Therapeutic potential of new Pt(II) and Ru(III) triazole-pyrimidine complexes against Leishmania donovani

We have already established an in vitro culture system using murine macrophages infected with Leishmania donovani in which the time course of parasite growth is determined quantitatively. We adopted this system for the screening of three triazole-pyrimidine derivatives that would ideally prove to be effective against L. donovani with no toxicity to the host cell. Amphotericin B deoxycholate was used as the standard drug and gave a IC50 value of 3.89 microg/ml. The three triazole-pyrimidine compounds assayed have been reported to be potent growth inhibitors of L. donovani promastigote and amastigote stages. Compounds SPIV and SPVI exhibited the highest toxicity for extracellular forms of parasites, with IC50 values of 19.95 and 21.61 microg/ml, respectively. The triazole-pyrimidine SPV, although to a lower degree, also showed pronounced effects against promastigote forms with IC50 of 33.14 microg/ml. Drug activity was higher against amastigote than against promastigote stages. The compounds SPIV and SPVI interfered with the synthesis of macromolecules, affecting primarily DNA at the lower concentration tested (5 microg/ml), while SPV also showed interference, though to a lesser extent, and at a higher concentration (15 microg/ml) the percentage of inhibition rose considerably. The synthesis or RNA and proteins was also depressed significantly by these compounds at administration rates of 15 microg/ml. Ultrastructural alterations were evident in the main organelles of L. donovani (nucleus, kinetoplast, mitochondria), after the addition of the three compounds at a concentration of 5 microg/ml, to the in vitro culture. The in vitro promastigote forms of L. donovani can degrade glucose to carbon dioxide, and part of the carbon skeleton of the glucose is excreted as end metabolites. The excretion of these metabolites, mainly acetate, was also inhibited by the three compounds assayed, suggesting that this could be due to a direct effect on some of the enzymes related to this fermentation pathway or to the inhibition exerted by the compounds on enzyme synthesis.     

Comparison of the efficacy of free and non-ionic-surfactant vesicular formulations of paromomycin in a murine model of visceral leishmaniasis

Non-ionic-surfactant vesicular (NIV) formulations of paromomycin have been tested in-vitro and in-vivo for their activity against Leishmania donovani. Production of NIV was dependent both on the surfactant used and on the concentration of paromomycin; only two of the surfactants studied formed vesicles at the highest paromomycin concentration (9 mg mL(-1)). At surfactant-lipid concentrations > or = 1.5 mM, suspensions of NIV (drug- or glucose-loaded) were cytotoxic to macrophages infected with L. donovani; high levels of nitrite were produced in cell supernatants. At surfactant-lipid concentrations < 1.5 mM, drug-loaded NIV were more effective than the same dose of free drug, in terms of the percentage of cells infected and the number of parasites/cell. At surfactant-lipid concentrations < or = 0.15 mM, drug-loaded NIV were ineffective in-vitro. In-vivo, treatment with decaethylene glycol mono n-hexadecyl ether paromomycin NIV was more effective than hexaethylene glycol mono n-hexadecyl ether paromomycin NIV, in terms of suppression of liver and spleen parasite burdens. Against liver parasites, both types of paromomycin-loaded NIV were more effective than free drug. Neither the NIV nor free forms of paromomycin caused significant suppression of bone-marrow parasites. The study shows that entrapment of paromomycin in NIV can be used to increase its antileishmanial activity in-vitro and in-vivo.     

Oxoaporphine alkaloids and quinones from Stephania dinklagei and evaluation of their antiprotozoal activities

Bioactivity-guided fractionation of Stephania dinklagei yielded six compounds including, two zwitterionic oxoaporphine alkaloids, N-methylliriodendronine, and 2-O,N-dimethylliriodendronine, two oxoaporphine alkaloids, liriodenine, and dicentrinone, one aporphine alkaloid, corydine, and one anthraquinone, aloe-emodin. Apart from corydine, the isolates have not been reported as constituents of S. dinklagei. N-Methylliriodendronine, and 2-O,N-dimethylliriodendronine are reported for the first time as natural products. All isolated compound were tested for antiprotozoal activity and cytotoxic activities in vitro. N-Methylliriodendronine was the most active against L. donovani amastigotes (IC50 = 36.1 microM). Liriodenine showed the highest activity against Leishmania donovani, and Plasmodium falciparum with IC50 values of 26.16 and 15 microM, respectively. Aloe-emodin was the only compound active (IC50 = 14 microM) against T. b. brucei.     

