Hypoxia modulates the effect of dihydroartemisinin on endothelial cells.

Artemisinin derivatives, the current cornerstone of malaria treatment, possess also anti-angiogenic and anti-tumor activity. Hypoxia plays a crucial role both in severe malaria (as a consequence of the cytoadherence of infected erythrocytes to the microvasculature) and in cancer (due to the restricted blood supply in the growing tumor mass). However, the consequences of hypoxia onto the effects of artemisinins is under-researched. This study aimed at assessing how the inhibition of microvascular endothelial cell (HMEC-1) growth induced by dihydroartemisinin (DHA, an antimalarial drug and the active metabolite of currently in-use artemisinins) is affected by oxygen tension. Low doses of DHA (achieved in the patients' plasma when treating malaria) were more inhibitory in hypoxia, whereas high doses (required for anti-angiogenic or anti-tumor activity) were more effective in normoxia. The peroxide bridge is essential for cellular toxicity (deoxyDHA was inactive). High doses of DHA caused HMEC-1 apoptosis and G2 cell cycle arrest. Effects were mediated by the generation of oxidative stress as demonstrated by DCF-DA fluorescence and membrane lipid peroxidation analysis. Overall, these results suggest that DHA inhibition of endothelial cell growth is related to the level of tissue oxygenation and drug concentration. This should be considered when studying both the effects of artemisinin derivatives as antimalarials and the potential therapeutic applications of these drugs as anti-tumor agents.

Old and new targets for innovative antimalarial compounds: the different strategies of the Italian Malaria Network.

Clinical treatment-failures to affordable drugs encouraged new investigation for discovery and development of new prophylactic and therapeutic interventions against malaria. The Drug Discovery Cluster (DDcl) of the Italian Malaria Network gathers several highly integrated and complementary laboratories from different Italian Institutions to identify, synthesise, screen in vitro and in vivo new antimalarial molecules directed against the intraerythrocytic stage of P. falciparum parasites and/or with transmission blocking activity to select lead compounds for further development. Complementary research activities, both in vitro and in the clinics, aim at investigating the pathogenetic mechanisms of severe malaria anaemia and the different manifestations of the disease in malaria-HIV co-infected patients to identify new therapies and improve survival.

Plasmodium falciparum parasitized red blood cells modulate the production of endothelin-1 by human endothelial cells.

AIM: Plasmodium falciparum (P. falciparum) malaria is the most important parasitic infection of humans, responsible for about 2,000,000 deaths every year. Cytoadherence of P. falciparum parasitized erythrocytes (pRBC) to vascular endothelium contributes to the pathogenesis of severe malaria causing microcirculatory obstruction and subsequent tissue hypoxia. Several cytokines and vasoactive mediators are involved in this process. The aim of this paper was to investigate the production of endothelin-1 (ET-1), a potent vasoconstrictor agent, by endothelial cells from large vessels (human umbilical vein endothelial cells, HUVEC) or the microvasculature (human microvascular endothelial cells, HMEC-1), co-cultured with different strains of P. falciparum pRBC under normoxic or hypoxic conditions. METHODS: HMEC-1, immortalized by SV 40 large Tontigen, were maintained in MCDB 131 medium supplement ed with 10% fetal calf serum, 10 ng/ml of epidermal growth factor, 1 microg/ml of hydrocortisone, 2 mM glutamine, 100 U/ml of penicillin, 100 microg/ml of streptomycin and 20 mM Hepes buffer. The levels of ET-1 in the supernatants were measured by immunoenzymatic assay. RESULTS: The results indicated that IL1-beta and hypoxia were able to induce ET-1 production by both HUVEC and HMEC-1. However, the co-incubation of HUVEC or HMEC-1 with pRBC induced a dose-dependent decrease of both constitutive and IL1- or hypoxia-induced ET-1 production. The inhibition was independent from the parasite strain used and from the origin of endothelial cells. CONCLUSION: These results show that pRBC by modulating both constitutive and stimulated ET-1 release from endothelial cells can induce modifications of the vascular tone in different anatomical districts. This could be of relevance in the pathogenesis of severe malaria.

