Cellular uptake of ODNs in HIV-1 human-infected cells: a role for viral particles in DNA delivery?

We have previously described how a 16 nucleotides ODN (termed 93del) is capable of inhibiting the activity of recombinant integrase in a cell-free system as well as HIV-1 replication in human-infected cells with IC(50) in the low nanomolar range. Intracellular HIV-1 replication was inhibited when the ODN was added at the onset of infection. These results raise several questions. Is a naked ODN able to enter the cell? Does the virus play a role in ODN entry? The uptake of several ODNs (93del, 60del(sc), TBA, T30923) was evaluated and then tracked by labeling the ODN with a fluorescent dye and assessing its intracellular localization by confocal microscopy. A significant level of cellular uptake of free ODN was observed in several cell lines: HeLa epithelial cells, Huh7 hepatic cells, and H9 lymphocytes, and was detected for all ODNs tested except for TBA. Striking differences were observed when naked ODNs were added to cell in the presence or absence of the virus. When HIV-1 virions were present a sharp increase in cellular fluorescence was observed. These results strongly suggest a role for HIV-1 virions in the uptake of certain ODNs.     

Intracellular oligonucleotide hybridization detected by fluorescence resonance energy transfer (FRET).

Fluorescence resonance energy transfer (FRET) was used to study hybrid formation and dissociation after microinjection of oligonucleotides (ODNs) into living cells. A 28-mer phosphodiester ODN (+PD) was synthesized and labeled with a 3' rhodamine (+PD-R). The complementary, antisense 5'-fluorescein labeled phosphorothioate ODN (-PT-F) was specifically quenched by addition of the +PD-R. In solution, the -PT-F/+PD-R hybrid had a denaturation temperature of 65 +/- 3 degrees C detected by both absorbance and FRET. Hybridization between the ODNs occurred within 1 minute at 17 microM and was not appreciably affected by the presence of non-specific DNA. The pre-formed hybrid slowly dissociated (T1/2 approximately 3 h) in the presence of a 300-fold excess of the unlabeled complementary ODN and could be degraded by DNAse I. Upon microinjection into the cytoplasm of cells, pre-formed fluorescent hybrids dissociated with a half-time of 15 minutes, which is attributed to the degradation of the phosphodiester. Formation of the hybrid from sequentially injected ODNs was detected by FRET transiently in the cytoplasm and later in the cell nucleus, where nearly all injected ODNs accumulate. This suggests that antisense ODNs can hybridize to an intracellular target, of exogenous origin in these studies, in both the cytoplasm and the nucleus.     

Effect of gamma-hydroxypropano deoxyguanosine, the major acrolein-derived adduct, on monomolecular quadruplex structure of telomeric repeat d(TTAGGG)(4)

The three oligodeoxyribonucleotides (ODNs) a-c, having the telomeric repeat d(TTAGGG)(4) sequence and incorporating gamma-hydroxypropano deoxyguanosine at different positions, were synthesized. Gel electrophoresis and CD analyses indicated that the ODNs assume monomolecular quadruplex structures in Na+ and in K+ buffers. The T(m) values, obtained by CD melting experiments, showed that the presence of the acrolein-dG adduct into the ODN b decreases the thermal stability of the monomolecular quadruplex structure in Na+ solution, whereas for a and c no significant effect could be detected in the same experimental conditions. On the contrary, all ODNs a-d show the same behaviour in K+ buffer. These findings are briefly discussed.     

Modulation of CpG oligodeoxynucleotide-mediated immune stimulation by locked nucleic acid (LNA)

Locked nucleic acid (LNA) is an RNA derivative that when introduced into oligodeoxynucleotides (ODN), mediates high efficacy and stability. CpG ODNs are potent immune stimulators and are recognized by toll-like receptor-9 (TLR9). Some phosphorothioate antisense ODNs bearing CpG dinucleotides have been shown to possess immune modulatory capacities. We investigated the effects of LNA substitutions on immune stimulation mediated by antisense ODN G3139 or CpG ODN 2006. LNA ODNs were tested for their ability to stimulate cytokine secretion from human immune cells or TLR9-dependent signaling. Phosphorothioate chimeric LNA/DNA antisense ODNs with phosphodiester-linked LNA nucleobases at both ends showed a marked decrease of immune modulation with an increasing number of 3' and 5' LNA bases. In addition, guanosine-LNA and cytosine-LNA or simply cytosine-LNA substitutions in the CpG dinucleotides of ODN 2006 led to strong decrease or near complete loss of immune modulation. TLR9-mediated signaling was similarly affected. These data indicate that increasing amounts of LNA residues in the flanks or substitutions of CpG nucleobases with LNA reduce or eliminate the immune stimulatory effects of CpG-containing phosphorothioate ODN.     

