Differential Anti-Fibrotic and Remodeling Responses of Human Dermal Fibroblasts to Artemisia sp., Artemisinin, and Its Derivatives.

Fibrosis is a ubiquitous pathology, and prior studies have indicated that various artemisinin (ART) derivatives (including artesunate (AS), artemether (AM), and dihydroartemisinin (DHA)) can reduce fibrosis in vitro and in vivo. The medicinal plant Artemisia annua L. is the natural source of ART and is widely used, especially in underdeveloped countries, to treat a variety of diseases including malaria. A. afra contains no ART but is also antimalarial. Using human dermal fibroblasts (CRL-2097), we compared the effects of A. annua and A. afra tea infusions, ART, AS, AM, DHA, and a liver metabolite of ART, deoxyART (dART), on fibroblast viability and expression of key fibrotic marker genes after 1 and 4 days of treatment. AS, DHA, and Artemisia teas reduced fibroblast viability 4 d post-treatment in up to 80% of their respective controls. After 4 d of treatment, AS DHA and Artemisia teas downregulated ACTA2 up to 10 fold while ART had no significant effect, and AM increased viability by 10%. MMP1 and MMP3 were upregulated by AS, 17.5 and 32.6 fold, respectively, and by DHA, 8 and 51.8 fold, respectively. ART had no effect, but A. annua and A. afra teas increased MMP3 5 and 16-fold, respectively. Although A. afra tea increased COL3A1 5 fold, MMP1 decreased >7 fold with no change in either transcript by A. annua tea. Although A. annua contains ART, it had a significantly greater anti-fibrotic effect than ART alone but was less effective than A. afra. Immunofluorescent staining for smooth-muscle α-actin (α-SMA) correlated well with the transcriptional responses of drug-treated fibroblasts. Together, proliferation, qPCR, and immunofluorescence results show that treatment with ART, AS, DHA, and the two Artemisia teas yield differing responses, including those related to fibrosis, in human dermal fibroblasts, with evidence also of remodeling of fibrotic ECM.

Dried whole-plant Artemisia annua slows evolution of malaria drug resistance and overcomes resistance to artemisinin.

Pharmaceutical monotherapies against human malaria have proven effective, although ephemeral, owing to the inevitable evolution of resistant parasites. Resistance to two or more drugs delivered in combination will evolve more slowly; hence combination therapies have become the preferred norm in the fight against malaria. At the forefront of these efforts has been the promotion of Artemisinin Combination Therapy, but despite these efforts, resistance to artemisinin has begun to emerge. In 2012, we demonstrated the efficacy of the whole plant (WP)--not a tea, not an infusion--as a malaria therapy and found it to be more effective than a comparable dose of pure artemisinin in a rodent malaria model. Here we show that WP overcomes existing resistance to pure artemisinin in the rodent malaria Plasmodium yoelii. Moreover, in a long-term artificial selection for resistance in Plasmodium chabaudi, we tested resilience of WP against drug resistance in comparison with pure artemisinin (AN). Stable resistance to WP was achieved three times more slowly than stable resistance to AN. WP treatment proved even more resilient than the double dose of AN. The resilience of WP may be attributable to the evolutionary refinement of the plant's secondary metabolic products into a redundant, multicomponent defense system. Efficacy and resilience of WP treatment against rodent malaria provides compelling reasons to further explore the role of nonpharmaceutical forms of AN to treat human malaria.

Root regulation of artemisinin production in Artemisia annua: trichome and metabolite evidence.

