Efficient transduction of pigtailed macaque hematopoietic repopulating cells with HIV-based lentiviral vectors

Lentiviral vectors are attractive for hematopoietic stem cell (HSC) gene therapy because they do not require mitosis for nuclear entry, they efficiently transduce hematopoietic repopulating cells, and self-inactivating (SIN) designs can be produced at high titer. Experiments to evaluate HIV-derived lentiviral vectors in nonhuman primates prior to clinical trials have been hampered by low transduction frequencies due in part to host restriction by TRIM5. We have established conditions for efficient transduction of pigtailed macaque (Macaca nemestrina) long-term repopulating cells using VSV-G–pseudotyped HIV-based lentiviral vectors. Stable, long-term, high-level gene marking was observed in 3 macaques using relatively low MOIs (5-10) in a 48-hour ex vivo transduction protocol. All animals studied had rapid neutrophil engraftment with a median of 10.3 days to a count greater than 0.5 x 10⁹/L (500/µL). Expression was detected in all lineages, with long-term marking levels in granulocytes at approximately 20% to 30%, and in lymphocytes at approximately 12% to 23%. All animals had polyclonal engraftment as determined by analysis of vector integration sites. These data suggest that lentiviral vectors should be highly effective for HSC gene therapy, particularly for diseases in which maintaining the engraftment potential of stem cells using short-term ex vivo transduction protocols is critical.     

Inhibitory effect on TNF-α-induced IL-8 secretion in HT-29 cell line by glyceroglycolipids from the leaves of Ficus microcarpa

Bioassay-guided fractionation based on the anti-inflammatory activity of a methanol extract of Ficus microcarpa leaves led to the isolation of seven galactolipids: 2(S)-3-O-octadeca-9Z,12Z,15Z-trienoylglyceryl-O-β-D-galactopyranoside (1), (2S)-2,3-O-dioctadeca-9Z,12Z,15Z-trienoylglyceryl-O-β-D-galactopyranoside (2), (2S)-2,3-O-dioctadeca-9Z,12Z-dienoylglyceryl-O-β-D-galactopyranoside (3), (2S)-3-O-octadeca-9Z,12Z,15Z-trienoylglyceryl-6′-O-(α-D-galactopyranosyl)-β-D-galactopyranoside (4), (2S)-2,3-O-dioctadeca-9Z,12Z,15Z-trienoylglyceryl-6’-O-(α-D-galactopyranosyl)-β-D-galactopyranoside (5), gingerglycolipid B (6), and (2S)-2,3-O-dioctadeca-9Z,12Z-dienoylglyceryl-6′-O-(α-D-galactopyranosyl)-β-D-galactopyranoside (7). Their chemical structures were elucidated by mass, 1D-, and 2D-NMR spectroscopic methods as well as chemical methods. The antiinflammatory effect of these compounds on TNF-α induced IL-8 secretion in the HT-29 cell line was evaluated. All above galactolipids showed significant inhibition ranging 40% at a concentration of 50 μM. The results suggest that galactolipids from the leaves of F. microcarpa may be used as potent anti-inflammatory agents.     

Cytotoxic oleane-type triterpene saponins from Glochidion eriocarpum

The anticancer activity of ten compounds from the aerial parts of Glochidion eriocarpum were evaluated on two human cancer cell lines, HL-60 and HCT-116. Compounds 1-4 displayed highly potent cytotoxic activity on the HCT-116 cancer cell line with IC(50) values ranging of 0.41～1.16 μM. Compounds 1-4 significantly inhibited the HL-60 cell line with IC(50) values ranging of 4.51～6.33 μM. These results suggested that the benzoyl group at the C-22 position in oleane-type triterpene saponins was essential for cytotoxicity towards tumor cells. Moreover, compounds 2 and 3 showed more potent cytotoxicity than compounds 1 and 4 against HL-60 and HCT-116 cells. With respect to the mechanism underlying cytotoxicity, compounds 1-4 increased chromatin condensation, a typical apoptotic characteristic in HL-60 and HCT-116 cells. In the mechanism of apoptosis induction, compounds 1-4 reduced Bcl-2 expression, whereas the expression of Bax was increased compared to controls in HCT-116 cells. In addition, compounds 1-4 decreased the level of procaspase-3. The cleavage of poly (ADP-ribose) polymerase (PARP), a vital substrate of effector caspase, was observed in HCT-116 cells. Furthermore, the induction of apoptosis was also accompanied by an activation of extracellular signal-regulated kinase (ERK) and p38 kinase in HCT-116 cells. These findings provide evidence demonstrating that the pro-apoptotic effects of compounds 1-4 are mediated through the activation of ERK and p38 in HCT-116 cells.     