Selective antimicrotubule activity of N1-phenyl-3,5-dinitro-N4,N4-di-n-propylsulfanilamide (GB-II-5) against kinetoplastid parasites

Analogs of the antimitotic herbicide oryzalin (3,5-dinitro-N4,N4-di-n-propylsulfanilamide) were recently prepared that were more potent in vitro than the parent compound against the kinetoplastid parasite Leishmania donovani (Bioorg Med Chem Lett 12:2395-2398, 2002). In the present work, we show that the most active molecule in the group, N1-phenyl-3,5-dinitro-N4,N4-di-n-propylsulfanilamide (GB-II-5), is a potent, selective antimitotic agent against kinetoplastid parasites. GB-II-5 possesses IC50 values of 0.41 and 0.73 microM in vitro against two strains of the related parasite Trypanosoma brucei but is much less toxic to J774 murine macrophages and PC3 prostate cancer cells, exhibiting IC50 values of 29 and 35 microM against these lines, respectively. Selectivity is also observed for GB-II-5 with purified leishmanial and mammalian tubulin. The assembly of 15 microM leishmanial tubulin is completely inhibited by 10 microM GB-II-5, whereas 40 microM GB-II-5 inhibits the assembly of 15 microM porcine brain tubulin by only 17%. In cultured L. donovani and T. brucei, treatment with 5 and 0.5 microM GB-II-5, respectively, causes a striking increase in the fraction of G2M cells compared with control. Given the potency and selectivity of this agent against kinetoplastid tubulin, GB-II-5 emerges as an exciting new antitrypanosomal and antileishmanial lead compound.     

In vitro screening of traditional South African malaria remedies against Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani, and Plasmodium falciparum

Three hundred extracts were prepared from plants traditionally used in South Africa to treat malaria and screened in vitro for activity against Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani, and Plasmodium falciparum. For the 43 extracts which inhibited the growth of one or more parasites to more than 95?% at 9.7?μg/mL, the IC?? values against all four protozoal parasites and cytotoxic IC??s against rat myoblast L6 cells were determined. Amongst the most notable results are the activities of AGATHOSMA APICULATA (IC?? of 0.3?μg/mL) against Plasmodium falciparum, as well as Salvia repens and Maytenus undata against Leishmania donovani with IC??s of 5.4?μg/mL and 5.6?μg/mL, respectively. This screening is the starting point for a HPLC-based activity profiling project in antiprotozoal lead discovery.     

Bioactive compounds from Celaenodendron mexicanum

Bioactivity-directed fractionation of the CHCl3-MeOH extract of the leaves of Celaenodendron mexicanum by means of the brine shrimp lethality test and chromatographic techniques led to the isolation of three carboxylic acid triterpenes, the new tirucalla-type triterpene, 3 alpha-hydroxytirucalla-7,24Z-dien-26-oic acid, 3-oxotirucalla-7,24Z-dien-26-oic acid, and epi-oleanolic acid, and three biflavonoids amentoflavone, podocarpusflavone A, and podocarpusflavone B. Four non-active compounds friedelin, maytensifolin B, 3 beta-hydroxyfriedelan-16-one, and celaenodendrolide were also obtained. epi-Oleanolic acid was the most active against brine shrimps with LC50 value of 23.3 microM. In addition, all isolates were tested for in vitro antiprotozoal and cytotoxic activities. 3-Oxotirucalla-7,24Z-dien-26-oic acid and epi-oleanolic acid showed the highest activity against Leishmania donovani promastigotes with IC50 values of 13.7 and 18.8 microM, respectively. Only 3-oxotirucalla-7,24Z-dien-26-oic acid showed activity against Trypanosoma brucei brucei bloodstream forms with IC50 value of 16.8 microM.     