MAIN CONCLUSION: Roots of plants with high artemisinin-producing leaves increased leaf production of artemisinin in low-producing plants and vice versa indicating roots are involved in controlling artemisinin biosynthesis in shoots. The anti-malarial sesquiterpene, artemisinin, is produced and stored in glandular trichomes (GLTs) of Artemisia annua. Evidence suggested roots, which produce no significant artemisinin nor precursor compounds, regulate production of artemisinin biosynthesis in the leaves. Using grafting, we studied the relationship between rootstock and scion by measuring GLTs and five artemisinic metabolites (artemisinin, deoxyartemisinin, dihydroartemisinic acid, artemisinic acid, arteannuin B) in scions of ungrafted, self-grafted, and cross-grafted plants among three cultivars: S and 15 both having GLTs with artemisinin at 1.49 and 0.57 %, respectively, and G producing neither GLTs nor detectable artemisinin. All artemisinin-producing self-grafts, e.g., S/S (scion/rootstock) and 15/15, produced more artemisinin than ungrafted plants, likely from grafting stress. S/S grafts also produced more GLTs. The 15/S grafts produced more artemisinin than S/15, suggesting rootstocks from high producing S plants stimulated artemisinin production in 15 scions. S/15 grafts yielded less artemisinin than S/S, but more than either 15/15 or ungrafted n15 and nS; S/15 grafts also had a lower density of GLTs than S/S, suggesting rootstock inhibition of the scion. The S rootstock induced trace artemisinin production in G scions, but did not induce GLT formation in G/S grafts. Different grafts exhibited different trichome morphologies and effects on artemisinic pathway flux. This study provides new information regarding the role of roots in GLT development and artemisinin production in this important medicinal plant.

Variations in key artemisinic and other metabolites throughout plant development in Artemisia annua L. for potential therapeutic use.

Dried leaves of Artemisia annua show promise as an inexpensive and sustainable antimalarial therapeutic, especially for use in developing countries. Along with the potent terpene, artemisinin, many other small molecules produced by the plant seem to aid in the therapeutic response. However, little is known about the ontogenic and phenological production of artemisinin in the plant, and its plethora of other important secondary metabolites. From a consistently high artemisinin-producing A. annua clone (SAM) we extracted and analyzed by GC/MS 22 different metabolites including terpenes, flavonoids, a coumarin, and two phenolic acids as they varied during leaf development and growth of the plant from the vegetative stage through the reproductive, full flower stage. As leaves developed, the maximum amount of most metabolites was in the shoot apical meristem. Artemisinin, on the other hand, maximized once leaves matured. Leaf and apical tissues (e.g. buds, flowers) varied in their metabolite content with growth stage with maximum artemisinin and other important secondary metabolites determined to be at floral bud emergence. These results indicated that plants at the floral bud stage have the highest level of artemisinin and other therapeutic compounds for the treatment of malaria.

Changes in key constituents of clonally propagated Artemisia annua L. during preparation of compressed leaf tablets for possible therapeutic use.

Artemisia annua L., long used as a tea infusion in traditional Chinese medicine, produces artemisinin. Although artemisinin is currently used as artemisinin-based combination therapy (ACT) against malaria, oral consumption of dried leaves from the plant showed efficacy and will be less costly than ACT. Many compounds in the plant have some antimalarial activity. Unknown, however, is how these plant components change as leaves are processed into tablets for oral consumption. Here we compared extracts from fresh and dried leaf biomass with compressed leaf tablets of A. annua. Using GC-MS, nineteen endogenous compounds, including artemisinin and several of its pathway metabolites, nine flavonoids, three monoterpenes, a coumarin, and two phenolic acids, were identified and quantified from solvent extracts to determine how levels of these compounds changed during processing. Results showed that compared to dried leaves, artemisinin, arteannuin B, artemisinic acid, chlorogenic acid, scopoletin, chrysoplenetin, and quercetin increased or remained stable with powdering and compression into tablets. Dihydroartemisinic acid, monoterpenes, and chrysoplenol-D decreased with tablet formation. Five target compounds were not detectable in any of the extracts of this cultivar. In contrast to the individually measured aglycone flavonoids, using the AlCl3 method, total flavonoids increased nearly fivefold during the tablet formation. To our knowledge this is the first study documenting changes that occurred in processing dried leaves of A. annua into tablets. These results will improve our understanding of the potential use of not only this medicinal herb, but also others to afford better quality control of intact plant material for therapeutic use.

