Cultured primary human keratinocytes are frequently employed for studies of immunological and inflammatory responses; however, interpretation of experimental data may be complicated by donor to donor ...variability, the relatively short culture lifetime, and variations between passages. To standardize the in vitro studies on keratinocytes, we investigated the use of HaCaT cells, a long-lived, spontaneously immortalized human keratinocyte line which is able to differentiate in vitro, as a suitable model to follow the release of inflammatory and repair mediators in response to TNFα or IL-1β. Different treatment conditions (presence or absence of serum) and differentiation stimuli (increase in cell density as a function of time in culture and elevation of extracellular calcium) were considered. ELISA and Multiplex measurement technologies were used to monitor the production of cytokines and chemokines. Taken together, the results highlight that Ca2+ concentration in the medium, cell density, and presence of serum influences at different levels the release of proinflammatory mediators by HaCaT cells. Moreover, HaCaT cells maintained in low Ca2+ medium and 80% confluent are similar to normal keratinocytes in terms of cytokine production suggesting that HaCaT cells may be a useful model to investigate anti-inflammatory interventions/therapies on skin diseases.
Artemisinin combination therapy (ACT) is recommended by the World Health Organization (WHO) as first line treatment for uncomplicated malaria both in adults and children. During pregnancy, ACT is ...considered safe only in the second and third trimester, since animal studies have demonstrated that artemisinin derivatives can cause foetal death and congenital malformation within a narrow time window in early embryogenesis. During this period, artemisinin derivatives induce defective embryonic erythropoiesis and vasculogenesis/angiogenesis in experimental models. However, clinical data on the safety profile of ACT in pregnant women have not shown an increased risk of miscarriage, stillbirth, or congenital malformation, nor low birth weight, associated with exposure to artemisinins in the first trimester. Although further studies are needed, the evidence collected up to now is prompting the WHO towards a change in the guidelines for the treatment of uncomplicated malaria, allowing the use of ACT also in the first trimester of pregnancy.
Malaria is still one of the most dangerous infectious diseases and the emergence of drug resistant parasites only worsens the situation. A series of new tetrahydro-β-carbolines were designed, ...synthesized by the Pictet-Spengler reaction, and characterized. Further, the compounds were screened for their in vitro antiplasmodial activity against chloroquine-sensitive (D10) and chloroquine-resistant (W2) strains of
. Moreover, molecular modeling studies were performed to assess the potential action of the designed molecules and toxicity assays were conducted on the human microvascular endothelial (HMEC-1) cell line and human red blood cells. Our studies identified
-(3,3-dimethylbutyl)-1-octyl-2,3,4,9-tetrahydro-1H-pyrido3,4-b indole-3-carboxamide (
) (a mixture of diastereomers) as the most promising compound endowed with the highest antiplasmodial activity, highest selectivity, and lack of cytotoxicity. In silico simulations carried out for (1
,3
)-
provided useful insights into its possible interactions with enzymes essential for parasite metabolism. Further studies are underway to develop the optimal nanosized lipid-based delivery system for this compound and to determine its precise mechanism of action.
The potential of natural and synthetic chalcones as therapeutic leads against different pathological conditions has been investigated for several years, and this class of compounds emerged as a ...privileged chemotype due to its interesting anti-inflammatory, antimicrobial, antiviral, and anticancer properties. The objective of our study was to contribute to the investigation of this class of natural products as anti-leishmanial agents. We aimed at investigating the structure-activity relationships of the natural chalcone lophirone E, characterized by the presence of benzofuran B-ring, and analogues on anti-leishmania activity. Here we describe an effective synthetic strategy for the preparation of the natural chalcone lophirone E and its application to the synthesis of a small set of chalcones bearing different substitution patterns at both the A and heterocyclic B rings. The resulting compounds were investigated for their activity against
promastigotes disclosing derivatives
and
,
as those endowed with the most interesting activities (IC
= 15.3, 27.2, 15.9 μM, respectively). The synthetic approaches here described and the early SAR investigations highlighted the potential of this class of compounds as antiparasitic hits, making this study worthy of further investigation.
