1,721,266 research outputs found
Antiprotozoal activity of novel diaryliminophenazines
Full text linkRecently, we synthesized a set of novel iminofenazines bearing a bicyclic basic head linked through an alkyl chain to the imino nitrogen in position 3 on the phenazine nucleus (Fig.1).
Most of these compounds inhibited the growth of different species of Leishmania promastigotes as well as of chloroquine sensitive (CQ-S) and chloroquine resistant (CQ-R) strains of P. falciparum with IC50 in the submicromolar range. Unfortunately, these compounds exhibited also a significant toxicity against the human endothelial cell line HMEC-1 with IC50 in the low micromolar range and with a consequent low selectivity index.
Figure1.Structures of the previously synthesized compounds.
To continue the studies on the antiprotozoal potentialities of this class of compounds and with the aim to improve their activity and selectivity on protozoa, we have now synthesized novel compounds characterized by the replacement of the aniline moiety in pos. 2 of the phenazine nucleus with an aminopyridine, and/or by a quaternarization of the basic nitrogen in the side chain with a methyl group (Fig.2).
Figure 2. Structures of the new compounds synthesized.
The in vitro activity of the new compounds on Leishmania promastigotes and on CQ-S and CQ-R strains of P. falciparum, as well as on the HMEC-1 cell line will be presented and discussed.
References
[1] A. Barteselli, M. Gavazzi, N. Basilico, S. Parapini, D. Taramelli, A. Sparatore. Clofazimine analogs with antileishmanial and antimalarial activities. XXII National Meeting on Medicinal Chemistry, Roma 2013
Activation of murine macrophages. I. Different pattern of activation by poly I:C than by lymphokine or LPS
No full textThe ability of poly I:C to activate mouse macrophages (M phi) to become tumoricidal was evaluated and compared with the ability of 2 other agents, lipopolysaccharide (LPS) and M phi-activating factor (MAF), to induce a tumoricidal state. All these agents were able to stimulate proteose-peptone-elicited M phi to kill RL male 1 tumor cells in an 18-hr 51Cr release cytotoxicity assay. High levels of cytotoxicity were obtained with concentrations as low as 1 microgram/ml of LPS or poly I:C and with 1/81 dilution of MAF. However, in the presence of reagents shown to contain less than 0.01 ng/ml of LPS by the LAL assay (LPS free), we found that poly I:C induced strong reactivity, whereas MAF was ineffective. The addition of 10 ng/ml of LPS during the stimulation period did not enhance the cytotoxicity induced by poly I:C, but it did restore MAF-induced, M phi-mediated cytotoxicity. In addition, poly I:C induced strong tumoricidal activity in resident M phi and in peritoneal exudate cells from the genetically defective C3H/HeJ mice that normally do not respond to LPS and MAF treatment. Therefore, it seems that although LPS is required as a second signal for MAF-induced cytotoxicity, such a second signal is not required for poly I:C-induced cytotoxicity. From the above results, it appears that poly I:C is a more powerful activating agent than LPS and MAF and either activates M phi via a different pathway or is effective on subpopulations of M phi that are not activated by the other agents
Sorveglianza molecolare della resistenza ai derivati dell’artemisinina e ad altri antimalarici in isolati di Plasmodium falciparum africani
Full text linkL’introduzione della terapia combinata a base di artemisinina (artemisinin-based combination therapy, ACT) ha rappresentato una svolta epocale nella lotta contro la malaria da Plasmodium falciparum, riducendo drasticamente la morbilità e mortalità da questa causate. Purtroppo, la diffusione della resistenza di P. falciparum all'artemisinina nel Sud-Est Asiatico costituisce una reale minaccia per il controllo e l’eliminazione della malaria, poiché rischia di vanificare i successi raggiunti nell’ultimo decennio, soprattutto in termini di sopravvivenza infantile. Numerosi studi, condotti su isolati di P. falciparum provenienti dal Sud-Est Asiatico, hanno dimostrato che la resistenza all’artemisinina è strettamente legata alla presenza di polimorfismi a singolo nucleotide (SNPs) nel gene kelch 13 (k13). Alcuni di questi SNPs sono anche stati descritti in Africa, ma mai nessuno è stato correlato ad