Behavior of Vibrio cholerae in hot foods.

Four food types held hot at 45 to 60 degrees C were deliberately contaminated with O1 and non-O1 Vibrio cholerae strains. These organisms were assayed for survival and recovery from the foods within 1 h of the time the food was kept hot. The results showed no growth of V. cholerae non-O1 on thiosulfate-citrate bile-sucrose agar plates after 24 h of incubation at 37 degrees C for food held hot at 50 to 60 degrees C. Growth was low for V. cholerae O1 and was not achieved in some instances in which foods were held at either 55 or 60 degrees C after 40 or 60 min of from the time the food was kept hot. Both organisms, however, were recovered equally from all food types held at all temperatures after 48 h of incubation. When incubated for an additional 24 h, the organisms grew to unusually small-sized colonies, measuring 0.1 to 0.3 mm in diameter, on the same agar plates that were negative for growth after an initial 24 h of incubation. It was concluded that V. cholerae survived the period of time held at hot temperatures. Although the organisms were not recovered from some foods when held at some of the temperatures and times after 24 h of incubation, they remained viable. An incubation period of 48 h at 37 degrees C was found to be appropriate for the recovery of V. cholerae from hot foods.     

Behavior of Vibrio cholerae in hot foods

Four food types held hot at 45 to 60 degrees C were deliberately contaminated with O1 and non-O1 Vibrio cholerae strains. These organisms were assayed for survival and recovery from the foods within 1 h of the time the food was kept hot. The results showed no growth of V. cholerae non-O1 on thiosulfate-citrate bile-sucrose agar plates after 24 h of incubation at 37 degrees C for food held hot at 50 to 60 degrees C. Growth was low for V. cholerae O1 and was not achieved in some instances in which foods were held at either 55 or 60 degrees C after 40 or 60 min of from the time the food was kept hot. Both organisms, however, were recovered equally from all food types held at all temperatures after 48 h of incubation. When incubated for an additional 24 h, the organisms grew to unusually small-sized colonies, measuring 0.1 to 0.3 mm in diameter, on the same agar plates that were negative for growth after an initial 24 h of incubation. It was concluded that V. cholerae survived the period of time held at hot temperatures. Although the organisms were not recovered from some foods when held at some of the temperatures and times after 24 h of incubation, they remained viable. An incubation period of 48 h at 37 degrees C was found to be appropriate for the recovery of V. cholerae from hot foods.     

CpG oligodeoxynucleotides modulate the osteoclastogenic activity of osteoblasts via Toll-like receptor 9

Regulation of osteoclastogenesis by lipopolysaccharide (LPS) is mediated via its interactions with toll-like receptor 4 (TLR4) on both osteoclast- and osteoblast-lineage cells. We have recently demonstrated that CpG oligodeoxynucleotides (CpG ODNs), known to mimic bacterial DNA, modulate osteoclastogenesis via interactions with osteoclast precursors. In the present study we characterize the interactions of CpG ODNs with osteoblasts, in comparison with LPS. We find that, similar to LPS, CpG ODNs modulate osteoclastogenesis in bone marrow cell/osteoblast co-cultures, although in a somewhat different pattern. Osteoblasts express receptors for both LPS and CpG ODN (TLR4 and TLR9, respectively). The osteoblastic TLR9 transmits signals into the cell as demonstrated by NFkappaB activation as well as by extracellular-regulated kinase (ERK) and p38 phosphorylation. Similar to LPS, CpG ODN increases in osteoblasts the expression of tumor necrosis factor (TNF)-alpha and macrophage-colony stimulating factor (M-CSF). The two TLR ligands do not affect osteoprotegerin expression in osteoblasts. CpG ODN does not significantly affect receptor activator of NFkappaB ligand (RANKL) expression, in contrast to LPS, which induces the expression of this molecule. In the co-cultures CpG ODN induces RANKL expression in osteoblasts as a result of the more efficient TNF-alpha induction. CpG ODN activity (modulation of osteoclastogenesis, gene expression, ERK and p38 phosphorylation, and nuclear translocation of NFkappaB) is specific, because the control oligodeoxynucleotide, not containing CpG, is inactive. Furthermore, these effects (unlike the LPS effects) are inhibited by chloroquine, suggesting a requirement for endosomal maturation/acidification, the classic CpG ODN mode of action. We conclude that CpG ODN, upon TLR9 ligation, induces osteoblasts osteoclastogenic activity.     