An O-methylflavone from Artemisia afra kills non-replicating hypoxic Mycobacterium tuberculosis.

ETHNOPHARMACOLOGICAL RELEVANCE: African wormwood (Artemisia afra Jacq. ex Willd.) has been used traditionally in southern Africa to treat illnesses causing fever and was recently shown to possess anti-tuberculosis activity. As tuberculosis is an endemic cause of fever in southern Africa, this suggests that the anti-tubercular activity of A. afra may have contributed to its traditional medicinal use. AIM OF THE STUDY: Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is a deadly and debilitating disease globally affecting millions annually. Emerging drug-resistant Mtb strains endanger the efficacy of the current therapies employed to treat tuberculosis; therefore, there is an urgent need to develop novel drugs to combat this disease. Given the reported activity of A. afra against Mtb, we sought to determine the mechanisms by which A. afra inhibits and kills this bacterium. MATERIALS AND METHODS: We used transcriptomics to investigate the impact of Artemisia spp. extracts on Mtb physiology. We then used chromatographic fractionation and biochemometric analyses to identify a bioactive fractions of A. afra extracts and identify an active compound. RESULTS: Transcriptomic analysis revealed that A. afra exerts different effects on Mtb compared to A. annua or artemisinin, suggesting that A. afra possesses other phytochemicals with unique modes of action. A biochemometric study of A. afra resulted in the isolation of an O-methylflavone (1), 5-hydroxy-7-methoxy-2-(4-methoxyphenyl)chromen-4-one, which displayed considerable activity against Mtb strain mc26230 in both log phase growth and metabolically downshifted hypoxic cultures. CONCLUSIONS: The present study demonstrated that an O-methylflavone constituent of Artemisia afra explains part of the activity of this plant against Mtb. This result contributes to a mechanistic understanding of the reported anti-tubercular activity of A. afra and highlights the need for further study of this traditional medicinal plant and its active compounds.

The effect of roots and media constituents on trichomes and artemisinin production in Artemisia annua L.

KEY MESSAGE : Rooting of Artemisia annua increases trichome size on leaves and helps drive the final steps of the biosynthesis of the sesquiterpene antimalarial drug, artemisinin. Artemisia annua produces the antimalarial drug, artemisinin (AN), which is synthesized and stored in glandular trichomes (GLTs). In vitro-grown A. annua shoots produce more AN when they form roots. This may be a function not of the roots, but rather media components such as the phytohormones, α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP), or salts and sucrose used to maintain either rooted or unrooted shoot cultures. We investigated how three main media components altered artemisinic metabolite production, pathway gene transcripts, and GLT formation in both mature and developing leaves in rooted and unrooted cultures. Although transcript levels of AN biosynthetic genes were not altered, AN levels were significantly different, and there were major differences in both artemisinic metabolite levels and trichomes in mature versus developing leaves. For example, NAA induced higher AN production in rooted shoots, but only in mature leaves. In developing leaves, BAP increased GLT density on the leaf surface. When both phytohormones were present, GLTs were larger on young developing leaves, but smaller on mature leaves. Furthermore, although other media components increased GLT density, their size decreased on young leaves, but there was no effect on mature leaves. Roots also appeared to drive conversion of artemisinic precursors towards end products. These results suggest that, while the presence of roots affects AN and trichome production, phytohormones and other media constituents used for in vitro culture of A. annua also exert an influence.

A methoxylated flavone from Artemisia afra kills Mycobacterium tuberculosis.