un’aumentata tolleranza o resistenza all'artemisinina (Ménard et al., 2016). La recente introduzione di strumenti di biologia molecolare di nuova generazione, volti alla rapida e precoce individuazione di parassiti resistenti, consente di perfezionare e rendere più accurate le strategie terapeutiche e profilattiche impiegate nei casi di malaria da P. falciparum. In questo lavoro di tesi, al fine di identificare la presenza di mutazioni puntiformi nel gene k13 di P. falciparum, sono stati analizzati, mediante PCR e sequenziamento diretto, 930 isolati di P. falciparum raccolti in quattro paesi africani nei quali la malaria continua a rappresentare un importante problema di salute pubblica: Camerun, Eritrea, Botswana, e Guinea Conakry. Per i campioni provenienti dal Camerun e dall’Eritrea, lo screening molecolare è stato effettuato tramite il sequenziamento di nuova generazione NGS, ovvero un sistema standardizzato di sorveglianza della farmaco-resistenza ad alto rendimento. Oltre all’analisi del gene Pfk13, è stata valutata l’eventuale presenza di mutazioni nei diversi geni marcatori di resistenza per gli antimalarici comunemente impiegati nelle combinazioni terapeutiche a base di artemisinina. Inoltre, in collaborazione con il CDC (Centre for Disease Control and Prevention) di Atlanta, è stato messo a punto un nuovo metodo di rilevazione dell’espressione del gene plasmepsina 2 di P. falciparum, coinvolto nella resistenza alla piperachina. I risultati dello studio di sorveglianza molecolare qui riportati non hanno evidenziato alcuna resistenza all'artemisinina. Tuttavia, per gli altri marcatori molecolari indagati, è stata identificata un’alta frequenza di varianti alleliche associate alla resistenza alle diverse classi di farmaci antimalarici. Questi risultati suggeriscono la forte necessità di effettuare una costante valutazione di efficacia terapeutica dei farmaci impiegati nel trattamento della malaria da P. falciparum, e un assiduo monitoraggio molecolare della farmaco-resistenza, al fine di sviluppare adeguate strategie di controllo e prevenzione della malaria
Gli ellagitannini di Punica granatum antagonizzano la risposta immune innata nella malaria
No full textIl pericarpo del frutto immaturo di Punica granatum (P.g.) L. è usato in una formulazione per la terapia e la profilassi malarica in Orissa, una regione dell’India. La malaria cerebrale è una complicanza grave dovuta alla citoaderenza di Plasmodium falciparum ai vasi cerebrali e all’eccesso di risposta infiammatoria, associata ad una sovraproduzione di mediatori tra cui metalloproteasi-9 (MMP-9) e TNF. Studi recenti hanno dimostrato l’attività antiplasmodio di P.g. in vitro [1]. Lo scopo del presente studio è stato di esplorare se oltre all’effetto antimalarico, P.g. potesse modulare la risposta immune dell’ospite. A tal fine si è preparata una frazione arricchita in tannini (P.g.-FRT) dall’estratto metanolico del pericarpo. L’espressione genica e la secrezione di MMP-9 sono state valutate in cellule THP-1 stimolate con il pigmento malarico (emozoina, 6 μg/ml). Per valutare se i meccanismi molecolari alla base dell’effetto coinvolgessero il fattore nucleare di trascrizione NF-kB, abbiamo valutato l’effetto delle molecole sul promotore di NF-kB in seguito a stimolo con emozoina. I saggi sono stati condotti su P.g.-FRT e sui costituenti principali della frazione: acido ellagico e punicalagina. Inoltre, è stato valutato anche l’effetto delle urolitine, i metaboliti intestinali degli ellagitannini. P.g.-FRT (50 e 100 μg/ml) inibisce la secrezione di MMP-9 indotta da emozoina rispettivamente del 78% e 95%; l’effetto osservato è ascrivibile alla presenza di punicalagina e acido ellagico poichè tali sostanze riducono la secrezione dell’enzima rispettivamente del 79% e 66% a 10 μM. L’effetto osservato sulla secrezione di MMP-9 sembra essere dovuto ad una diminuzione dell’espressione genica in quanto FRT e i composti puri, alle medesime concentrazioni, diminuiscono i livelli di mRNA di MMP-9 e inibiscono l’attività del promotore di MMP-9. Anche le urolitine (25 μM) inibiscono l’espressione e la secrezione di MMP-9. FRT, acido ellagico e punicalagina riducono l’attività del promotore di NF-kB, suggerendo un coinvolgimento di questo fattore di trascrizione nei meccanismi alla base dell’attività biologica osservata.
