Determination of the UV Inactivation Constant Under 280 nm UV LED Irradiation for SARS-CoV-2.

The ongoing emergency provoked by the SARS-CoV-2 pandemic demands the development of technologies to mitigate the spread of infection, and UV irradiation is a technique that can efficiently address this issue. However, proper use of UV equipment for disinfection requires an understanding of how the effects on SARS-CoV-2 are dependent on certain parameters. In this work, we determined the UV-C inactivation constant k for SARS-CoV-2 using an LED source at λ = 280 nm. Specifically, a Log3 reduction was measured after irradiation for 24 min with a delivered UV-C dose of 23 J m-2 . By multitarget model fitting, n = 2 and k = 0.32 ± 0.02 m2 J-1 were obtained. A lag time for the inactivation effect was also observed, which was attributed to the low irradiation levels used to perform the study. The combination of k and delay time allows for reliable estimation of disinfection times in small, closed environments.

In Vitro SARS-CoV-2 Infection of Microvascular Endothelial Cells: Effect on Pro-Inflammatory Cytokine and Chemokine Release.

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.

Total Synthesis of the Natural Chalcone Lophirone E, Synthetic Studies toward Benzofuran and Indole-Based Analogues, and Investigation of Anti-Leishmanial Activity.

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 Leishmania infantum promastigotes disclosing derivatives 1 and 28a,b as those endowed with the most interesting activities (IC50 = 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.

A rapid spectrophotometric method to identify inhibitors of human erythropoiesis.

Erythropoiesis is a complex physiological process by which erythroid progenitors proliferate and differentiate into nonnucleated red blood cells. Several methods can be used to monitor in vitro the differentiation of erythroid precursors, and hence the toxic effects of drugs, chemicals, or pollutants. One of the most commonly available assay of erythropoiesis is the microscopic observation of differentiated cells after benzidine staining, which forms a blue complex with hemoglobin. However, this method is laborious and does not provide accurate results since it heavily relies on the reader's interpretation. Moreover, benzidine is a carcinogen and a highly reactive molecule which forces the reader to microscopically count differentiated and non-differentiated cells within a short time frame (5 min). Here we have developed a simple, inexpensive, in-vitro spectrophotometric assay to measure erythroid differentiation using K562 cell line as a model. Materials needed included 96-well round-bottomed microplates and a microplate reader. Remarkably, carcinogenic benzidine was replaced by its isomeric tetramethyl derivative, the 3,3', 5,5'- tetramethylbenzidine (TMB), which presents several advantages: it is cheap, not mutagenic and a ready-to-use chromogenic substrate. A small volume (50 µl) of TMB added to the samples forms a blue complex in 15 min, and the reaction can be easily stopped and stabilized by the addition of H2SO4. The yellow precipitate is then solubilized, and the absorbance is measured at 450 nm. In addition, the suitability of the assay to determine the effects of compounds on erythroid differentiation was further tested with known inhibitors (artemisinin derivatives) of K562 differentiation. Overall, the reported methodology permits to measure in an accurate and reproducible manner the K562 differentiation and can be used for medium throughput screenings (MTS) of compounds or environmental toxics with potential erythro-toxicity and ability to inhibit erythroid differentiation.

Phagocytosis and activation of bone marrow-derived macrophages by Plasmodium falciparum gametocytes.

BACKGROUND: The innate immune response against various life cycle stages of the malaria parasite plays an important role in protection against the disease and regulation of its severity. Phagocytosis of asexual erythrocytic stages is well documented, but little and contrasting results are available about phagocytic clearance of sexual stages, the gametocytes, which are responsible for the transmission of the parasites from humans to mosquitoes. Similarly, activation of host macrophages by gametocytes has not yet been carefully addressed. METHODS: Phagocytosis of early or late Plasmodium falciparum gametocytes was evaluated through methanol fixed cytospin preparations of immortalized mouse C57Bl/6 bone marrow-derived macrophages treated for 2 h with P. falciparum and stained with Giemsa, and it was confirmed through a standardized bioluminescent method using the transgenic P. falciparum 3D7elo1-pfs16-CBG99 strain. Activation was evaluated by measuring nitric oxide or cytokine levels in the supernatants of immortalized mouse C57Bl/6 bone marrow-derived macrophages treated with early or late gametocytes. RESULTS: The results showed that murine bone marrow-derived macrophages can phagocytose both early and late gametocytes, but only the latter were able to induce the production of inflammatory mediators, specifically nitric oxide and the cytokines tumour necrosis factor and macrophage inflammatory protein 2. CONCLUSIONS: These results support the hypothesis that developing gametocytes interact in different ways with innate immune cells of the host. Moreover, the present study proposes that early and late gametocytes act differently as targets for innate immune responses.

