Purified mouse dopamine neurons thrive and function after transplantation into brain but require novel glial factors for survival in culture

Cell replacement therapy in Parkinson's disease depends on a reliable source of purified dopamine (DA) neurons (PDN) and the identification of factors relevant to their survival. Our goal was to genetically tag and purify by flow cytometry embryonic midbrain DA neurons from a transgenic mouse line carrying 11 kb of human tyrosine hydroxylase promoter driving expression of the enhanced green fluorescent protein(GFP) for studies in vivo and in vitro. A 99% purification of GFP+ cells was achieved. When transplanted into 6-hydroxydopamine-treated rat striatum, PDN survived, became well-integrated and produced recovery from amphetamine-induced motor behaviors. However, when grown in culture, PDN died within days of plating. No known growth factors prevented PDN death as did incubation with novel factors in glia/glial-conditioned media. We conclude that GFP-tagged DA neurons can be purified to homogeneity and can survive and function when grown with glial factors in vitro or after transplantation in vivo.     

青蒿素哌喹片治疗无并发症恶性疟的剂量探索试验

目的探索青蒿素哌喹片治疗无并发症恶性疟的安全有效的适宜剂量。方法收治7～65岁男性和女性病人共100例,按区组随机化方案分成2组,成人总量1400 mg组年龄(22±s12)岁,1750 mg组年龄(21±12)岁。分别于0h和24h给药一次,比较2组的平均原虫转阴和退热时间、28d治愈率及原虫复燃率。结果2组的平均原虫转阴时间分别是(61±19)h和(57±21)h,平均退热时间为(20±15)h和(18±10)h,P>0.05;28 d治愈率是80%和96%,原虫复燃率为20%和4%,总量1750 mg组显著优于1400 mg组(P<0.05)。2组耐受性均良好,未发现明显的不良反应。结论推荐青蒿素哌喹片的临床治疗剂量为总量1750 mg,每日1次,分2d服完为1个疗程。     

Identification of a novel piscine Cryptosporidium genotype and Cryptosporidium parvum in cultured rainbow trout (Oncorhynchus mykiss)

This study reports for the first time the presence and molecular characterization of Cryptosporidium in farmed rainbow trout (Oncorhynchus mykiss Walbaum, 1792). A total of 360 fish, with no apparent clinical signs of disease, were collected and classified into groups according to their size. Cryptosporidium oocysts were detected by immunofluorescence microscopy in 33 specimens (9.2%), which were located in pyloric caeca samples (42.4%), intestinal scrapings (39.4%), or at both locations (18.2%). In the smallest (youngest) fish group, a higher percentage of positive samples were detected in the pyloric caeca relative to the intestinal location (58.8 vs. 17.6%; P = 0.01), including a cluster with more than 10 oocysts observed in the pyloric caeca of one specimen. PCR amplification and sequencing of fragments of SSU-rDNA and hsp70 genes identified a novel Cryptosporidium piscine genotype (genotype 9) in two specimens and Cryptosporidium parvum in seven fish, including the specimen in which the oocyst cluster was observed. Moreover, Cryptosporidium oocysts were detected in farm water samples (41.7 and 16.7% from influent and effluent, respectively). Although Giardia was not found in gastrointestinal samples, Giardia cysts were observed in 50.0 and 33.3% of the influent and effluent water samples, respectively. The results support the existence of natural infections by C. parvum in freshwater cultured fish, suggesting that the rainbow trout could shed infectious oocysts in aquatic environments and it may be a potential source of human infection when this edible fish is handled.

     

Purified mouse dopamine neurons thrive and function after transplantation into brain but require novel glial factors for survival in culture

Cell replacement therapy in Parkinson's disease depends on a reliable source of purified dopamine (DA) neurons (PDN) and the identification of factors relevant to their survival. Our goal was to genetically tag and purify by flow cytometry embryonic midbrain DA neurons from a transgenic mouse line carrying 11 kb of human tyrosine hydroxylase promoter driving expression of the enhanced green fluorescent protein (GFP) for studies in vivo and in vitro. A 99% purification of GFP(+) cells was achieved. When transplanted into 6-hydroxydopamine-treated rat striatum, PDN survived, became well-integrated and produced recovery from amphetamine-induced motor behaviors. However, when grown in culture, PDN died within days of plating. No known growth factors prevented PDN death as did incubation with novel factors in glia/glial-conditioned media. We conclude that GFP-tagged DA neurons can be purified to homogeneity and can survive and function when grown with glial factors in vitro or after transplantation in vivo.     