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is a deadly and debilitating disease globally affecting millions annually. Emerging drug-resistant Mtb strains endanger the efficacy of the current combination therapies employed to treat tuberculosis; therefore, there is an urgent need to develop novel drugs to combat this disease. Artemisia afra is used traditionally in southern Africa to treat malaria and recently has shown anti tuberculosis activity. This genus synthesizes a prodigious number of phytochemicals, many of which have demonstrated human health effects. Transcriptomic analysis revealed that A. afra exerts different effects on Mtb compared to A. annua or the well-known antimalarial artemisinin, suggesting other phytochemicals present in A. afra with unique modes of action. A biochemometric study of A. afra resulted in the isolation of a methoxylated flavone (1), which displayed considerable activity against Mtb strain mc26230. Compound 1 had an MIC of 312.5 µg/mL and yielded no viable colonies after 6 days of treatment. In addition, 1 was effective in killing hypoxic Mtb cultures, with no viable cultures after 2 days of treatment. This suggested that A. afra is a source of potentially powerful anti-Mtb phytochemicals with novel mechanisms of action.

The flavonoids casticin and artemetin are poorly extracted and are unstable in an Artemisia annua tea infusion.

A number of flavonoids including casticin and artemetin from Artemisia annua have shown synergism with artemisinin against Plasmodium falciparum, but it is unclear if the flavonoids are also extracted into a tea infusion of the plant. Using a tea infusion preparation protocol that was reported to be highly effective for artemisinin extraction, we measured casticin and artemetin extraction. There was only a 1.8 % recovery of casticin in the infusion while artemetin was undetectable. After 24 hr storage at room temperature, casticin yield declined by 40 %. These results show that although a tea infusion of the plant may extract artemisinin, the polymethoxylated flavonoids casticin and artemetin are poorly extracted and lost with storage at room temperature and thus, the tea infusion appears to lose synergistic value.

Artemisia extracts differ from artemisinin effects on human hepatic CYP450s 2B6 and 3A4 in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: The Chinese medicinal herb, Artemisia annua L., has been used for >2,000 yr as traditional tea infusions to treat a variety of infectious diseases including malaria, and its use is spreading globally (along with A. afra Jacq. ex Willd.) mainly through grassroots efforts. AIM OF THE STUDY: Artemisinin is more bioavailable delivered from the plant, Artemisia annua L. than the pure drug, but little is known about how delivery via a hot water infusion (tea) alters induction of hepatic CYP2B6 and CYP3A4 that metabolize artemisinin. MATERIALS AND METHODS: HepaRG cells were treated with 10 µM artemisinin or rifampicin (positive control), and teas (10 g/L) of A. annua SAM, and A. afra SEN and MAL with 1.6, 0.05 and 0 mg/g DW artemisinin in the leaves, respectively; qPCR and Western blots were used to measure CYP2B6 and CYP3A4 responses. Enzymatic activity of these P450s was measured using human liver microsomes and P450-Glo assays. RESULTS: All teas inhibited activity of CYP2B6 and CYP3A4. Artemisinin and the high artemisinin-containing tea infusion (SAM) induced CYP2B6 and CYP3A4 transcription, but artemisinin-deficient teas, MAL and SEN, did not. Artemisinin increased CYP2B6 and CYP3A4 protein levels, but none of the three teas did, indicating a post-transcription inhibition by all three teas. CONCLUSIONS: This study showed that Artemisia teas inhibit activity and artemisinin autoinduction of CYP2B6 and CYP3A4 post transcription, a response likely the effect of other phytochemicals in these teas. Results are important for understanding Artemisia tea posology.

Biomass production of hairy roots of Artemisia annua and Arachis hypogaea in a scaled-up mist bioreactor.

