Self-neutralizing oligonucleotides with enhanced cellular uptake.

There is tremendous potential for oligonucleotide (ON) therapeutics, but low cellular penetration due to their polyanionic nature is a major obstacle. We addressed this problem by developing a new approach for ON charge neutralization in which multiple branched charge-neutralizing sleeves (BCNSs) are attached to the internucleoside phosphates of ON by phosphotriester bonds. The BCNSs are terminated with positively charged amino groups, and are optimized to form ion pairs with the neighboring phosphate groups. The new modified ONs can be prepared by standard automated phosphoramidite chemistry in good yield and purity. They possess good solubility and hybridization properties, are not involved in non-standard intramolecular aggregation, have low cytotoxicity, adequate chemical stability, improved serum stability, and above all, display significantly enhanced cellular uptake. Thus, the new ON derivatives exhibit properties that make them promising candidates for the development of novel therapeutics or research tools for modulation of the expression of target genes.

Direct detection of the bacterial stress response in intact samples of platelets by differential impedance.

BACKGROUND: We have previously described a new rapid approach that relies on monitoring intentionally stressed bacteria in contaminated platelet concentrates (PCs). This earlier work included human cell lysis with Triton X-100 and filtration as steps in the sample preparation. This study was undertaken to develop an improved and time-saving protocol that enables direct bacterial detection in PCs without lysis and filtration. STUDY DESIGN AND METHODS: Apheresis- or whole blood-derived PCs were spiked with 17 model bacteria and tested at final concentrations from 10(3) to 10(6) colony-forming units (CFUs)/mL. The contaminated PCs were treated with a chemical compound that induces a stress response in bacteria and monitored using differential impedance sensing to detect and record subtle changes in the dielectric permittivities of the contaminated platelet (PLT) samples. RESULTS: No measurable responses from sterile PLT samples were observed during exposure to the compounds used as stressors. In contrast, distinct response profiles were obtained without exception for all 17 bacterial species for all bacterial concentrations tested. Bacterial presence was established within 5 to 10 minutes for high inocula (10(6) and 10(5) CFUs/mL) while low inocula (10(4) and 10(3) CFUs/mL) were usually detectable within 20 minutes. The entire testing process routinely took less than 30 minutes from the point of sampling to the time that the final results are available. CONCLUSIONS: The results described here demonstrate that monitoring the development of stress in bacteria is a fast and simple way to detect 10(3) CFUs/mL or more bacteria in complex cellular blood products such as PCs.

Rapid microbiological testing: monitoring the development of bacterial stress.

The ability to respond to adverse environments effectively along with the ability to reproduce are sine qua non conditions for all sustainable cellular forms of life. Given the availability of an appropriate sensing modality, the ubiquity and immediacy of the stress response could form the basis for a new approach for rapid biological testing. We have found that measuring the dielectric permittivity of a cellular suspension, an easily measurable electronic property, is an effective way to monitor the response of bacterial cells to adverse conditions continuously. The dielectric permittivity of susceptible and resistant strains of Escherichia coli and Staphylococcus aureus, treated with gentamicin and vancomycin, were measured directly using differential impedance sensing methods and expressed as the Normalized Impedance Response (NIR). These same strains were also heat-shocked and chemically stressed with Triton X-100 or H(2)O(2). The NIR profiles obtained for antibiotic-treated susceptible organisms showed a strong and continuous decrease in value. In addition, the intensity of the NIR value decrease for susceptible cells varied in proportion to the amount of antibiotic added. Qualitatively similar profiles were found for the chemically treated and heat-shocked bacteria. In contrast, antibiotic-resistant cells showed no change in the NIR values in the presence of the drug to which it is resistant. The data presented here show that changes in the dielectric permittivity of a cell suspension are directly correlated with the development of a stress response as well as bacterial recovery from stressful conditions. The availability of a practical sensing modality capable of monitoring changes in the dielectric properties of stressed cells could have wide applications in areas ranging from the detection of bacterial infections in clinical specimens to antibiotic susceptibility testing and drug discovery.

New approach for drug susceptibility testing: monitoring the stress response of mycobacteria.