A CpG oligodeoxynucleotide enhances the immune response to rabies vaccination in mice

Background

Rabies is a fatal disease that is preventable when post exposure prophylaxis (PEP) is administered in a timely fashion. CpG oligodeoxynucleotides (ODNs) can trigger cells that express Toll-like receptor 9, and their immunopotentiation activity in an inactivated aluminum-adjuvanted rabies vaccine for dogs has been identified using mouse and dog models.

Methods

A human diploid cell rabies vaccine (HDCV) of humans and a CpG ODNs with cross-immunostimulatory activity in humans and mice were used to evaluate the immunogenicity and protective efficacy of CpG ODN in a mouse model that simulates human PEP.

Results

HDCV combined with CpG ODN (HDCV–CpG) stimulated mice to produce rabies virus-specific neutralizing antibody (RVNA) earlier and increased the seroconversion rate. Compared with HDCV alone, either HDCV–1.25 μg CpG or HDCV–5 μg CpG increased the levels of RVNA. In particular, 5 μg CpG ODN per mouse significantly boosted the levels of RVNA compared with HDCV alone. IFN-γ producing splenocytes generated in the HDCV-5 μg CpG group were significantly increased compared to the group treated with HDCV alone. When the immunization regimen was reduced to three injections or the dose was reduced to half of the recommended HDCV combined with CpG ODN, the RVNA titers were still higher than those induced by HDCV alone. After viral challenge, 50% of mice immunized with a half-dose HDCV–CpG survived, while the survival rate of mice immunized with HDCV alone was 30%.

Conclusions

The immunopotentiation activity of CpG ODNs for a commercially available human rabies vaccine was first evaluated in a mouse model on the basis of the Essen regimen. Our results suggest that the CpG ODN used in this study is a potential adjuvant to rabies vaccines for human use.

     

Highly efficient cellular uptake of c-myb antisense oligonucleotides through specifically designed polymeric nanospheres.

c-myb antisense oligonucleotides (AS ODNs) were reversibly immobilized to a novel polymeric core shell nanosphere and their cellular uptake and inhibitory effect on HL60 leukemia cell proliferation studied. The nanosphere surface was so designed as to directly bind ODNs via ionic interactions and reversibly release them inside the cells. Compared with the cellular uptake of free oligonucleotide, the use of AS ODN (immobilized to the nanospheres) produced a 50-fold increase in the intracellular concentration. Specifically, a single dose of 320 nM of AS ODN immobilized to the nanospheres was capable of inhibiting HL60 cell proliferation with the same degree of efficiency obtained using a 50-fold higher dose of free AS ODN. Flow cytometric experiments with fluoresceinated ODNs showed a temperature-dependent uptake, which was detectable as early as 2 h after the beginning of treatment. The inhibitory effect on cell proliferation was maintained for up to 8 days of culture. Moreover, the level of c-Myb protein decreased by 24% after 2 days and by 60% after 4 days of treatment, thus indicating a continuous and sustained release of non-degraded AS ODN from the nanospheres inside the cells.     