There is great concern regarding the rapid emergence and spread of drug-resistance in Plasmodium falciparum, the parasite responsible for the most severe form of human malaria. Parasite populations ...resistant to some or all the currently available antimalarial treatments are present in different world regions. Considering the need for novel and integrated approaches to control malaria, combinations of drugs were tested on P. falciparum. The primary focus was on doxycycline, an antibiotic that specifically targets the apicoplast of the parasite. In combination with doxycycline, three different drugs known to inhibit efflux pumps (verapamil, elacridar and ivermectin) were tested, with the assumption that they could increase the intracellular concentration of the antibiotic and consequently its efficacy against P. falciparum. We emphasize that elacridar is a third-generation ABC transporters inhibitor, never tested before on malaria parasites. In vitro experiments were performed on asexual stages of two strains of P. falciparum, chloroquine-sensitive (D10) and chloroquine-resistant (W2). Incubation times on asynchronous or synchronous cultures were 72h or 96h, respectively. The antiplasmodial effect (i.e. the IC50) was determined by measuring the activity of the parasite lactate dehydrogenase, while the interaction between drugs was determined through combination index (CI) analyses. Elacridar achieved an IC50 concentration comparable to that of ivermectin, approx. 10-fold lower than that of verapamil, the other tested ABC transporter inhibitor. CI results showed synergistic effect of verapamil plus doxycycline, which is coherent with the starting hypothesis, i.e. that ABC transporters represent potential targets, worth of further investigations, towards the development of companion molecules useful to enhance the efficacy of antimalarial drugs. At the same time, the observed antagonistic effect of doxycycline in combination with ivermectin or elacridar highlighted the importance of drug testing, to avoid the de-facto generation of a sub-dosage, a condition that facilitates the development of drug resistance.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
•L. infantum infection reduces nigericin or Aβ42-stimulated NLRP3 inflammasome activation.•L. infantum infection induces an anti-inflammatory milieu in Aβ42-stimulated cells.•A side effect of L. ...infantum infection could be control over Alzheimer’s disease.
Activation of the NLRP3 inflammasome has been shown to play a major role in the neuroinflammation that accompanies Alzheimer’s disease (AD); interventions that down regulate the NLRP3 inflammasome could thus be beneficial in AD. Parasite infections were recently shown to be associated with improved cognitive functions in Apolipoprotein E4 (ApoE4)-expressing members of an Amazonian tribe. We verified in an in vitro model whether Leishmania infantum infection could reduce NLRP3. Results obtained in an initial experimental model in which PBMC were LPS primed and nigericin-stimulated showed that L. infantum infection significantly reduced ASC-speck formation (i.e. intracellular inflammasome proteins assembly), as well as the production of activated caspase 5 and IL-1β, but increased that of activated caspase 1 and IL-18. Moreover, L. infantum infection induced the generation of an anti-inflammatory milieu by suppressing the production of TNFα and increasing that of IL-10. These results were replicated when cells that had been LPS-primed were stimulated with Aβ42 and infected with L. infantum. Results herein indicate that Leishmania infection favors an anti-inflammatory milieu, which includes the down-regulation of NLRP3 inflammasome activation, possibly to facilitate its survival inside host cells. A side effect of Leishmaniasis would be the hampering of neuroinflammation; this could play a protective role against AD development.
The sun-dried rind of the immature fruit of pomegranate (Punica granatum) is presently used as a herbal formulation (OMARIA, Orissa Malaria Research Indigenous Attempt) in Orissa, India, for the ...therapy and prophylaxis of malaria. The pathogenesis of cerebral malaria, a complication of the infection by Plasmodium falciparum, is an inflammatory cytokine-driven disease associated to an up-regulation and activity of metalloproteinase-9 and to the increase of TNF production. The in vitro anti-plasmodial activity of Punica granatum (Pg) was recently described. The aim of the present study was to explore whether the anti-malarial effect of OMARIA could also be sustained via other mechanisms among those associated to the host immune response.
From the methanolic extract of the fruit rind, a fraction enriched in tannins (Pg-FET) was prepared. MMP-9 secretion and expression were evaluated in THP-1 cells stimulated with haemozoin or TNF. The assays were conducted in the presence of the Pg-FET and its chemical constituents ellagic acid and punicalagin. The effect of urolithins, the ellagitannin metabolites formed by human intestinal microflora, was also investigated.