Gli effetti benefici del pericarpo di P.g. nel trattamento della malaria sono quindi in relazione sia all’attività diretta sul parassita sia all’inibizione di uno dei meccanismi pro-infiammatori coinvolti nell’insorgenza della malaria cerebrale. Riferimenti [1] M. Dell’Agli, G.V. Galli, Y. Corbett, D. Taramelli, L. Lucantoni, A. Habluetzel, O. Maschi, D. Caruso, S. Giavarini, S. Romeo, D. Bhattacharya, E. Bosisio Journal of Ethnopharmacology, 2009, 125, pag. 27
In vitro induction of tumoricidal and suppressor macrophages by lymphokines: possible feedback regulation
No full textProteose-peptone-induced macrophages (pM phi) from C57BL/6 (B6) mice were treated in vitro with lymphokine, and 2 functions were evaluated: the ability to suppress lymphokine production and the cytotoxic capacity against tumor target cells. When 10% to 20% pM phi treated with lymphokine were added to a lymphokine-producing system, strong inhibition of MIF and MAF production occurred. The suppression was not specific, since pM phi inhibited the production of lymphokine obtained by either mitogenic stimulation of normal spleen cells (NSC) or alloantigen stimulation of immune spleen cells (ISC). The suppressor cells were adherent, phagocytic, Thy 1.2 negative, with monocyte-macrophage-like morphology. Lymphokine-treated pM phi were also tumoricidal when tested in 18 hr microcytotoxicity assay against RL male 1 lymphoma target cells. However, using endotoxin-free reagents (less than or equal to 0.01 ng/ml of endotoxin by the LAL assay), we found that small amounts of lipopolysaccharide (LPS) were required, in addition to lymphokine, to induce tumoricidal activity in pM phi. In contrast, noncytotoxic pM phi stimulated with lymphokine alone were also able to inhibit lymphokine production, although not as effectively as pM phi stimulated by lymphokine plus LPS. Therefore, we concluded that lymphokine treatment itself is sufficient to induce M phi to become suppressor cells, whereas an additional signal is necessary to induce cytolytic activity. We suggest that there is a negative feedback mechanism of control on the lymphokine-producing cells through the activation of suppressor M phi by lymphokine
RNA synthesis in activated macrophages I. Poly(I) X poly(C)-induced triggering of cytolytic activity is associated with decrease in RNA synthesis
No full textThe effects of polyinosinic, polycytidylic acid [poly(I) X poly(C)] on the activation and RNA metabolism in murine peritoneal macrophages (M phi) elicited by proteose-peptone (pM phi) was investigated. Poly(I) X poly(C) triggered the cytolytic activity of pM phi and augmented their glucose oxidation. In contrast, a profound depression of [3H]uridine incorporation into RNA was observed in poly(I) X poly(C)-activated pM phi. The degree of depression of RNA labeling paralleled the dose of poly(I) X poly(C) used to activate the pM phi and the expression of tumoricidal activity. This decrease in [3H]uridine incorporation into M phi RNA could not be accounted for by decreased permeability of the activated M phi to [3H]uridine, or by instability of the labeled RNA. Moreover, analysis of the specific activity of the intracellular uridine triphosphate (UTP) pool and studies on the labeling of M phi RNA with [32P] orthophosphate indicated that the decreased RNA labeling was not due to changes in the specific activity of UTP. We concluded that poly(I) X poly(C)-activated pM phi exhibit a depressed rate of RNA synthesis. We suggest that the rate of RNA synthesis may be investigated as a potential new indicator for M phi activation
Endotoxin requirement for macrophage activation by lymphokines in a rapid microcytotoxicity assay