Spectral and physical characterization of the inverted terminal repeat DNA structure from adenoassociated virus 2

An oligodeoxynucleotide (ODN) that includes elements found in secondary structures at the 5'- and 3'- ends of adenoassociated virus 2 virion DNA was synthesized by ligation of three overlapping ODNs. The most stable secondary structure was calculated to be branched, with a 61 bp duplex stem, terminating in a three-way junction with 9 bp arms. The electrophoretic mobility of the ODN is slower than expected for normal duplex DNA of the same size, suggesting a bent or branched conformation. CD spectra indicate that the ITR structure is largely B form DNA, although there is a slight blue shift compared to the spectra of the isolated stem and loop elements. Thermal melting experiments indicate that the hairpin is significantly more stable than the isolated stem and loop elements. Singular value decomposition of UV spectra obtained as a function of temperature indicates that four species contribute to changes in the spectra upon denaturation, indicating that the melting is not a simple two-state process. Characterization of the branched ODN by differential scanning calorimetry permits estimation of the enthalpy of melting by a model-free analysis, yielding DeltaHcal= 614 kcal mol-1. This value agrees with the enthalpy computed for the most stable secondary structure.     

Optimisation of conditions for the extraction of casearins from Casearia sylvestris using response surface methodology

Optimal conditions for the extraction of casearins from Casearia sylvestris were determined using response surface methodology. The maceration and sonication extraction techniques were performed using a 3 x 3 x 3 full factorial design including three acidity conditions, three solvents of different polarities and three extraction times. The yields and selectivities of the extraction of casearins were significantly influenced by acidity conditions. Taking into account all variables tested, the optimal conditions for maceration extraction were estimated to involve treatment with dichloromethane saturated with ammonium hydroxide for 26 h. Similar yields and selectivities for casearins were determined for sonication extraction using the same solvent but for the much shorter time of 1 h. The best results for stabilisation of the fresh plant material were obtained using leaves that had been oven dried at 40 degrees C for 48 h.     

Acridine-modified,clamp-forming antisense oligonucleotides synergize with cisplatin to inhibit c-Myc expression and B16-F0 tumor progression

The c-myc protooncogene plays a key role in the abnormal growth regulation of melanoma cells. We have targeted three polypurine sequences within the mouse myc mRNA with acridine-modified, clamp-forming antisense oligonucleotides (AS ODNs) in an effort to inhibit growth of murine melanoma cells. These ODNs are unique in that they hybridize to the target mRNA by both Watson–Crick and Hoogsteen hydrogen bond interactions, forming a triple-stranded structure. At a concentration of 3 µM E1C, E2C and E3C inhibit B16-F0 proliferation by 76, 66 and 78%, respectively. Both immunofluorescent staining and western blot analysis corroborate a proportional reduction in c-Myc expression by all three ODNs. There were clear distinctions in the ability of these ODNs to inhibit tumor progression in C57BL/6 mice as a function of Myc expression. There was no synergy demonstrated between ODN E1C with cisplatin (DDP), which inhibited tumor growth by 77% alone and 82% in combination. Although E2C inhibited growth by 54%, its effect was decreased to 32% with DDP, when compared with controls. E3C, on the other hand, demonstrated a synergistic effect with DDP, inhibiting growth by 72% in combination, but only by 1% as a single agent. Immunofluorescence analysis of tumors for each group revealed a concomitant reduction in c-Myc expression in tumors from mice treated with the most active clamp ODN alone (E1C) or clamp ODN + DDP (E1C/E3C + DDP). Western blot analysis confirmed this decrease in target protein expression. Our results document the growth-inhibitory activity of two myc-targeting antisense clamp ODNs; E1C, which has activity as a single agent, and E3C, which has in vivo synergy with DDP pretreatment. These data confirm the antiproliferative effects of these novel ODNs and document an interesting synergy with the chemotherapeutic agent DDP.     

Parameters of binding and transport of oligodeoxynucleotide, which includes BCL2 mRNA translation start, to the K562 cells

