum were 3.386 mg/ml and 0.128, mg/ml respectively. The concentrations of somatic protein and Fasciola gigantica and Eurytrema pancreaticum were 8.028 mg/ml and 0.534 mg/ml, respectively. It can be concluded that the protein concentration from somatic tissue of Fasciola gigantica and Eurytrema pancreaticum is higher than the protein concentration from excretory/secretory of Fasciola gigantica and Eurytrema pancreaticum. The protein concentration from excerory/secretory antigen of Fasciola gigantica is higher than the protein concentration from excretory/secretory antigen of Eurytrema pancreaticum. ____________________________________________________________________________________________________________________ Key words: Fasciola gigantica, Eurytrema pancreaticum, protein concentation

Toxocara canis: anthelmintic activity of quinone derivatives in murine toxocarosis

SUMMARYHuman toxocarosis is a chronic tissue parasitosis most often caused by Toxocara canis. The seroprevalence can reach up to 50%, especially among children and adolescents. The anthelmintics used in the treatment have moderate efficacy. The aim of this study was to evaluate the in vitro and in vivo anthelmintic activity of quinones and their derivatives against T. canis larvae and the cytotoxicity of the larvicidal compounds. The compounds were evaluated at 1 mg mL−1 concentration in microculture plates containing third stage larvae in an Roswell Park Memorial Institute (RPMI) 1640 environment, incubated at 37 °C in 5% CO2 tension for 48 h. Five naphthoxiranes were selected for the cytotoxicity analysis. The cell viability evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays using murine peritoneal macrophages isolated from C57BL/6 mice revealed that the naphthoxiranes (1 and 3) were less cytotoxic at a concentration of 0·05 mg mL−1. The efficacy of naphthoxiranes (1 and 3) was examined in murine toxocarosis also. The anthelmintic activity was examined by evaluating the number of larvae in the brain, carcass, liver, lungs, heart, kidneys and eyes. Compound (3) demonstrated anthelmintic activity similar to that of albendazole by decreasing the number of larvae in the organs of mice and thus could form the basis of the development of a new anthelmintic drug.

Uji Sitotoksisitas Ekstrak Metanol Daun Sisik Naga (Drymoglossum piloselloides Presl.) terhadap Sel Leukemia P388

Penelitian mengenai uji sitotoksisitas ekstrak metanol daun sisik naga (Drymoglossum piloselloides Presl.) terhadap sel leukemia P388 telah dilakukan. Penelitian ini bertujuan untuk mengetahui sitotoksisitas ekstrak metanol daun sisik naga terhadap sel leukemia P388 berdasarkan penghambatan pertumbuhan sel 50% (IC50). Metoda yang dilakukan menggunakan uji MTT (Microculture Tetrazolium Technique) pada sel kanker leukemia P388. Sel dikultur menggunakan media RPMI (Roswell Park Memorial Institute). Pertumbuhan sel diukur melalui absorbansi formazan pada panjang gelombang 540 nm pada berbagai konsentrasi dari 0,1 µg/mL sampai 100 µg/mL ekstrak sampel. IC50 ditentukan dengan persamaan logaritma antara nilai absorbansi dengan konsentrasi ekstrak. Pengolahan data digunakan program Originlab 9.0 32-bit (Originlab Corporation USA). Hasil penelitian menunjukkan bahwa ekstrak metanol daun sisik naga memiliki efek sitotoksik terhadap sel leukemia P388 yang ditunjukkan dengan penghambatan pertumbuhan sel leukemia sebanyak 50% adalah 19,32 µg/mL.The research about cytotoxicity assay of sisik naga (Drymoglossum piloselloides Presl.) leaf methanol extract on leukemia cells P388 has been done. This study aimed to determine the cytotoxicity of the methanol extract of sisik naga leaf against leukemia cells P388 based on the inhibition of 50% growth (IC50). The MTT (Microculture Tetrazolium Technique) test was used in this experiment. Leukemia cells were cultured on RPMI (Roswell Park Memorial Institute) medium. The cell growth was determined by measuring the formazan absorbance in variation of concentration 0,1 µg/mL to 100 µg/mL of sample extract at 540 nm. IC50 determined by logarithmic equation of absorbance values with concentration of extract. Data analysis used the program Originlab 9.0 32-bit (Originlab Corporation USA). The result showed that methanol extract of sisik naga leaf had cytotoxic effects against leukemia cells and inhibition of 50% leukemia cell growth was 19.32 µg/mL.