Anti-plasmodial and anti-leishmanial activity of conformationally restricted pentamidine congeners

A library of 52 pentamidine congeners in which the flexible pentyldioxy linker in pentamidine was replaced with various restricted linkers was tested for in-vitro activity against two Plasmodium falciparum strains and Leishmania donovani. The tested compounds were generally more effective against P. falciparum than L. donovani. The most active compounds against the chloroquine-sensitive (D6, Sierra Leone) and -resistant (W2, Indochina) strains of P. falciparum were bisbenzamidines linked with a 1,4-piperazinediyl or 1, 4-homopiperazinediyl moiety, with IC50 values (50% inhibitory concentration, inhibiting parasite growth by 50% in relation to drug-free control) as low as 7 nM based on the parasite lactate dehydrogenase assay. Seven piperazine-linked bisbenzamidines substituted at the amidinium nitrogens with a linear alkyl group of 3-6 carbons (22, 25, 27, 31) or cycloalkyl group of 4, 6 or 7 carbons (26, 32, 34) were more potent (IC50<40 nM) than chloroquine or pentamidine as anti-plasmodial agents. The most active anti-leishmanial agents were 4,4'-[1,4-phenylenebis(methyleneoxy)]bisbenzenecarboximidamide (2, IC50 approximately 0.290 microM) and 1,4-bis[4-(1H-benzimidazol-2-yl)phenyl] piperazine (44, IC50 approximately 0.410 microM), which were 10- and 7-fold more potent than pentamidine (IC50 approximately 2.90 microM). Several of the more active anti-plasmodial agents (e.g. 2, 31, 33, 36-38) were also potent anti-leishmanial agents, indicating broad antiprotozoal properties. However, a number of analogues that showed potent anti-plasmodial activity (1, 18, 21, 22, 25-28, 32, 43, 45) were not significantly active against the Leishmania parasite. This indicates differential modes of anti-plasmodial and anti-leishmanial actions for this class of compounds. These compounds provide important structure-activity relationship data for the design of improved chemotherapeutic agents against parasitic infections.     

Novel 3-nitro-1H-1,2,4-triazole-based amides and sulfonamides as potential antitrypanosomal agents

A series of novel 3-nitro-1H-1,2,4-triazole-based (and in some cases 2-nitro-1H-imidazole-based) amides and sulfonamides were characterized for their in vitro antitrypanosomal and antileishmanial activities as well as mammalian toxicity. Out of 36 compounds tested, 29 (mostly 3-nitro-1H-1,2,4-triazoles) displayed significant activity against Trypanosoma cruzi intracellular amastigotes (IC(50) ranging from 28 nM to 3.72 μM) without concomitant toxicity to L6 host cells (selectivity 66-2782). Twenty-three of these active compounds were more potent (up to 58-fold) than the reference drug benznidazole, tested in parallel. In addition, nine nitrotriazoles which were moderately active (0.5 μM ≤ IC(50) < 6.0 μM) against Trypanosoma brucei rhodesiense trypomastigotes were 5-31-fold more active against bloodstream-form Trypanosoma brucei brucei trypomastigotes engineered to overexpress reduced nicotinamide adenine dinucleotide dependent nitroreductase. Finally, three nitrotriazoles displayed a moderate activity against the axenic form of Leishmania donovani . Therefore, 3-nitro-1H-1,2,4-triazole-based amides and sulfonamides are potent antitrypanosomal agents.     

Arsonoliposomes: effect of arsonolipid acyl chain length and vesicle composition on their toxicity towards cancer and normal cells in culture.

Arsonolipid-containing liposomes were investigated in order to characterize the influence of the lipid acyl-chain length and liposome composition on cytotoxicity. Three types of cancer cells (HL-60, C6 and GH3), and two types of normal cells (HUVEC and RAME) were used. Liposomes containing the lauroyl, myristoyl and stearoyl side chain arsonolipids (with different lipid compositions) were incubated with a given number of cells and cell viability was estimated (MTT assay and trypan blue exclusion). Morphological studies were also performed in some cases. In addition, the interaction between some of the prepared arsonoliposomes and HUVEC cells was assessed. Results reveal that all the studied arsonoliposomes cause a dose dependent inhibition of survival in all three malignant cell lines studied (initiated at 10(-6) M). The corresponding toxicity against normal cells (HUVEC and RAME) is much lower for all arsonoliposomes, except for the lauroyl side chain arsonoliposomes which were demonstrated to be relatively toxic towards normal cells, especially RAME. The microscopic observations that these vesicles possibly cause apoptosis of most cell types studied, as well as the different speed of their cytotoxic activity, imply a different mechanism of action for this arsonoliposome type. Taking the results of this study in conjunction with our previous results on arsonoliposome physical stability and cytotoxicity, it is recommended that palmitoyl-arsonolipid arsonoliposomes be used for further investigations in vivo towards the development of an anticancer product.     