Hairy roots have the potential to produce a variety of valuable small and large molecules. The mist reactor is a gas phase bioreactor that has shown promise for low-cost culture of hairy roots. Using a newer, disposable culture bag, mist reactor performance was studied with two species, Artemisia annua L. and Arachis hypogaea (peanut), at scales from 1 to 20 L. Both species of hairy roots when grown at 1 L in the mist reactor showed growth rates that surpassed that in shake flasks. From the information gleaned at 1 L, Arachis was scaled further to 4 and then 20 L. Misting duty cycle, culture medium flow rate, and timing of when flow rate was increased were varied. In a mist reactor increasing the misting cycle or increasing the medium flow rate are the two alternatives for increased delivery of liquid nutrients to the root bed. Longer misting cycles beyond 2-3 min were generally deemed detrimental to growth. On the other hand, increasing the medium flow rate to the sonic nozzle especially during the exponential phase of root growth (weeks 2-3) was the most important factor for increasing growth rates and biomass yields in the 20 L reactors. A. hypogaea growth in 1 L reactors was µ = 0.173 day(-1) with biomass yield of 12.75 g DW L(-1). This exceeded that in shake flasks at µ = 0.166 day(-1) and 11.10 g DW L(-1). Best growth rate and biomass yield at 20 L was µ = 0.147 and 7.77 g DW L(-1), which was mainly achieved when medium flow rate delivery was increased. The mist deposition model was further evaluated using this newer reactor design and when the apparent thickness of roots (+hairs) was taken into account, the empirical data correlated with model predictions. Together these results establish the most important conditions to explore for future optimization of the mist bioreactor for culture of hairy roots.

Bench to batch: advances in plant cell culture for producing useful products.

Despite significant efforts over nearly 30 years, only a few products produced by in vitro plant cultures have been commercialized. Some new advances in culture methods and metabolic biochemistry have improved the useful potential of plant cell cultures. This review will provide references to recent relevant reviews along with a critical analysis of the latest improvements in plant cell culture, co-cultures, and disposable reactors for production of small secondary product molecules, transgenic proteins, and other products. Some case studies for specific products or production systems are used to illustrate principles.

DMSO triggers the generation of ROS leading to an increase in artemisinin and dihydroartemisinic acid in Artemisia annua shoot cultures.

The antimalarial sesquiterpene, artemisinin, is in short supply; demand is not being met, and the role of artemisinin in the plant is not well established. Prior work showed that addition of dimethyl sulfoxide (DMSO) to seedlings increased artemisinin in their shoots and this study further investigated that serendipitous observation. When in vitro-cultured Artemisia annua rooted shoots were fed different amounts of DMSO (0-2.0% v/v), artemisinin levels doubled and showed biphasic optima at 0.25 and 2.0% DMSO. Both artemisinin and its precursor, dihydroartemisinic acid, increased with the former continuing 7 days after DMSO treatment. There was no stimulation of artemisinin production in DMSO-treated unrooted shoots. The first gene in the artemisinin biosynthetic pathway, amorphadiene synthase, showed no increase in transcript level in response to DMSO compared to controls. In contrast, the second gene in the pathway, CYP71AV1, did respond to DMSO but at a level of transcripts inverse to artemisinin levels. When rooted shoots were stained for the reactive oxygen species (ROS), H2O2, ROS increased with increasing DMSO concentration; unrooted shoots produced no ROS in response to DMSO. Both the increases in DMSO-induced ROS response and corresponding artemisinin levels were inhibited by addition of vitamin C. Together these data show that at least in response to DMSO, artemisinin production and ROS increase and that when ROS is reduced, so also is artemisinin suggesting that ROS may play a role in artemisinin production in A. annua.

Production of mouse interleukin-12 is greater in tobacco hairy roots grown in a mist reactor than in an airlift reactor.