Synergistic and antagonistic interactions between haemozoin and bacterial endotoxin on human and mouse macrophages.

Haemozoin (malaria pigment) is a birefringent crystalline material made of Fe (III) Protoporphyrin IX dimers that derives from the degradation of haemoglobin by intraerythrocytic Plasmodia. At schizont rupture, it accumulates indigested inside phagocytic cells altering their immunological properties. Both pro-inflammatory and immunosuppressive activities have been associated with pigment-fed monocyte-macrophages or dendritic cells. These conflicting results were attributed to the source of macrophages or the different preparations of pigment. However, the interactions of malaria pigment with other phagocytes stimuli, such as bacterial endotoxin (LPS) or interferon-gamma have not been fully analysed, yet. The purpose of this study was to compare the immunological properties of native haemozoin (HZ), freshly extracted from Plasmodium falciparum cultures, versus beta-haematin (BH), the synthetic crystals identical to native haemozoin, and to evaluate the relationship between haemozoin and endotoxin on the immune response of different macrophages populations. The results indicate that the iron-porphyrin moiety of both native and synthetic pigment can exert either a synergistic or antagonistic effect with LPS that is related to the length and sequence of treatment, the source of macrophages and is associated with the generation of oxidative stress. These data rise the question of whether and how in vivo concomitant gram(-) bacteremia may affect the pathogenesis and/or the immune response of malaria infections and vice versa.

Ultrastructural characteristics, biological activity and pharmacological relevance of synthetic malaria pigment (beta-haematin).

Malaria pigment (haemozoin, HZ) is the detoxification product of haemoglobin-derived haem of intraerythrocytic malaria parasites. At schizont rupture, haemozoin accumulates inside host phagocytic cells. The chemical structure and the spectroscopic characteristics of haemozoin are identical to those of beta-haematin (BH), a synthetic pigment obtained from Ferriprotoporphyrin IX (Fe (III) PPIX) in acidic conditions. The process of BH formation is the target of quinoline antimalarials. Here, we summarise the results of our studies on the ultrastructural characteristics, biological and pharmacological relevance of synthetic vs. native haemozoin. 1) By electron microscopy, native HZ and synthetic BH appear as dark brown crystals, morphologically indistinguishable and are internalised by phagocytes at the same extent. 2) Both HZ and BH modulate the production of cytokines (TNF and NO) and increase the susceptibility to lipid peroxidation of mouse or human phagocytes. The antioxidant status of the phagocytes regulates the susceptibility to BH/HZ-mediated effects. 3) The process of BH formation from Fe(III)PPIX, hence haem detoxification, can be inhibited by electrochemically-reduced, Fe(II)PPIX molecules. Maintaining iron in the reduced state can thus be considered a new pharmacological target. This was confirmed by the observation that thiol-reducing agents (NAC, cystein) were able to inhibit BH formation and were toxic to parasites in vitro.

Phagocytosis of hemozoin (native and synthetic malaria pigment), and Plasmodium falciparum intraerythrocyte-stage parasites by human and mouse phagocytes.

Hemozoin, the detoxification product of hemoglobin heme, piles up as electron-dense material in the food vacuole (FV) of intraerythrocytic malaria parasites (malaria pigment). In infected individuals, pigment is internalized by both circulating and resident phagocytes, thus modulating their functions. Synthetic beta-hematin, prepared in vitro from hematin (ferriprotoporphyrin IX hydroxide) in acidic condition, is spectroscopically identical to hemozoin. In this electron microscopy study, native and synthetic hemozoin also prove to be morphologically indistinguishable (large polygonal crystals with apparent transverse banding) and to undergo the same process when internalized by phagocytes (primarily a direct uptake of crystals, similar to what is described for asbestos fibers). On the contrary, whole parasites appear to follow a classical endocytic pathway. This suggests that there may be differences between the ingestion of free particles and whole parasites in terms of modulation of phagocytes' functions.

Standardization of the physicochemical parameters to assess in vitro the beta-hematin inhibitory activity of antimalarial drugs.