No full textA short term microcytotoxicity assay is described for studying the activation of M0 by lymphokines. Proteose-peptone-induced macrophages were purified by adherence in flat-bottomed microtiter plates and then activated by MAF-containing supernatants for 18 h. 51Cr-labeled tumor target cells were added for an additional 18 h, and then the supernatants were harvested and the % isotope release quantitated. When endotoxin-free medium and FBS were used, we found that small amounts of LPS were absolutely required for macrophage activation in this assay. The advantages of this technique included (a) good reproducibility, (b) the requirement for small number of M0, and (c) the potential of standardizing the assay and thereby testing a large number of samples. Moreover, this assay may have particular value for investigations of M0 activation by exogenous stimuli, since M0 that were not pretreated with activating agents did not exhibit cytotoxicity
Measurement of macrophage-mediated cytotoxicity against adherent and non-adherent target cells by release of 11 indium-oxine
No full textThis report describes the utilization of 111indium-oxine chelate ([111In]Ox) for studies of macrophage-mediated cytotoxicity. [111In]Ox efficiently labeled both non-adherent and adherent tumor targets with no decrease in cell viability. Spontaneous release of intracellularly incorporated [111In]Ox was very slow (0.25-0.50%/h) from most targets, making isotope-release assays of at least 48 h feasible. In addition, released [111In]Ox was not reutilized. In contrast to its low spontaneous release from intact cells, incorporated [111In]Ox was rapidly released from tumor targets after interaction with activated macrophages. Levels of [111In]Ox released in response to cytolytic macrophages correlated well with those observed for the 51Cr and [3H]TdR radiolabels. Therefore, [111In]Ox can be utilized for relatively short-term (less than 20 h) assays with lymphoma targets, as well as for longer-term assays with adherent cells. This should facilitate the testing, with the same radioisotope-release assay, of a wide range of tumor targets for susceptibility to macrophage-mediated cytotoxicity
Suppression of lymphokine production: II. Macrophage-dependent inhibition of production of macrophage activating factor
No full textThe ability of different populations of macrophages to affect the production of macrophage activating factor (MAF) by stimulated T lymphocytes was investigated. We found that activated macrophages, infiltrating MSV-induced regressing tumors or macrophages recovered from the peritoneum of mice injected with Corynebacterium parvum, were able to actively suppress the production of MAF. MAF production by antigen-stimulated MSV-immune or -alloimmune spleen cells and by normal spleen cells stimulated by Con A was susceptible to macrophage-dependent suppression to a similar extent. In contrast, resident macrophages or those elicited by light mineral oil or proteose-peptone did not affect MAF production. While suppressor macrophages added at the time of the lymphocyte stimulation inhibited MAF production, the same cells added 4-6 hr after stimulation were ineffective. Therefore, it seems that the macrophages suppressed the early events of lymphocyte activation leading to MAF production. Suppressor macrophages, by inhibiting MAF production, may limit the expansion of the cytotoxic activity. This regulation of macrophage functions, mediated by the effects of suppressor macrophages on T lymphocytes, could be responsible for an insufficient antitumor cytotoxic response by macrophages
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