Surveillance of the efficacy of artesunate and mefloquine combination for the treatment of uncomplicated falciparum malaria in Cambodia

Artesunate and mefloquine combination treatment has been used since 2000 in Cambodia as the first-line drug for the treatment of uncomplicated falciparum malaria. In order to assess its efficacy and safety, the national malaria control programme conducted 14 therapeutic efficacy studies with the drug combination between 2001 and 2004 at nine sites. In 2001 and 2002, co-blister packs of artesunate and mefloquine were used, whereas in 2003 and 2004, drugs were given individually from a bulk pack at a total dose of 12 mg/kg of artesunate and 25 mg/kg of mefloquine over 3 days. A total of 1025 patients were enrolled over the 4 years and 977 were follow-up during the period of 28 days. The PCR-corrected cure rates ranged from 85.7% to 100% with an overall cure rate of 95.8% (920/960). The studies in 2002 showed also that co-blister packs used on the basis of age and not on the basis of weight could lead to underdosed regimens but without any detectable effect on the treatment outcome. The follow-up period was extended from 28 to 42 days in three sites in 2004. A total of 219 among 255 were follow-up until day 42. The cure rate decreased but not significantly from 90.1% (73/81) with 28 days follow-up to 79.3% (46/58) with 42 days follow-up in Pailin, whereas the cure rate remained at 100% in the two other sites. Side effects were common, especially dizziness, but were mild and transient and patients recovered without any medical intervention.     

Decreasing pfmdr1 Copy Number Suggests that Plasmodium falciparum in Western Cambodia Is Regaining In Vitro Susceptibility to Mefloquine

Dihydroartemisinin-piperaquine is the current frontline artemisinin combination therapy (ACT) for Plasmodium falciparum malaria in Cambodia but is now failing in several western provinces. To investigate artesunate plus mefloquine (AS+MQ) as a replacement ACT, we measured the prevalence of multiple pfmdr1 copies—a molecular marker for MQ resistance—in 844 P. falciparum clinical isolates collected in 2008 to 2013. The pfmdr1 copy number is decreasing in Western Cambodia, suggesting that P. falciparum is regaining in vitro susceptibility to MQ.     

Ex Vivo Susceptibility of Plasmodium falciparum to Antimalarial Drugs in Western,Northern, and Eastern Cambodia, 2011-2012: Association with Molecular Markers

In 2008, dihydroartemisinin (DHA)-piperaquine (PPQ) became the first-line treatment for uncomplicated Plasmodium falciparum malaria in western Cambodia. Recent reports of increased treatment failure rates after DHA-PPQ therapy in this region suggest that parasite resistance to DHA, PPQ, or both is now adversely affecting treatment. While artemisinin (ART) resistance is established in western Cambodia, there is no evidence of PPQ resistance. To monitor for resistance to PPQ and other antimalarials, we measured drug susceptibilities for parasites collected in 2011 and 2012 from Pursat, Preah Vihear, and Ratanakiri, in western, northern, and eastern Cambodia, respectively. Using a SYBR green I fluorescence assay, we calculated the ex vivo 50% inhibitory concentrations (IC₅₀s) of 310 parasites to six antimalarials: chloroquine (CQ), mefloquine (MQ), quinine (QN), PPQ, artesunate (ATS), and DHA. Geometric mean IC₅₀s (GMIC₅₀s) for all drugs (except PPQ) were significantly higher in Pursat and Preah Vihear than in Ratanakiri (P ≤ 0.001). An increased copy number of P. falciparum mdr1 (pfmdr1), an MQ resistance marker, was more prevalent in Pursat and Preah Vihear than in Ratanakiri and was associated with higher GMIC₅₀s for MQ, QN, ATS, and DHA. An increased copy number of a chromosome 5 region (X5r), a candidate PPQ resistance marker, was detected in Pursat but was not associated with reduced susceptibility to PPQ. The ex vivo IC₅₀ and pfmdr1 copy number are important tools in the surveillance of multidrug-resistant (MDR) parasites in Cambodia. While MDR P. falciparum is prevalent in western and northern Cambodia, there is no evidence for PPQ resistance, suggesting that DHA-PPQ treatment failures result mainly from ART resistance.