Intraerythrocytic plasmodia form hemozoin as a detoxification product of hemoglobin-derived heme. An identical substance, beta-hematin (BH), can be obtained in vitro from hematin at acidic pH. Quinoline-antimalarials inhibit BH formation. Standardization of test conditions is essential for studying the interaction of compounds with this process and screening potential inhibitors. A spectrophotometric microassay of heme polymerization inhibitory activity (HPIA) (Basilico et al., Journal of Antimicrobial Chemotherapy 42, 55-60, 1998) previously reported was used to investigate the effect of pH and salt concentration on BH formation. The yield of BH formation decreased with pH. Moreover, under conditions used in the above HPIA assay (18 h, 37 degrees C, pH = 2.7), several salts including chloride and phosphate inhibited the process. Aminoquinoline drugs formulated as salts (chloroquine-phosphate, primaquine-diphosphate), but not chloroquine-base, also inhibited the reaction. Interference by salts was highest at low pH and decreased at higher pH (pH 4). Here, we describe different assay conditions that eliminate these problems (BHIA, beta-hematin inhibitory activity). By replacing hematin with hemin as the porphyrin and NaOH solution with DMSO as solvent, the formation of BH was independent of pH up to pH 5.1. No interference by salts was observed over the pH range 2.7-5.1. Dose-dependent inhibition of BH formation was obtained with chloroquine-base, chloroquine-phosphate, and chloroquine-sulfate at pH 5.1. Primaquine was not inhibitory. The final product, characterized by solubility in DMSO, consists of pure BH by FT-IR spectroscopy. The BHIA assay (hemin in DMSO, acetate buffer pH 5 +/- 0.1, 18 h at 37 degrees C) is designed to screen for those molecules forming pi-pi interactions with hematin and thus inhibiting beta-hematin formation.

Macrophage preconditioning with synthetic malaria pigment reduces cytokine production via heme iron-dependent oxidative stress.

Hemozoin (malaria pigment), a polymer of hematin (ferri-protoporphyrin IX) derived from hemoglobin ingested by intraerythrocytic plasmodia, modulates cytokine production by phagocytes. Mouse peritoneal macrophages (PM) fed with synthetic beta-hematin (BH), structurally identical to native hemozoin, no longer produce tumor necrosis factor alpha (TNFalpha) and nitric oxide (NO) in response to lipopolysaccharide (LPS). Impairment of NO synthesis is due to inhibition of inducible nitric oxide synthase (iNOS) production. BH-mediated inhibition of PM functions cannot be ascribed to iron release from BH because neither prevention by iron chelators nor down-regulation of iron-regulatory protein activity was detected. Inhibition appears to be related to pigment-induced oxidative stress because (a) thiol compounds partially restored PM functions, (b) heme oxygenase (HO-1) and catalase mRNA levels were up-regulated, and (c) free radicals production increased in BH-treated cells. The antioxidant defenses of the cells determine the response to BH: microglia cells, which show a lower extent of induction of HO-1 and catalase mRNAs and lower accumulation of oxygen radicals, are less sensitive to the inhibitory effect of BH on cytokine production. Results indicate that BH is resistant to degradation by HO-1 and that heme-iron mediated oxidative stress may contribute to malaria-induced immunosuppression. This study may help correlate the different clinical manifestations of malaria, ranging from uncomplicated to severe disease, with dysregulation of phagocyte functions and promote better therapeutic strategies to counteract the effects of hemozoin accumulation.

A novel endogenous antimalarial: Fe(II)-protoporphyrin IX alpha (heme) inhibits hematin polymerization to beta-hematin (malaria pigment) and kills malaria parasites.