Synthesis and biological evaluation of benzhydryl-based antiplasmodial agents possessing Plasmodium falciparum chloroquine resistance transporter (PfCRT) inhibitory activity.

Due to the surge in resistance to common therapies, malaria remains a significant concern to human health worldwide. In chloroquine (CQ)-resistant (CQ-R) strains of Plasmodium falciparum, CQ and related drugs are effluxed from the parasite's digestive vacuole (DV). This process is mediated by mutant isoforms of a protein called CQ resistance transporter (PfCRT). CQ-R strains can be partially re-sensitized to CQ by verapamil (VP), primaquine (PQ) and other compounds, and this has been shown to be due to the ability of these molecules to inhibit drug transport via PfCRT. We have previously developed a series of clotrimazole (CLT)-based antimalarial agents that possess inhibitory activity against PfCRT (4a,b). In our endeavor to develop novel PfCRT inhibitors, and to perform a structure-activity relationship analysis, we synthesized a new library of analogues. When the benzhydryl system was linked to a 4-aminoquinoline group (5a-f) the resulting compounds exhibited good cytotoxicity against both CQ-R and CQ-S strains of P. falciparum. The most potent inhibitory activity against the PfCRT-mediated transport of CQ was obtained with compound 5k. When compared to the reference compound, benzhydryl analogues of PQ (5i,j) showed a similar activity against blood-stage parasites, and a stronger in vitro potency against liver-stage parasites. Unfortunately, in the in vivo transmission blocking assays, 5i,j were inactive against gametocytes.

Human polyomaviruses genomes in clinical specimens of colon cancer patients.

Colon cancer is the third cause of cancer death in the developed countries. Some environmental factors are involved in its pathogenesis, including viral infections. The possible involvement of human polyomaviruses (HPyVs) in colon cancer pathogenesis has been previously reported, leading to inconsistent conclusions. Clinical specimens were collected from 125 colon cancer patients. Specifically, 110 tumor tissues, 55 negative surgical margins, and 39 peripheral blood samples were analyzed for the presence of six HPyVs: JC polyomavirus (JCPyV), BK polyomavirus (BKPyV), Merkel cell PyV (MCPyV), HPyV -6, -7, and -9 by means of DNA isolation and subsequent duplex Real Time quantitative polymerase chain reaction. HPyVs genome was detected in 33/204 samples (16.2%): the significant higher positivity was found in tumor tissues (26/110, 23.6%), followed by negative surgical margins (3/55, 5.5%, p < .05), and peripheral blood mononuclear cells (PBMCs) (4/39; 10.3%). HPyVs load was statistically higher only in the tumor tissues compared to negative surgical margins (p < .05). Specifically, MCPyV was detected in 19.1% (21/110) of tumor tissues, 3.6% (2/55) of negative surgical margins (p < .05), and 7.7% (3/39) of PBMCs; HPyV-6 in 2.7% (3/110) of tumor tissues, and 1.8% (1/55) of negative surgical margins; one tumor tissue (1/110, 0.9%) and one PBMCs sample (1/39, 2.6%) were positive for BKPyV; JCPyV was present in 0.9% (1/110) of tumor tissues. HPyV-7 and 9 were not detected in any sample. High prevalence and load of MCPyV genome in the tumor tissues might be indicative of a relevant rather than bystander role of the virus in the colon tumorigenesis.

Human Endogenous Retroviruses Long Terminal Repeat Methylation, Transcription, and Protein Expression in Human Colon Cancer.