Interaction of oligodeoxynucleotides (ODN), 18-mer, which included sequence of BCL2 mRNA translation start, with K562 cells has been studied. The kinetic curves of interaction showed that oligonucleotide total binding with the cells at 37 degrees C and low oligonucleotide concentration (< or = 30 nM), as well as under lipofection, were composed of two processes: 18-mer surface binding with cell membranes and its non-proportional internalization into the cells. The last, in turn, consisted of three consequent steps. The enhanced extent and rate of oligonucleotide internalization was diminished after first hour incubation and later they were increased again. This reflected rising additional binding sites that provided internalization. At chosen time-points the internalization of ODN into cells, been proceeded at 37 degrees C, were at most abruptly abrogated by cooling down. ODN to K562 cell membrane binding constants and specific number of binding sites have been determined. Time-intervals, providing equilibrium for each successive stage of multistep ODN bound/free determination, were maintained. It was established that receptor binding with increased binding constant (more than 2 x 10(9) M(-1)) promoted ODN internalization. Oligonucleotide binding and internalization with prolonged incubation were also up-regulated due to priming new binding sites of higher affinity. Lipofection enhanced ODN binding to cell membrane but conserved the main features of ligand-receptor interaction. During lipofection constants and ODN binding site numbers increased without changing the overall time-pattern of the process, observed for ODN without liposomes. Extent and rate of internalization of ODN in liposomal formulation did not differ substantially from ODN in solution.     

Phosphodiester and phosphorothioate oligonucleotide condensation and preparation of antisense nanoparticles

Protamine is a cationic peptide with a molecular mass of approx. 4000 Da that is able to condense DNA. In the present study it was used to complex antisense oligonucleotides (ODNs) and to form solid particles with initial diameters of 90-150 nm. The reaction was very rapid and occurred by simple mixing of diluted solutions of the polycation with the oligonucleotide. The aggregation was dependent on the oligonucleotide chain length and the protamine/ODN mass ratio. Particle formation required a minimal chain length of nine nucleotides and a mass ratio of 0.5:1. The particle surface charge and the number of particles depended on the mass ratio. With increasing amounts of the peptide, the number of particles and the zeta potential increased. Both negatively and positively charged particles improved the stability of oligonucleotides against DNase I digestion. Above a mass ratio of 2.5:1 no degradation was found. The uptake of unbound rhodamine-labelled ODNs and its complexes with protamine was determined with Vero cells under in vitro cell culture conditions at 37 degrees C and 4 degrees C. At 37 degrees C the cellular uptake increased with increasing mass ratio. The internalized oligonucleotides were localized in the cytoplasm and in the nucleus of the cells. When Vero cells were treated with these samples at 4 degrees C for 4 h, no fluorescence could be detected inside the cells. Therefore, our data indicate an energy dependent endocytotic uptake mechanism. In contrast, spermine and spermidine, which are also known condensation agents, did not aggregate with oligonucleotides into nanoparticles under the same conditions.     

Delivery of antisense oligonucleotide to the cornea by iontophoresis

We wished to evaluate the potential of iontophoresis to promote the delivery of antisense oligonucleotides (ODN) directed at the vascular endothelial growth factor (VEGF)-R2 receptor (KDR/Flk) to the cornea of the rat eye. Fluorescence (CY5)-labeled ODNs in phosphate-buffered saline (PBS) (20 microM) were locally administered to rat eyes, and their fate within the anterior segment was studied. Thirty-four male, 5-week-old Wistar rats were used for all experiments. The rats were divided in four groups. In group I (12 rats, 12 eyes), the ODNs (20 microM) were delivered by iontophoresis (300 microA for 5 minutes) using a specially designed corneal applicator. In group II (12 rats, 12 eyes), the ODNs (20 microM) were delivered using the same applicator, but no electrical current was applied. In group III (6 rats, 6 eyes), a corneal neovascular reaction was induced prior to the application of ODNs (20 microM), and iontophoresis electrical current was delivered as for group I rats. Group IV (4 rats, 4 eyes) received ODN (60 microM) iontophoresis application (300 microA for 5 minutes) and were used for ODN integrity studies. The animals were killed 5 minutes, 90 minutes, and 24 hours after a single ODN application and studied. Topically applied ODNs using the same iontophoresis applicator but without current do not penetrate the cornea and remain confined to the superficial epithelial layer. ODNs delivered with transcorneoscleral iontophoresis penetrate into all corneal layers and are also detected in the iris. In corneas with neovascularization, ODNs were particularly localized within the vascular endothelial cells of the stroma. ODNs extracted from eye tissues 24 hours after iontophoresis remained unaltered. The iontophoresis current did not cause any detectable ocular damage under these conditions. Iontophoresis promotes the delivery of ODNs to the anterior segment of the eye, including all corneal layers. Iontophoresis of ODNs directed at VEGF-R2 may be used for the design of specific antiangiogenic strategy in diseases of the cornea.     