The polymerization of hemoglobin-derived ferric-protoporphyrin IX [Fe(III)PPIX] to inert hemozoin (malaria pigment) is a crucial and unique process for intraerythrocytic plasmodia to prevent heme toxicity and thus a good target for new antimalarials. Quinoline drugs, i.e., chloroquine, and non-iron porphyrins have been shown to block polymerization by forming electronic pi-pi interactions with heme monomers. Here, we report the identification of ferrous-protoporphyrin IX [Fe(II)PPIX] as a novel endogenous anti-malarial. Fe(II)PPIX molecules, released from the proteolysis of hemoglobin, are first oxidized and then polymerized to hemozoin. We obtained Fe(II)PPIX on preparative scale by electrochemical reduction of Fe(III)PPIX, and the reaction was monitored by cyclic voltammetry. Polymerization assays at acidic pH were conducted with the resulting Fe(II)PPIX using a spectrophotometric microassay of heme polymerization adapted to anaerobic conditions and the products characterized by infrared spectroscopy. Fe(II)PPIX (a) did not polymerize and (b) produced a dose-dependent inhibition of Fe(III)PPIX polymerization (IC(50) = 0.4 molar equiv). Moreover, Fe(II)PPIX produced by chemical reduction with thiol-containing compounds gave similar results: a dose-dependent inhibition of heme polymerization was observed using either L-cysteine, N-acetylcysteine, or DL-homocysteine, but not with L-cystine. Cyclic voltammetry confirmed that the inhibition of heme polymerization was due to the Fe(II)PPIX molecules generated by the thiol-mediated reduction of Fe(III)PPIX. These results point to Fe(II)PPIX as a potential endogenous antimalarial and to Fe(III)PPIX reduction as a potential new pharmacological target.

A fine balance between oxidised and reduced haem controls the survival of intraerythrocytic plasmodia.

The polymerisation of haemoglobin-derived ferri-protoporphyrin IX (Fe(III)PPIX) to haemozoin (malaria pigment) is a crucial process for intraerythrocytic plasmodia to prevent haem toxicity. It is also the target of in-use antimalarial drugs and newer compounds. This reaction and the inhibition thereof can be reproduced and studied in vitro.

Macrophage populations of different origins have distinct susceptibilities to lipid peroxidation induced by beta-haematin (malaria pigment).

We investigated the susceptibility of peritoneal mouse macrophages and macrophage and microglia cell lines to the peroxidative activity of beta-haematin, the synthetic polymer identical to native malaria pigment. The extent of lipid peroxidation, measured as production of thiobarbituric acid reactive substances (TBARS), was greater for peritoneal macrophages than for cell lines and microglia cells. TBARS production apparently was not attributable to the release of free iron from the protoporphyrin moiety, but related to lower glutathione content and different lipid composition of the cell membrane. These findings offer a new interpretation for the contentious immunomodulatory effects of beta-haematin reported for phagocytes of different origins.

The effect of synthetic malaria pigment (beta-haematin) on adhesion molecule expression and interleukin-6 production by human endothelial cells.

The effects of synthetic malaria pigment (beta-haematin, BH) on the expression of the intercellular adhesion molecule 1 (ICAM-1) and platelet endothelial cell adhesion molecule 1 (PECAM-1) and the production of interleukin-6 (IL-6) by human microvascular endothelial cells were measured using flow cytometry analysis and immunoenzymatic assay. BH alone did not affect basal levels of ICAM-1, PECAM-1 or IL-6. When added to cell cultures before or with, but not after, lipopolysaccharide or tumour necrosis factor alpha, BH at 1-100 micrograms/mL induced a dose-dependent inhibition of ICAM-1 and PECAM-1 expression and IL-6 production. Cell viability and human leucocyte antigen A,B,C expression remained unaffected. Similar, though more variable, results were obtained using human umbilical vein endothelial cells. These results suggested that accumulation of pigment within endothelial cells following repeated malaria infection reduces local inflammation and parasite sequestration through inhibition of either cytokine production or parasitized erythrocyte receptors on endothelial cells.

A microtitre-based method for measuring the haem polymerization inhibitory activity (HPIA) of antimalarial drugs.