Colon cancer is the fourth most common malignancy in both incidence and mortality in developed countries. Infectious agents are among the risk factors for colon cancer. Variations in human endogenous retrovirus (HERV) transcript and protein levels are associated with several types of cancers, but few studies address HERV expression in colon cancer. Fifty-eight patients with advanced-stage colon cancer were enrolled in this study. HERV-H, -K (HML-2), -P LTRs, Alu, and LINE-1 methylation levels and transcription of HERV-H, -K (HML-2), and -P env and HERV-K pol genes in normal adjacent and tumor tissues were investigated by pyrosequencing and RT-qPCR, respectively. Expression of the HERV-K (HML-2) Pol and Env proteins in selected tissues was examined by Western blotting. Associations between HERV transcript expression and methylation levels and between clinical characteristics and HERV expression were evaluated. Compared to adjacent normal tissues, LINE-1 was hypomethylated in tumor tissues (p < 0.05), whereas Alu, HERV-K (HML-2), and -H LTRs showed a decreasing trend in tumor tissue compared to normal tissue, though without a significant difference. The transcription levels of HERV env and pol genes were similar. However, the HERV-K (HML-2) Pol protein was more highly expressed (p < 0.01) in surrounding normal tissues, but the HERV-K (HML-2) Env protein was only expressed in tumor tissues. Although HERV LTR methylation and gene expression did not show significant differences between tumor and normal tissues, HERV protein expression differed greatly. Pol protein expression in normal cells may induce reverse transcription and subsequent integration into the host genome, likely favoring cell transformation; in contrast, the Env protein in tumor tissue may contribute to cancer progression through cell-to-cell fusion.

Human endogenous retroviruses env gene expression and long terminal repeat methylation in colorectal cancer patients.

Human endogenous retroviruses (HERV) are remnants of exogenous retroviral infections, representing 8% of the human genome. Their regulation is based on the DNA methylation of promoters, the long terminal repeats (LTRs). Transcripts from HERV have been associated with cancers, but reports concerning HERV expression in colorectal cancer remain sporadic. Sixty-three patients with advanced stages of colorectal cancer were enrolled in this study. The expressions of HERV env gene, and HERV-H, -K, -R and -P LTRs and Alu, LINE-1 methylation levels, were investigated in the tumor, normal adjacent tissues, and, where possible, blood and plasmatic extracellular vesicles (EVs). Associations among HERV env expression, methylation status and clinical characteristics were evaluated. No differences were observed in HERV env gene expression levels among the clinical specimens, while Alu, LINE-1, HERV-H and -K LTRs were demethylated in the tumor compared to the normal adjacent tissues (p < 0.05).The HERV env gene was expressed in the EVs at of 54% (-H), 38% (-K), 31% (-R) patients. Association was not found between HERV env expression and LTR methylation, but significant higher expression of HERV-P and -R env was found in tumor tissues arising from the right colon. Our findings do not demonstrate significant overexpression of the studied HERV in colorectal cancer, but their association with tumor localization and specificity of the changes in DNA methylation of retroelements are shown. HERV sequences were packaged in the EVs and might be transferred from one cell to another.

Effect of Hypoxia on Gene Expression in Cell Populations Involved in Wound Healing.

Wound healing is a complex process regulated by multiple signals and consisting of several phases known as haemostasis, inflammation, proliferation, and remodelling. Keratinocytes, endothelial cells, macrophages, and fibroblasts are the major cell populations involved in wound healing process. Hypoxia plays a critical role in this process since cells sense and respond to hypoxic conditions by changing gene expression. This study assessed the in vitro expression of 77 genes involved in angiogenesis, metabolism, cell growth, proliferation and apoptosis in human keratinocytes (HaCaT), microvascular endothelial cells (HMEC-1), differentiated macrophages (THP-1), and dermal fibroblasts (HDF). Results indicated that the gene expression profiles induced by hypoxia were cell-type specific. In HMEC-1 and differentiated THP-1, most of the genes modulated by hypoxia encode proteins involved in angiogenesis or belonging to cytokines and growth factors. In HaCaT and HDF, hypoxia mainly affected the expression of genes encoding proteins involved in cell metabolism. This work can help to enlarge the current knowledge about the mechanisms through which a hypoxic environment influences wound healing processes at the molecular level.

Merkel Cell Polyomavirus Is Associated with Anal Infections in Men Who Have Sex with Men.

BACKGROUND: Viral infections of the anal/rectal tract of men who have sex with men (MSM) have been poorly studied. METHODS: In total, 158 swab samples (81 anal/rectal, 65 throat/oral and 12 urethral) were collected from 126 MSM. DNA was isolated and subjected to real-time PCR assays for the detection of the sexually transmitted (ST) pathogens Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasmas ssp, human papillomavirus (HPV) and six human polyomaviruses (HPyVs; JCPyV, BKPyV, Merkel cell PyV⁻MCPyV-, HPyV-6, HPyV-7 and HPyV-9). RESULTS: C. trachomatis (31/126, 24.6%) and M. genitalium (30/126, 23.8%) were the most frequently detected ST pathogens. Thirty-one/126 (24.6%) patients were positive for at least one HPyV. The significantly (p < 0.05) prevalent HPyV in the anal tract was MCPyV, which was amplified in 27/81 (33.3%) samples, followed by HPyV-6, which was amplified in 6/81 (7.4%) swabs. Coinfections with MCPyV and C. trachomatis or Mycoplasmas were found in 4/21 (19.0%) and 5/21 (23.8%) anal/rectal swabs, respectively. Three/4 MCPyV-C. trachomatis coinfected patients were symptomatic. CONCLUSIONS: Based on the high prevalence of MCPyV in the anal/rectal swabs from MSM patients and on the well-known oncogenic properties of MCPyV, sexual transmission and possible involvement of HPyVs in the pathogenesis of diseases of the anal canal should be further studied.