HL-91-Technetium-99m: A new marker of viability in ischemic myocardium

BACKGROUND: Technetium 99m-HL91 is a new hypoxia imaging agent that demonstrates increased uptake in ischemic, viable myocardium. This study was performed to determine whether HL91 is taken up by nonviable myocardium. METHODS: Twenty-three Krebs-Henseleit buffer-perfused, isolated rat hearts were studied. Tc-99m-HL91 300 microCi was infused over 10 minutes, followed by a 60-minute clearance. Myocardial activity was monitored by use of an NaI crystal. Four groups were studied: control (flow = 12 mL/min, n = 7), low flow (flow = 1 mL/min, n = 6), no flow/reflow (60 minutes no flow/60 minutes reflow before Tc-99m-HL91 infusion, flow = 12 mL/min, n = 5), and cyanide-treated (before Tc-99m-HL91 infusion, flow = 12 mL/min, n = 5). Injury was assessed by creatine kinase, transmission electron microscopy, and triphenyltetrazolium chloride. RESULTS: Control (no injury) and cyanide-treated (severe injury) hearts demonstrated low uptake (6.3+/-0.5 mean+/-SEM and 5.7+/-1.2 microCi, respectively) and low 60-minute retention (13.8%+/-2.2% and 13.7%+/-3.9%, respectively). Low-flow hearts (minimal injury) demonstrated markedly increased uptake (43.5+/-2.8 microCi, P < .01) and increased 60-minute retention (33.2%+/-2.9%, P < .01) compared with control. No-flow/reflow hearts (moderate injury) demonstrated intermediate uptake (8.7+/-0.5 microCi, P < .05 to control), although retention was not significantly different (18.9%+/-3.5%, P = ns). Severely and rapidly injured myocardium demonstrated Tc-99m-HL91 peak uptake and retention indistinguishable from normal. Moderately injured myocardium demonstrated uptake intermediate between severely injured and low-flow-induced ischemic, viable myocardium. CONCLUSION: Thus Tc-99m-HL91 is not taken up or retained in nonviable and irreversibly injured myocardium.     

A potential inhibitor of rat aortic vascular smooth muscle cell proliferation from the pollen of <Emphasis Type="Italic">Typha angustata</Emphasis>

By various chromatographic methods, three flavonoids, (2S)-naringenin (1), isorhamnetin 3-O-(2-O-α-l-rhamnopyranosyl) β-d-glucopyranoside (2), typhaneoside (3), and two sterol glycosides, β-sitosterol-3-O-(6-octadecanoyl) β-d-glucopyranoside (4) and β-sitosterol-3-O-(6-octadeca-9Z,12Z-dienoyl) β-d-glucopyranoside (5), were isolated from the pollen of Typha angustata. Their structures were determined on the basis of spectroscopic analyses. The flavonoids (1–3) were evaluated for their effects on the viability and proliferation of rat aortic smooth muscle cells. (2S)-naringenin (1) significantly inhibited cell proliferation in a dose-dependent manner without cytotoxic at concentrations of 30, and 50 μM; it reduced the number of cells following PDGF-BB treatment to 1.83 ± 0.30 × 10⁴ and 2.20 ± 0.60 × 10⁴ cells/well, respectively. These findings suggest that (2S)-naringenin has antiproliferative effects on aortic smooth muscle cells.     

Concurrent activation of c-myc and inactivation of bcl-2 by chromosomal translocation in a lymphoblastic lymphoma cell line

We have characterized a chromosomal translocation in a cell line (SU-DUL5) established from a patient with lymphoblastic lymphoma in which the c-myc gene on chromosome 8 was juxtaposed to a t(14;18). Cytogenetic analysis of this cell line showed 14q+, 18q-, and 8p+q+ marker chromosomes in the absence of t(14;18). Genomic Southern blot analysis showed juxtaposition of the immunoglobulin heavy chain joining region (JH) with chromosome 18 near the minor breakpoint cluster region (mcr) of the bcl-2 gene. There was also a rearranged c-myc gene detectable with a 5' c-myc probe. Molecular cloning studies showed that the c-myc gene was joined to chromosome 18 DNA. Nucleotide sequence analysis of cloned breakpoint DNA revealed that the crossover between chromosomes 8 and 18 occurred at the 3' end of the bcl-2 gene resulting in replacement of the bcl-2 gene on the 14q+ chromosome with the c-myc gene. As a result of this translocation the SU-DUL5 cell line contains no detectable bcl-2 mRNA or protein but has abundant levels of c-myc mRNA. Our data suggest that bcl-2 inactivation occurred simultaneously with c-myc translocation in a B cell lymphoblastic lymphoma.     

Hyponatraemia, rhabdomyolysis, alterations in blood pressure and persistent mydriasis in patients envenomed by Malayan kraits (Bungarus candidus) in southern Viet Nam

Between 1998 and 2007, 42 patients admitted to Choray hospital, Ho Chi Minh City, and to two hospitals in adjacent regions in southern Viet Nam brought the Malayan kraits (Bungarus candidus) that had been responsible for biting them. Half of the patients had been bitten while they were asleep. Fang marks and numbness were the only local features of the bites. Common signs of neurotoxic envenoming included bilateral ptosis, persistently dilated pupils, limb weakness, breathlessness, hypersalivation, dysphonia and dysphagia. Thirty patients (71.4%) required endotracheal intubation of whom all but one were mechanically ventilated. Fourteen patients (33.3%) developed hypertension, 13 (31.0%) shock, 31 (73.8%) hyponatraemia (plasma sodium concentration < 130 mEq/l) and 30 (71.4%) showed evidence of mild rhabdomyolysis (peak plasma creatine kinase concentration 1375 ± 140 u/l). None developed acute kidney injury. All the patients were treated with a new monospecific B. candidus antivenom. There were no fatalities.Hyponatraemia has been reported previously in victims of Chinese kraits (Bungarus multicinctus) in northern Viet Nam and rhabdomyolysis in patients envenomed by B. niger in Bangladesh. These features of envenoming pose new problems for the management of krait bite cases in South east Asia and should stimulate a search for the causative venom toxins.