Pg-FET and its constituents inhibited the secretion of MMP-9 induced by haemozoin or TNF. The effect occurred at transcriptional level since MMP-9 mRNA levels were lower in the presence of the tested compounds. Urolithins as well inhibited MMP-9 secretion and expression. Pg-FET and pure compounds also inhibited MMP-9 promoter activity and NF-kB-driven transcription.
The beneficial effect of the fruit rind of Punica granatum for the treatment of malarial disease may be attributed to the anti-parasitic activity and the inhibition of the pro-inflammatory mechanisms involved in the onset of cerebral malaria.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In the novel pandemic of Coronavirus Disease 2019, high levels of pro-inflammatory cytokines lead to endothelial activation and dysfunction, promoting a pro-coagulative state, thrombotic events, and ...microvasculature injuries. The aim of the present work was to investigate the effect of SARS-CoV-2 on pro-inflammatory cytokines, tissue factor, and chemokine release, with Human Microvascular Endothelial Cells (HMEC-1). ACE2 receptor expression was evaluated by western blot analysis. SARS-CoV-2 infection was assessed by one-step RT-PCR until 7 days post-infection (p.i.), and by Transmission Electron Microscopy (TEM). IL-6, TNF-α, IL-8, IFN-α, and hTF mRNA expression levels were detected by RT-PCR, while cytokine release was evaluated by ELISA. HMEC-1 expressed ACE2 receptor and SARS-CoV-2 infection showed a constant viral load. TEM analysis showed virions localized in the cytoplasm. Expression of IL-6 at 24 h and IFN-α mRNA at 24 h and 48 h p.i. was higher in infected than uninfected HMEC-1 (p < 0.05). IL-6 levels were significantly higher in supernatants from infected HMEC-1 (p < 0.001) at 24 h, 48 h, and 72 h p.i., while IL-8 levels were significantly lower at 24 h p.i. (p < 0.001). These data indicate that in vitro microvascular endothelial cells are susceptible to SARS-CoV-2 infection but slightly contribute to viral amplification. However, SARS-CoV-2 infection might trigger the increase of pro-inflammatory mediators.
Blocking Plasmodium falciparum transmission to mosquitoes has been designated a strategic objective in the global agenda of malaria elimination. Transmission is ensured by gametocyte-infected ...erythrocytes (GIE) that sequester in the bone marrow and at maturation are released into peripheral blood from where they are taken up during a mosquito blood meal. Release into the blood circulation is accompanied by an increase in GIE deformability that allows them to pass through the spleen. Here, we used a microsphere matrix to mimic splenic filtration and investigated the role of cAMP-signalling in regulating GIE deformability. We demonstrated that mature GIE deformability is dependent on reduced cAMP-signalling and on increased phosphodiesterase expression in stage V gametocytes, and that parasite cAMP-dependent kinase activity contributes to the stiffness of immature gametocytes. Importantly, pharmacological agents that raise cAMP levels in transmissible stage V gametocytes render them less deformable and hence less likely to circulate through the spleen. Therefore, phosphodiesterase inhibitors that raise cAMP levels in P. falciparum infected erythrocytes, such as sildenafil, represent new candidate drugs to block transmission of malaria parasites.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The clinical manifestations of cerebral malaria (CM) are well correlated with underlying major pathophysiological events occurring during an acute malaria infection, the most important of which, is ...the adherence of parasitized erythrocytes to endothelial cells ultimately leading to sequestration and obstruction of brain capillaries. The consequent reduction in blood flow, leads to cerebral hypoxia, localized inflammation and release of neurotoxic molecules and inflammatory cytokines by the endothelium. The pharmacological regulation of these immunopathological processes by immunomodulatory molecules may potentially benefit the management of this severe complication. Adjunctive therapy of CM patients with an appropriate immunomodulatory compound possessing even moderate anti-malarial activity with the capacity to down regulate excess production of proinflammatory cytokines and expression of adhesion molecules, could potentially reverse cytoadherence, improve survival and prevent neurological sequelae. Current major drug discovery programmes are mainly focused on novel parasite targets and mechanisms of action. However, the discovery of compounds targeting the host remains a largely unexplored but attractive area of drug discovery research for the treatment of CM. This review discusses the properties of the plant immune-modifier curcumin and its potential as an adjunctive therapy for the management of this complication.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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