The malaria parasite metabolizes haemoglobin and detoxifies the resulting haem by polymerizing it to form haemozoin (malaria pigment). A polymer identical to haemozoin, beta-haematin, can be obtained in vitro from haematin at acidic pH. Quinoline-containing anti-malarials (e.g. chloroquine) inhibit the formation of either polymer. Haem polymerization is an essential and unique pharmacological target. To identify molecules with haem polymerization inhibitory activity (HPIA) and quantify their potency, we developed a simple, inexpensive, quantitative in-vitro spectrophotometric microassay of haem polymerization. The assay uses 96-well U-bottomed polystyrene microplates and requires 24 h and a microplate reader. The relative amounts of polymerized and unpolymerized haematin are determined, based on solubility in DMSO, by measuring absorbance at 405 nm in the presence of test compounds as compared with untreated controls. The final product (a solid precipitate of polymerized haematin) was validated using infrared spectroscopy and the assay proved reproducible; in this assay, activity could be partly predicted based on the compound's chemical structure. Both water-soluble and water-insoluble compounds can be quantified by this method. Although the throughput of this assay is lower than that of radiometric methods, the assay is easier to set up and cheaper, and avoids the problems related to radioactive waste disposal.

[Exclusive radical radiotherapy of laryngeal tumors: 10-year experience]. / La radioterapia radicale esclusiva nei tumori della laringe: esperienza decennale.

Exclusive irradiation is used to treat primary neoplasms localized to the larynx because it is known to be able to cure, or at least control, the disease in a high percentage of cases without affecting speech. We report our ten-year experience in the Radiotherapy Department of Chieti Hospital. From 1985 to 1994, exclusive radiotherapy was used to treat 87 patients bearing histologically proved epidermoid carcinomas of the larynx. The patients average age was 67 years. The primary site was the glottis in 64 cases and the supraglottic and subglottic areas in 21 and 2 cases, respectively. The lymph nodes were clinically positive in 8 patients (9%) and negative in 79 (91%). The minimum follow-up was 20 months. All the patients were treated with cobalt 60 beams; the daily dose was 2 Gy, fractionation was 5 days a week. Average tumor dose was 64 Gy (range: 55-70 Gy). Disease-free survival actuarial curves show 72% five-year survival for glottic cancer (75% for N0, 80% for T1 and 61% for T2-T3-T4 cases) and 21% for supraglottic cancer (25% for N0 cases). To conclude, irradiation is confirmed to be a useful tool to treat early laryngeal cancer, while new combinations of surgery, chemotherapy and irradiation are needed to treat locally advanced cancer.

Non-iron porphyrins inhibit beta-haematin (malaria pigment) polymerisation.

Infrared spectroscopy was used to evaluate the effect of non-iron porphyrins (protoporphyrin IX and haematoporphyrin) on haematin polymerisation to beta-haematin at acidic pH. Both molecules effectively inhibited the reaction, with haematoporphyrin 6 times as active as protoporphyrin IX. We postulated that the interaction between the pi electron system of porphyrin rings leads to the formation of pi-pi adducts, which inhibit polymer elongation in the same way as antimalarial drugs (e.g., chloroquine); the presence of hydroxyl groups able to bind haem iron enhances activity.

Production of cytokines by alveolar and peritoneal macrophages stimulated by Aspergillus fumigatus conidia or hyphae.

Aspergillus fumigatus is an opportunistic pathogen responsible for severe, invasive infections in neutropenic hosts. Lung clearance of A. fumigatus conidia seems to be mediated by phagocytic cells and oxygen radicals. It is not known if cytokines or nitrogen radicals are also involved. We tested for the production of TNF alpha, IL-1 and nitric oxide (NO) after stimulation of mouse macrophages with the fungus. We found that both cytokines, but not NO, were produced in a dose-dependent manner during the first 24 h of culture. Except for a faster kinetic, no appreciable differences were seen between alveolar and peritoneal macrophages. Furthermore, both hyphae and conidia, either alive or killed, were capable of inducing cytokines production. However, among different Aspergillus spp. only A. fumigatus and A. flavus seemed to induce significant amount of TNF alpha and IL-1, whereas A. terreus and A. niger were less effective. In no case could we detect production of NO. Finally, macrophages from dexamethasone-treated mice failed to produce cytokines in response to A. fumigatus conidia. These results indicate that in normal hosts inflammatory cytokines contribute to the natural response against Aspergillus infections and suggest that the impairment of cytokine production, in immunodepressed patients, may favour the growth and spread of the fungus.