Effect of temperature on spore germination and vegetative cell growth of Clostridium botulinum.

Spore germination and vegetative growth of Clostridium botulinum type E strain VH at 2 to 50 degrees C were studied. At all of these temperatures, germination began immediately after the addition of the spores to the germination medium. Microscopic observations during germination revealed three types of spores: phase bright (ungerminated), phase variable (partially germinated), and phase dark (fully germinated). At all temperatures except 50 degrees C, there was a pronounced lag between the initial appearance of phase-variable spores and their eventual conversion to phase-dark spores. The number of partially germinated spores increased steadily, reaching 40 to 60% by 18 to 21 h of incubation. During this time, phase-dark, fully germinated spores developed slowly and did not exceed 28% in any of the samples. At 18 to 26 h of incubation, the rate of full germination increased abruptly four-fold. There was extensive and relatively rapid germination at 2 degrees C, the lowest temperature tested, yielding about 60% phase-variable spores by 18 h, which became phase-dark by 26 h of incubation. The optimum temperature for partial and full germination was consistently 9 degrees C. Germination at 50 degrees C was exceptionally rapid and was completed within 1 to 2 h, although 40% remained phase bright. Vegetative cells showed detectable growth at 6 to 41 degrees C, with a distinct optimum at 32.5 degrees C. No growth occurred at 50 degrees C, and only marginal growth was observed at 6 to 14 degrees C. The psychrophilic nature of the germination process coupled with the cold tolerance of vegetative growth appears to give C. botulinum type E an advantage in cold climates as well as in cold-stored foods.     

Stable expression of functional CBP70 lectin during heat shock

CBP70 is a glycoslylated lectin that interacts through either glycan-lectin or protein-protein interactions. In addition, depending on its cellular localization, this lectin has different partners, for example, galectin-3, an 82-kDa ligand in the nucleus, or Bcl-2 in the cytoplasm. In this study, we observed the persistence of plurilocalized lectin CBP70 after two heat-shock treatments conducted either under mild conditions, i.e., incubating the cells for 1 h at 42 degrees C then for 1, 3, 5, 7, or 9 h at 37 degrees C, or harsh conditions, i.e., incubation at 42 degrees C for 1, 2, 4, 6, 8, or 10 h. By combining the information collected from biochemical, fluorocytometric, confocal, and affinity-chromatography analyses, we concluded that CBP70 persisted in HL60 cells and its N-acetylglucosamine-binding sites remained active after all the heat-shock treatments tested. These data and the previously published findings reviewed in this report concur in supporting the hypothesis that CBP70 could function as an organizer of multimeric assembly, leading to the formation of various complexes in different cellular compartments, according to the needs of the cell.     

Predictive models of the combined effects of curvaticin 13, NaCl and pH on the behaviour of Listeria monocytogenes ATCC 15313 in broth

Thirty-three strains of Listeria monocytogenes belonging to different serotypes were tested for their sensitivity to curvaticin 13, an antilisterial bacteriocin produced by Lactobacillus curvatus SB13, using the well diffusion method in Institut Pasteur agar plates at 37 degrees C. No relationship between serotype and sensitivity was observed. The sensitivity of this species was strain-dependent and a large variation in tolerance to curvaticin 13 was observed. The combined effects of curvaticin 13 (0-160 AU ml-1), NaCl (0-6% w/v), pH values (5.0-8.2) and incubation time (0-24 h) were investigated on L. monocytogenes ATCC 15313 in trypcase soy-yeast extract broth at 22 degrees C. For this study, two Doehlert matrices were used in order to investigate the main effects of these factors and their different interactions. The results were analysed using the Response Surface Methodology. Curvaticin 13 had a major inhibitory effect and the response was NaCl concentration-, time- and pH-dependent. This inhibitory activity was the same at pH values between 6.6 and 8.2. Curvaticin 13 was bactericidic at acidic pH values, but the surviving cells resumed growth. For a short incubation time (12 h), the effectiveness of curvaticin 13 was maximal in the absence of NaCl. For longer incubation times (12-48 h), with high NaCl (6%) and curvaticin 13 concentrations (160 AU ml-1), the inhibition of L. monocytogenes was greater than that observed with NaCl or curvaticin 13 alone.