We compared the growth and productivity of a tobacco line of hairy roots that produces murine interleukin 12 (mIL-12) grown in three different culture systems: shake flasks, an airlift reactor, and a scalable mist reactor. Of the total mIL-12 produced by cultures grown in shake flasks ( approximately 434.8 microg L(-1)), almost 21% was recovered from the medium. In contrast to roots harvested from shake flasks and the mist reactor, roots were not uniformly distributed in the airlift reactor. Roots formed a dense ring around the wall of the reactor and surrounding the central rising column of fine aeration bubbles. Root quality was also better in both the shake flasks and mist reactor than in the airlift reactor. There were more pockets of dark roots in the airlift reactor suggesting some of the roots were nutrient starved. Although the best root growth (7 g DW L(-1)) was in the shake flasks, both reactors produced about the same, but less dry mass, nearly 5 g DW L(-1). Total mIL-12 concentration was highest in the mist reactor at 5.3 microg g(-1) FW, but productivity, 31 microg g(-1) FW day(-1) was highest in shake flasks. Roots grown in the mist reactor produced about 49.5% more mIL-12 than roots grown in the airlift reactor. Protease activity in the media increased steadily during culture of the roots in all three systems. The comparisons of protease activity, protein and mIL-12 levels done in the shake flask system suggest that the increase in proteases associated with progression into stationary phase is most detrimental to mIL-12 concentration. This is the first description of the design and operation of a scalable version of a mist bioreactor that uses a plastic bag. This also the first report of reasonable production levels of functional mIL-12, or any protein, produced by hairy roots grown in a mist reactor. Results will prove useful for further optimization and scale-up studies of plant-produced therapeutic proteins.

Artemisia annua and Artemisia afra tea infusions vs. artesunate-amodiaquine (ASAQ) in treating Plasmodium falciparum malaria in a large scale, double blind, randomized clinical trial.

BACKGROUND AND OBJECTIVE: Prior small-scale clinical trials showed that Artemisia annua and Artemisia afra infusions, decoctions, capsules, or tablets were low cost, easy to use, and efficient in curing malaria infections. In a larger-scale trial in Kalima district, Democratic Republic of Congo, we aimed to show A. annua and/or A. afra infusions were superior or at least equivalent to artesunate-amodiaquine (ASAQ) against malaria. METHODS: A double blind, randomized clinical trial with 957 malaria-infected patients had two treatment arms: 472 patients for ASAQ and 471 for Artemisia (248 A. annua, 223 A. afra) remained at end of the trial. ASAQ-treated patients were treated per manufacturer posology, and Artemisia-treated patients received 1 l/d of dry leaf/twig infusions for 7 d; both arms had 28 d follow-up. Parasitemia and gametocytes were measured microscopically with results statistically compared among arms for age and gender. RESULTS: Artemisinin content of A. afra was negligible, but therapeutic responses of patients were similar to A. annua-treated patients; trophozoites cleared after 24  h, but took up to 14 d to clear in ASAQ-treated patients. D28 cure rates defined as absence of parasitemia were for pediatrics 82, 91, and 50% for A. afra, A. annua and ASAQ; while for adults cure rates were 91, 100, and 30%, respectively. Fever clearance took 48  h for ASAQ, but 24  h for Artemisia. From D14-28 no Artemisia-treated patients had microscopically detectable gametocytes, while 10 ASAQ-treated patients remained gametocyte carriers at D28. More females than males were gametocyte carriers in the ASAQ arm but were unaffected in the Artemisia arms. Hemoglobin remained constant at 11 g/dl for A. afra after D1, while for A. annua and ASAQ it decreased to 9-9.5  g/dl. Only 5.0% of Artemisia-treated patients reported adverse effects, vs. 42.8% for ASAQ. CONCLUSION: A. annua and A. afra infusions are polytherapies with better outcomes than ASAQ against malaria. In contrast to ASAQ, both Artemisias appeared to break the cycle of malaria by eliminating gametocytes. This study merits further investigation for possible inclusion of Artemisia tea infusions as an alternative for fighting and eradicating malaria.

Effect of Artemisia annua and Artemisia afra tea infusions on schistosomiasis in a large clinical trial.