Interplay between Plasmodium falciparum haemozoin and L-arginine: implication for nitric oxide production.

BACKGROUND: Plasmodium falciparum haemozoin, a detoxification product of digested haemoglobin from infected erythrocytes, is released into the bloodstream upon schizont rupture and accumulates in leukocytes. High levels of haemozoin correlate with disease severity. Some studies have shown that concentrations of the substrate of inducible nitric oxide synthase (iNOS), L-arginine, as well as nitric oxide are low in patients infected with P. falciparum malaria. The present study investigates, in vitro, the role of P. falciparum haemozoin on nitric oxide production, iNOS expression in macrophages, and the possible interaction between L-arginine and haemozoin. METHODS: Plasmodium falciparum haemozoin was obtained from in vitro cultures through magnetic isolation. Phagocytosis of haemozoin by immortalized bone marrow derived macrophages was detected by confocal reflection combined with fluorescence microscopy. Nitrite concentrations in the supernatants was evaluated by Griess assay as a standard indication of nitric oxide production, while iNOS expression was detected on cell extracts by western blotting. Detection of L-arginine in haemozoin-treated or untreated media was achieved by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Haemozoin synergizes in vitro with interferon-gamma to produce nitric oxide. However, when mouse macrophages were stimulated with haemozoin, a proportional increase of nitric oxide was observed up to 25 µM of haemozoin, followed by a decrease with doses up to 100 µM, when nitric oxide release was completely abrogated. This was not due to reactive oxygen species production, nor to an effect on iNOS activity. Interestingly, when at 24 h, haemozoin-treated macrophages were washed and incubated in fresh medium for further 24 h, the nitric oxide production was restored in a dose-response manner. Similar results were seen when L-arginine-enriched media was used in the stimulation. Moreover, muramyldipeptide, a strong nitric oxide inducer, was unable to activate macrophages to release nitric oxide in the presence of haemozoin-treated medium. By LC-MS/MS a complete depletion of L-arginine was observed in this haemozoin-treated, conditioned medium. CONCLUSIONS: It is proposed that haemozoin interacts with L-arginine reducing its availability for iNOS, and thus decreasing nitric oxide production. The clinical (or pathological) implications of these results are discussed.

Open Source Drug Discovery with the Malaria Box Compound Collection for Neglected Diseases and Beyond.

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts.

Reinvestigating Old Pharmacophores: Are 4-Aminoquinolines and Tetraoxanes Potential Two-Stage Antimalarials?

The syntheses and antiplasmodial activities of various substituted aminoquinolines coupled to an adamantane carrier are described. The compounds exhibited pronounced in vitro and in vivo activity against Plasmodium berghei in the Thompson test. Tethering a fluorine atom to the aminoquinoline C(3) position afforded fluoroaminoquinolines that act as intrahepatocytic parasite inhibitors, with compound 25 having an IC50 = 0.31 µM and reducing the liver load in mice by up to 92% at 80 mg/kg dose. Screening our peroxides as inhibitors of liver stage infection revealed that the tetraoxane pharmacophore itself is also an excellent liver stage P. berghei inhibitor (78: IC50 = 0.33 µM). Up to 91% reduction of the parasite liver load in mice was achieved at 100 mg/kg. Examination of tetraoxane 78 against the transgenic 3D7 strain expressing luciferase under a gametocyte-specific promoter revealed its activity against stage IV-V Plasmodium falciparum gametocytes (IC50 = 1.16 ± 0.37 µM). To the best of our knowledge, compounds 25 and 78 are the first examples of either an 4-aminoquinoline or a tetraoxane liver stage inhibitors.

Dextran-shelled oxygen-loaded nanodroplets reestablish a normoxia-like pro-angiogenic phenotype and behavior in hypoxic human dermal microvascular endothelium.