The heme moiety of malaria pigment (beta-hematin) mediates the inhibition of nitric oxide and tumor necrosis factor-alpha production by lipopolysaccharide-stimulated macrophages.

To investigate the effect of the heme moiety of malaria pigment, hemozoin, on phagocyte functions, mouse macrophages were fed with insoluble beta-hematin, the synthetic heme-polymer chemically identical to the native pigment, or the soluble monomer, hematin. Production of inflammatory cytokines, interleukin 1 (IL1), tumor necrosis factor alpha (TNF alpha), and nitric oxide (NO) was assayed in the supernatants after stimulation with lipopolysaccharide. The results indicate that both beta-hematin and hematin induce a dose-dependent inhibition of macrophage production of TNF alpha and NO, but not of IL1. One-hour pretreatment with soluble hematin inhibited production of cytotoxic mediators by more than 50% compared to controls, while 6-hr exposure was necessary for insoluble beta-hematin to induce the same level of inhibition. However, the same treatment did not modify the production of TNF alpha and NO by mouse microglia cell lines. The inhibition was partially counterbalanced by adding sulphydryl group donors such as 2-mercaptoethanol, glutathione, or N-acetyl-cysteine during the preincubation time. The results of the present study confirm the inhibitory role of malaria pigment and show that such effect is due to the heme moiety and may be selective for the production of cytotoxic mediators by specific phagocytes. The implications of these findings in the control of malaria infection and disease and in the pathogenesis of severe malaria are discussed.

Effects of pidotimod on macrophage functions in methylprednisolone-treated mice.

CD-1 mice were treated with methylprednisolone (mPDN) 2-5 mg/kg s.c., for 11 or 6 days, in order to achieve an immunosuppressed state. For the same length of time a group of mice also received pidotimod ((R)-3-[(S)-(5-oxo-2-pyrrolidinyl) carbonyl]-thiazolidine-4-carboxylic acid. PGT/1A, CAS 121808-62-6) i.p. at 100 or 10 mg/kg. At the end of treatment, peritoneal macrophages (MO) were recovered, purified by adherence to plastic and activated in vitro with different stimuli. After 24 h of incubation, the supernatants were collected and assayed for the presence of tumor necrosis factor-alpha (TNF-alpha) and nitrite (NO2-), which is the stable derivative of nitric oxide (NO) in acqueous solution. It is well known that TNF-alpha and NO represent two out of many molecules secreted by activated MO which are essential for killing microorganisms and for natural response to infections. It was observed that MO from mPDN-treated mice were unable to produce sufficient levels of both TNF-alpha and NO when stimulated in vitro with lipopolysaccharide, IFN-gamma or conidia from an opportunistic fungus, Aspergillus fumigatus, confirming that corticosteroids are able to inhibit the antimicrobial activity of MO. However, MO from mice received mPDN plus pidotimod fully recovered the capacity to produce TNF-alpha and NO in response to the same stimuli. Optimal dose of pidotimod was 100 mg/kg. In addition, pidotimod was also able to reconstitute the cellularity of the peritoneum and of the spleens of mice immunodepressed by mPDN.(ABSTRACT TRUNCATED AT 250 WORDS)

[Radiotherapy of inoperable, loco-regional epidermoid carcinoma of the lung. 5-year experience]. / Radioterapia del carcinoma epidermoidale del polmone inoperabile in fase loco-regionale. Esperienza di cinque anni.

Results of chest radiotherapy on 48 patients with inoperable squamous-cell carcinoma are reported; 25 patients had continuous treatment whereas the remaining 23 patients had split-course. Objective remission was obtained in 71% of the cases; mean survival was 11.7 months for the whole series, and 14 months for responders. An advantage in objective remission rate for patients having a dose of 60 Gy or more was found. Split-course resulted in similar survival and objective remission rate but was less toxic than continuous treatment. In 43% of cases the disease progressed only loco-regionally. Radiotherapy was interrupted because of toxicity in 23% of cases and there was only a case of life-threatening toxicity.