BACKGROUND AND OBJECTIVE: Schistosomiasis (bilharzia), a serious neglected tropical disease affecting millions, has few cost-effective treatments, so two Artemisia wormwood species, A. annua and A. afra, were compared with the current standard praziquantel (PZQ) treatment in an 800 patient clinical trial, August-November of 2015. METHODS: The double blind, randomized, superiority clinical trial had three treatment arms: 400 for PZQ, 200 for A. annua, and 200 for A. afra. PZQ-treated patients followed manufacturer posology. Artemisia-treated patients received 1 l/d of dry leaf/twig tea infusions divided into 3 aliquots daily, for 7 days with 28-day follow-up. RESULTS: Of 800 enrolled patients having an average of >700 Schistosoma mansoni eggs per fecal sample, 780 completed the trial. Within 14 days of treatment, all Artemisia-treated patients had no detectable eggs in fecal smears, a result sustained 28 days post treatment. Eggs in fecal smears of PZQ-treated patients were undetectable after D21. More males than females who entered the trial had melena, but both genders responded equally well to treatment; by D28 melena disappeared in all patients. In all arms, eosinophil levels declined by about 27% from D0 to D28. From D0 to D28 hemoglobin increases were greater in PZQ and A. afra-treated patients than in A. annua-treated patients. Hematocrit increases were greater from D0 to D28 for patients treated with either PZQ or A. annua compared to those treated with A. afra. Gender comparison showed that A. afra-treated males had significantly greater hemoglobin and hematocrit increases by D28 than either PZQ or A. annua-treated males. In contrast, PZQ and A. afra-treated females had greater hemoglobin and hematocrit increases than A. annua-treated females. Both adults and pediatric patients treated with A. annua responded better compared to PZQ treatment. CONCLUSION: Both A. annua and A. afra provided faster effective treatment of schistosomiasis and should be considered for implementation on a global scale.

Artemisia annua dried leaf tablets treated malaria resistant to ACT and i.v. artesunate: Case reports.

BACKGROUND: Dried leaf Artemisia annua (DLA) has shown efficacy against Plasmodium sp. in rodent studies and in small clinical trials. Rodent malaria also showed resiliency against the evolution of artemisinin drug resistance. PURPOSE: This is a case report of a last resort treatment of patients with severe malaria who were responding neither to artemisinin combination therapy (ACT) nor i.v. artesunate. STUDY DESIGN: Of many patients treated with ACTs and i.v. artesunate during the 6 mon study period, 18 did not respond and were subsequently treated with DLA Artemisia annua. METHODS: Patients were given a dose of 0.5g DLA per os, twice daily for 5d. Total adult delivered dose of artemisinin was 55mg. Dose was reduced for body weight under 30kg. Clinical symptoms, e.g. fever, coma etc., and parasite levels in thick blood smears were tracked. Patients were declared cured and released from hospital when parasites were microscopically undetectable and clinical symptoms fully subsided. RESULTS: All patients were previously treated with Coartem® provided through Santé Rurale (SANRU) and following the regimen prescribed by WHO. Of 18 ACT-resistant severe malaria cases compassionately treated with DLA, all fully recovered. Of the 18, this report details two pediatric cases. CONCLUSIONS: Successful treatment of all 18 ACT-resistant cases suggests that DLA should be rapidly incorporated into the antimalarial regimen for Africa and possibly wherever else ACT resistance has emerged.

Adhesion of plant roots to poly-L-lysine coated polypropylene substrates.

The ability to immobilize plant tissue in a bioreactor is an important process tool. We have shown that roots of several species rapidly attach to poly-L-lysine coated polypropylene mesh in a liquid environment. Using transformed roots of Artemisia annua as a model, the attachment process was found to be enhanced by sheep serum, but not BSA and inhibited by excess Mn(2+), but unaffected by Ca(2+) or Mg(2+). Attempts to characterize the molecule(s) responsible for binding using lectins and antibodies showed that the binding site does not appear to be glycosylated or vitronectin-like. This method of rapid attachment should prove useful for controlled immobilization of roots in bioreactors.