In chronic wounds, hypoxia seriously undermines tissue repair processes by altering the balances between pro-angiogenic proteolytic enzymes (matrix metalloproteinases, MMPs) and their inhibitors (tissue inhibitors of metalloproteinases, TIMPs) released from surrounding cells. Recently, we have shown that in human monocytes hypoxia reduces MMP-9 and increases TIMP-1 without affecting TIMP-2 secretion, whereas in human keratinocytes it reduces MMP-2, MMP-9, and TIMP-2, without affecting TIMP-1 release. Provided that the phenotype of the cellular environment is better understood, chronic wounds might be targeted by new oxygenating compounds such as chitosan- or dextran-shelled and 2H,3H-decafluoropentane-cored oxygen-loaded nanodroplets (OLNs). Here, we investigated the effects of hypoxia and dextran-shelled OLNs on the pro-angiogenic phenotype and behavior of human dermal microvascular endothelium (HMEC-1 cell line), another cell population playing key roles during wound healing. Normoxic HMEC-1 constitutively released MMP-2, TIMP-1 and TIMP-2 proteins, but not MMP-9. Hypoxia enhanced MMP-2 and reduced TIMP-1 secretion, without affecting TIMP-2 levels, and compromised cell ability to migrate and invade the extracellular matrix. When taken up by HMEC-1, nontoxic OLNs abrogated the effects of hypoxia, restoring normoxic MMP/TIMP levels and promoting cell migration, matrix invasion, and formation of microvessels. These effects were specifically dependent on time-sustained oxygen diffusion from OLN core, since they were not achieved by oxygen-free nanodroplets or oxygen-saturated solution. Collectively, these data provide new information on the effects of hypoxia on dermal endothelium and support the hypothesis that OLNs might be used as effective adjuvant tools to promote chronic wound healing processes.

Salinomycin and other ionophores as a new class of antimalarial drugs with transmission-blocking activity.

The drug target profile proposed by the Medicines for Malaria Venture for a malaria elimination/eradication policy focuses on molecules active on both asexual and sexual stages of Plasmodium, thus with both curative and transmission-blocking activities. The aim of the present work was to investigate whether the class of monovalent ionophores, which includes drugs used in veterinary medicine and that were recently proposed as human anticancer agents, meets these requirements. The activity of salinomycin, monensin, and nigericin on Plasmodium falciparum asexual and sexual erythrocytic stages and on the development of the Plasmodium berghei and P. falciparum mosquito stages is reported here. Gametocytogenesis of the P. falciparum strain 3D7 was induced in vitro, and gametocytes at stage II and III or stage IV and V of development were treated for different lengths of time with the ionophores and their viability measured with the parasite lactate dehydrogenase (pLDH) assay. The monovalent ionophores efficiently killed both asexual parasites and gametocytes with a nanomolar 50% inhibitory concentration (IC50). Salinomycin showed a fast speed of kill compared to that of standard drugs, and the potency was higher on stage IV and V than on stage II and III gametocytes. The ionophores inhibited ookinete development and subsequent oocyst formation in the mosquito midgut, confirming their transmission-blocking activity. Potential toxicity due to hemolysis was excluded, since only infected and not normal erythrocytes were damaged by ionophores. Our data strongly support the downstream exploration of monovalent ionophores for repositioning as new antimalarial and transmission-blocking leads.

Antiplasmodial and anti-inflammatory activities of Canthium henriquesianum (K. Schum), a plant used in traditional medicine in Burkina Faso.