Preliminary screening of antiprotozoal activity of Jasonia glutinosa aerial parts

As a part of a research on anti-protozoan agents, the acetonic extracts from Jasonia glutinosa aerial parts (Asteraceae) were tested in vitro against Entamoeba histolytica, Leishmania donovani (promastigote forms) and Trichomonas vaginalis. The 20% acetone extract was not effective at the assayed concentrations. The pure acetone extract was found to be active against Leishmania donovani and Entamoeba histolytica.     

2H-benzimidazole 1,3-dioxide derivatives: a new family of water-soluble anti-trypanosomatid agents

Three series of benzimidazole N-oxide derivatives were developed and were examined for their activity against trypanosomatid parasites (Trypanosoma cruzi and Leishmania spp.). 2H-benzimidazole 1,3-dioxides displayed remarkable in vitro activities against both parasites, with derivatives 28, 29, and 32 being the most potent (IC50 < 5 microM) against the epimastigote form of T. cruzi and 28, 33, and 35 the most potent against the promastigote form of Leishmania spp. Unspecific cytotoxicity was evaluated using murine macrophages, and derivative 33 was not toxic at a concentration 30 times that of its IC50 against T. cruzi that was completely toxic for Leishmania spp., implying that the series of 2H-benzimidazole 1,3-dioxides is selective toward both trypanosomatid parasites. Derivatives 33 and 35 were submitted to an in vivo assay using an acute model of Chagas' disease and a short-term treatment (30 mg/kg/day orally administrated as aqueous solution, during 10 days). While in the control (untreated) and Benznidazole (50 mg/kg/day) groups survival fraction was 60.0% and 87.5%, respectively, none of the animals treated with derivatives 33 and 35 died. From the preliminary structure-activity relationship studies reduction potential and electrophilicity were found relevant to anti-T. cruzi activity. Active compounds are better electrophiles and more easily reduced than inactive ones.     

Structure-activity study on the in vitro antiprotozoal activity of glutathione derivatives

A series of N-, S-, and COOH-blocked glutathione derivatives were evaluated against the pathogenic parasites Trypanosoma brucei, Trypanosoma cruzi, and Leishmania donovani in vitro, to identify the determinants necessary for activity and for further development into an active lead structure. The results show that N,S-blocked glutathione diesters are the most effective inhibitors of T. brucei with structures 14-16 being the most active, 14 having an IC(50) approximately 1.9 microM. The toxicity effects observed for glutathione derivatives 12, 14, and 16 have been correlated to the K562 antileukemic activity of these compounds and their inhibitory effects on the glyoxalase system of the host. Diester compounds based on S-2,4-dinitrophenylglutathione (17-22) were found to be significantly better inhibitors of T. brucei with ED(50)'s in the range 16-0.19 microM. Compounds 19 and 20 were the two best inhibitors, with an ED(50) of approximately 1.07 and 0.19 microM, respectively; however 20 displayed toxicity in parasitic assays. Monoesters, monoamides, and diamides tested generally exhibited low in vitro activity. The compounds did not inhibit glutathionylspermidine synthetase and trypanothione reductase enzyme targets in the unique trypanothione pathway of these parasites. Diester compounds per se were considered to be ineffective inhibitors of trypanothione metabolism suggesting that these compounds might act as prodrugs, being hydrolyzed in situ into a variety of glutathione derivatives which include combinations of monoesters, free acids, and amines, some of which are inhibitors of trypanothione metabolism.     

In-vitro and in-vivo studies on a topical formulation of sitamaquine dihydrochloride for cutaneous leishmaniasis

The efficacy of topical formulations of the 8-aminoquinoline, sitamaquine dihydrochloride, in both in-vitro and in in-vivo models of cutaneous leishmaniasis is reported. In-vitro parasite assays confirmed that sitamaquine dihydrochloride was active against a range of Leishmania species that cause either cutaneous or visceral leishmaniasis, with ED50 values against amastigotes over the range of 2.9 to 19.0 microM. A range of topical sitamaquine dihydrochloride formulations (anhydrous gel, emulsions) were developed for studies on experimental cutaneous leishmaniasis using only topically acceptable excipients or those currently undergoing regulatory approval. An uptake study into murine skin confirmed in-vitro skin penetration and retention. Several formulations were tested in-vivo against Leishmania major cutaneous lesions in BALB/c mice. None of the sitamaquine dihydrochloride formulations tested appeared to either slow lesion progression or reduce parasite burden.     

Bisphosphonates inhibit the growth of Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Plasmodium falciparum: a potential route to chemotherapy

We have investigated the effects in vitro of a series of bisphosphonates on the proliferation of Trypanosoma cruzi, Trypanosoma brucei rhodesiense, Leishmania donovani, Toxoplasma gondii, and Plasmodium falciparum. The results show that nitrogen-containing bisphosphonates of the type used in bone resorption therapy have significant activity against parasites, with the aromatic species having in some cases nanomolar or low-micromolar IC(50) activity values against parasite replication (e.g. o-risedronate, IC(50) = 220 nM for T. brucei rhodesiense; risedronate, IC(50) = 490 nM for T. gondii). In T. cruzi, the nitrogen-containing bisphosphonate risedronate is shown to inhibit sterol biosynthesis at a pre-squalene level, most likely by inhibiting farnesylpyrophosphate synthase. Bisphosphonates therefore appear to have potential in treating parasitic protozoan diseases.     

Synthesis and evaluation of antiparasitic activities of new 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine derivatives

A series of new 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine derivatives, prepared by two synthetic routes, were in vitro assayed against three Trypanosoma strains, Leishmania donovani, and Plasmodium falciparum K1. Seven out of 17 compounds showed moderate to very good activity against blood stage T. b. rhodesiense, with 10 and 17 exhibiting highest potency (IC50 of 1.0 and 1.1 microM, respectively). Interestingly, the beta-diketone precursors 1-3 had good antitrypanosomal activity toward the insect stage, with IC50 values of 1.0-3.4 microM. Among different compounds with moderate activity against T. cruzi, compound 17 showed the lowest IC50 value of 9.5 microM; thus, the series seemed to act selectively toward the different Trypanosoma parasites. Eight compounds were moderately active against L. donovani, with 2, 3, and 12 being the most promising ones (IC50 values of 2.3-5.2 microM), whereas compound 14 was the only derivative with good activity against P. falciparum (IC50 of 3.7 microM).     

Antiprotozoal activity of sesquiterpenes from jasonia glutinosa

Two sesquiterpene alcohols, [11 R]-eudesm-4(14)-en-5?,11,12-triol ( 1 ) and [11R]-eudesm-4(14)-en-5a,11-12-triol ( 2 ), from Jasonia glutinosa aerial parts (Asteraceae), have been tested using an in vitro technique against Leishmania donovani (promastigote forms) and Plasmodium falciparum . Sesquiterpene 1 was not effective at the assayed concentrations. Sesquiterpene 2 was found to be active against Leishmania donovani and Plasmodium falciparum .     

Antiprotozoal Activity of Sesquiterpenes from Jasonia Glutinosa

Two sesquiterpene alcohols, [11 R]-eudesm-4(14)-en-5ß,11,12-triol ( 1 ) and [11R]-eudesm-4(14)-en-5a,11-12-triol ( 2 ), from Jasonia glutinosa aerial parts (Asteraceae), have been tested using an in vitro technique against Leishmania donovani (promastigote forms) and Plasmodium falciparum . Sesquiterpene 1 was not effective at the assayed concentrations. Sesquiterpene 2 was found to be active against Leishmania donovani and Plasmodium falciparum .     