ETHNOPHARMACOLOGICAL RELEVANCE: Canthium henriquesianum (K. Schum) is traditionally used in Burkina Faso for the treatment of malaria, but has not been properly investigated, yet. The aim of this study was to characterize in vitro the antiplasmodial and the anti-inflammatory activity of extracts from Canthium henriquesianum (K. Schum). In parallel, extracts of Gardenia sokotensis (Hutch) and Vernonia colorata (Willd), also traditionally used together in Burkina Faso and already reported with antimalarial activity, were compared. MATERIALS AND METHODS: Plant extracts were tested in vitro for antimalarial activity against chloroquine susceptible (D10) and resistant (W2) strains of Plasmodium falciparum using the lactate dehydrogenase assay. Cell cytotoxicity was assessed on human dermal fibroblast (HDF) by the MTT assay. The selectivity index (SI) was used as the ratio of the activity against the parasites compared to the toxicity of the plant extract against HDF. In vitro cytokine production was assessed by ELISA technique. RESULTS: Canthium henriquesianum aqueous extract had a moderate antimalarial activity (IC50<50 µg/ml) with a good selectivity index (SI=HDF/D10>7). Canthium henriquesianum diisopropyl ether extract was the most potent inhibitor of parasite growth with an IC50 9.5 µg/ml on W2 and 8.8 µg/ml on D10 and limited toxicity (SI>2). Gardenia sokotensis and Vernonia colorata aqueous extracts were shown to be significantly less active (IC50≥50 µg/ml) with substantial toxicity. In addition, when the production of IL-1ß and TNFα by lipopolysaccharide (LPS) or hemozoin (malaria pigment) stimulated human THP-1 monocytes was assayed, it was found that the extract of Canthium henriquesianum induced a dose-dependent inhibition of IL-1ß, but not of TNFα production, thus confirming its traditional use as antipyretic. By NMR analysis, the chromone was identified as the mostly represented compound in the diisopropyl ether extract of Canthium henriquesianum. Chromone however, was less active as antimalarial than the crude extract and it did not inhibit cytokine production at not toxic doses, indicating that other molecules in the total extracts contribute to the antiplasmodial and anti-inflammatory activity. CONCLUSION: Canthium henriquesianum seems to possess antimalarial activity in vitro and the ability to inhibit the production of the pyrogenic cytokine IL-1ß.

Dihydroartemisinin inhibits the human erythroid cell differentiation by altering the cell cycle.

Artemisinin derivatives such as dihydroartemisinin (DHA) induce significant depletion of early embryonic erythroblasts in animal models. We have reported previously that DHA specifically targets pro-erythroblasts and basophilic erythroblasts, when human CD34+ stem cells are differentiated toward the erythroid lineage, indicating that a window of susceptibility to artemisinins may exist also in human developmental erythropoiesis during pregnancy. To better investigate the toxicity of artemisinin derivatives, the structure-activity relationship was evaluated against the K562 leukaemia cell line, used as a model for differentiating early human erythroblasts. All artemisinins derivatives, except deoxyartemisinin, inhibited both spontaneous and induced erythroid differentiation, confirming that the peroxide bridge is responsible for the erythro-toxicity. On the contrary, cell growth was markedly reduced by DHA, artemisone and artesunate but not by artemisinin, 10-deoxoartemisinin or deoxy-artemisinin. The substituent at position C-10 is responsible only for the anti-proliferative effect, since 10-deoxoartemisinin did not reduce cell growth but arrested the differentiation of K562 cells. In particular, the results showed that DHA resulted the most potent and rapidly acting compound of the drug family, causing (i) the decreased expression of GpA surface receptors and the down regulation the γ-globin gene; (ii) the alteration of S phase of cell cycle and (iii) the induction of programmed cell death of early erythroblasts in a dose dependent manner within 24h. In conclusion, these findings confirm that the active metabolite DHA is responsible for the erythro-toxicity of most of artemisinins used in therapy. Thus, as long as no further clinical data are available, current WHO recommendations of avoiding malaria treatment with artemisinins during the first trimester of pregnancy remain valid.

Differential effects on angiogenesis of two antimalarial compounds, dihydroartemisinin and artemisone: implications for embryotoxicity.

Artemisinin derivatives are highly effective and well-tolerated antimalarial drugs that now form the basis of antimalarial combination therapies recommended by the World Health Organization. Although not yet reported to be a problem in clinical use, neurotoxicity and embryotoxicity are displayed by the compound class in in vitro and in vivo experimental models, in particular by dihydroartemisinin, the main metabolite of all current clinical artemisinins. Embryotoxicity appears to be connected with defective angiogenesis and vasculogenesis in certain stages of embryo development. This may prevent the use of artemisinin derivatives in malaria during pregnancy, when both mother and fetus are at high risk of death. Artemisone is a novel 10-alkylamino derivative which is not metabolised to dihydroartemisinin. It was selected as a clinical drug candidate on the basis of its high efficacy against Plasmodium falciparum in vitro and its lack of detectable neurotoxicity in both in vitro and in vivo screens. Here we describe the results of a comparative study of the anti-angiogenic properties of both artemisone and dihydroartemisinin in different model systems. We evaluated the proliferation of human endothelial cells and their migration on a fibronectin matrix, the sprouting of new vessels from rat aorta sections grown in collagen and the production of pro-angiogenic cytokines such as vascular endothelial growth factor (VEGF) and interleukin-8 (CXCL-8). The data show that artemisone is significantly less anti-angiogenic than dihydroartemisinin in all the experimental models, suggesting that it will be safer to use than the current clinical artemisinins during pregnancy.