In vitro action of platinum (II) and platinum (IV) complexes on Trypanosoma cruzi and Leishmania donovani

The in vitro activities of 22 neutral Pt(II) complexes, 4 Pt(IV) complex salts, and one Pt(II) complex salt on epimastigote forms of Trypanosoma cruzi and promastigote forms of Leishmania donovani were studied. Only 2 out of the 18 complexes with cis-Pt(DDH)Xn structure completely inhibited the growth of the epimastigote forms of T. cruzi and the promastigote of L. donovani. Against T. cruzi the cis-Pt(L)n(Cl)2 complexes showed no activities, but the complex in which the (L) ligand was stilbamidine did show a limited activity against L. donovani. Of the Pt(IV)complex salts, [Pt X6]H2(L) were very active against the epimastigote forms of T. cruzi. However, the Pt(II) complex salt [cis-Pt Cl4]H2 (pentamidine) showed no activity. The actions of the Pt(IV) complex salts on the promastigote forms of L. donovani were different and only those salts in which the (L) ligand was pentamidine or stilbamidine induced parasite growth inhibition. The one Pt(II) salt tested showed no antiparasitic activity. At the same time, the known antiparasitic activities of the nifurtimox and pentamidine molecules were confirmed.     

QSAR study on the contribution of log P and E(s) to the in vitro antiprotozoal activity of glutathione derivatives

A series of N-S-blocked glutathione monoester and diester derivatives based on N-benzyloxycarbonyl-S-(2,4-dinitrophenyl)glutathione were evaluated for activity against the pathogenic parasites Trypanosoma brucei brucei, Trypanosoma cruzi, and Leishmania donovani in vitro.Only monoesters 7-9 with a log P value of >2.7 were active inhibitors of T.b. brucei bloodstream form trypomastigotes. Diester compounds 10-15 and 17-27 in most cases were better inhibitors of T.b. brucei than monoester compounds, and some displayed high activity against T. cruzi 14 and L. donovani 17, 19, 29. Compounds 14, 24, and 25 were the most active compounds identified against T.b. brucei having ED(50) values of <0.4 microM. Analysis of the inhibition data (ED(50)) vs calculated log P and E(s) values provided evidence to support membrane penetration and steric factors as the key component in the activity of these compounds. The optimum values for log P and E(s) determined were 5.8 and -0.70, respectively. A QSAR equation relating log(1/ED(50)) vs log P and E(s) was determined and interpreted within the proposed mechanism of activity for these compounds.     

Antileishmanial activity of hydrolyzable tannins and their modulatory effects on nitric oxide and tumour necrosis factor-alpha release in macrophages in vitro.

A series of 27 hydrolyzable tannins and related compounds was tested for antiparasitic effects against both extracellular promastigote and intracellular amastigote Leishmania donovani organisms. In parallel, the compounds were evaluated for their immunomodulatory effects on macrophage functions, including release of nitric oxide (NO), tumour necrosis factor-alpha (TNF-alpha) and interferon (IFN)-like properties using several functional assays. Of the series of polyphenols tested, only gallic acid (54 microM NO) and its methyl ester (32 microM NO) induced murine macrophage-like RAW 264.7 cells to release NO in appreciable amounts (IFN-gamma/LPS 119 microM NO). The in vitro TNF-inducing potential of the polyphenols examined increased in the order of oligomeric ellagitannins (EC(50) > 25 microg/ml) < monomeric ellagitannins, gallotannins (EC(50) 8.5 to > 25 microg/ml) < C-glucosidic ellagitannins, dehydroellagitannins (EC(50) 0.6 - 2.8 microg/ml) at the host cell subtoxic concentration of 50 microg/ml. Furthermore, promastigotes of Leishmania donovani were assayed in the presence of these polyphenols and the results showed that none of the compounds was significantly toxic (EC(50) > 25 microg/ml) to the extracellular forms. In contrast, all polyphenols showed pronounced antileishmanial activities (EC(50) < 0.4 - 12.5 versus 7.9 microg/ml for Pentostam) against intracellular amastigotes of L. donovani residing within RAW cells. Noteworthy, most compounds exhibited low cytotoxicity against the murine host cells (EC(50) >25 microg/ml). Furthermore, some ellagitannins and the majority of dehydroellagitannins induced potent interferon-like activities as reflected by inhibition of the cytopathic effect of encephalomyocarditis virus on fibroblast L929 cells. This is the first report on hydrolyzable tannins as a new class of natural products with leishmanicidal activity including their potential for inducing the release of NO, TNF and IFN-like activity in macrophage-like RAW cells.