Methods currently used for in vitro drug susceptibility testing are based on the assessment of bacterial growth-related processes. This reliance on cellular reproduction leads to prolonged incubation times, particularly for slowly growing organisms such as mycobacteria. A new rapid phenotypic method for the drug susceptibility testing of mycobacteria is described. The method is based on the detection of the physiological stress developed by susceptible mycobacterial cells in the presence of an antimicrobial compound. The induced stress was quantified by differential monitoring of the dielectric properties of the bacterial suspension, an easily measurable electronic property. The data presented here characterize the stress developed by Mycobacterium tuberculosis cells treated with rifampin (rifampicin), isoniazid, ethambutol, and pyrazinamide. Changes in the dielectric-based profiles of the drug-treated bacteria revealed the respective susceptibilities in near real time, and the susceptibilities were well correlated with conventional susceptibility test data.

Monitoring the physiologic stress response: a novel biophysical approach for the rapid detection of bacteria in platelet concentrate.

BACKGROUND: Currently approved culture-based methods for the bacterial testing of platelet concentrates (PCs) require an extended period of time to obtain results. A new approach based on the monitoring of the bacterial response to physiologic stress is presented. Because the stress response is independent of the growth rate, decisive results can be obtained in near real time. STUDY DESIGN AND METHODS: PCs were spiked with Gram-positive Staphylococcus epidermidis and Propionibacterium acnes and Gram-negative Escherichia coli organisms commonly implicated in posttransfusion septic reactions. All suspensions were treated with Triton X-100 at concentrations that caused human cell lysis while acting as a stressor for bacterial cells. Normalized impedance response (NIR) profiles of these suspensions were recorded using the BioSense Technologies differential impedance sensing platform specifically designed to monitor subtle changes in the dielectric properties of biologic suspensions. Further development of the approach included measurement of NIRs for spiked PCs treated with nonlysing chemical stressors. RESULTS: NIR profiles for bacteria conventionally growing under optimal conditions were characterized by an overall increase in the NIR values. In contrast, the corresponding responses from chemically stressed bacteria revealed immediate and continuous decreases in value-enabling bacterial detection in less than 30 minutes. CONCLUSION: These pilot experiments demonstrated that monitoring of the bacterial stress response is a fast and effective way to detect bacteria in PCs. In addition, special culture conditions are not required for detecting anaerobes and fastidious species.

Inactivation of protozoan parasites in red blood cells using INACTINE PEN110 chemistry.

BACKGROUND: The transmission of parasites, including Babesia, plasmodia, and Trypanosoma cruzi, via transfusions is an important public health concern. INACTINE technology is a pathogen-reduction process that utilizes PEN110, an electrophilic agent that inac-tivates a wide range of pathogens by disrupting nucleic acid replication. The present study investigated the effect of PEN110 treatment on the viability of protozoa in RBCs. STUDY DESIGN AND METHODS: B. microti-parasitized RBCs from infected hamsters were treated with PEN110 and inoculated to naïve animals. Parasitemia was detected by blood smears and PCR. Human RBCs infected with P. falciparum were treated with PEN110 and incubated with fresh RBCs. P. falciparum multiplication was detected by blood smears. Human RBCs spiked with T. cruzi and treated with PEN110 were analyzed for the presence of live parasites using in-vitro infectivity assay or by inoculating susceptible mice. RESULTS: Treatment of RBCs infected with B. microti or P. falciparum with 0.01 to 0.1 percent (vol/vol) PEN110 resulted in parasite inactivation to below the limit of detection during 24 hours. T. cruzi inoculated into human RBCs was inactivated below the limit of detection by 0.1 percent PEN110 after 3 hours. CONCLUSION: The study demonstrates that treatment of blood with PEN110 is highly effective in eradicating transfusion-transmitted protozoan parasites.

Inactivation of mycoplasma species in blood by INACTINE PEN110 process.

BACKGROUND: Mycoplasmas have been associated with multiple acute and chronic diseases. Mycoplasma genome is found in the blood of 10 to 15 percent of subjectively healthy individuals. If blood borne and viable in donated blood, mycoplasmas could potentially be transfusion transmissible. The INACTINE PEN110 technology is a pathogen reduction process that is in Phase 3 clinical studies. The present study investigated the ability of this process to eradicate mycoplasmas in human blood. STUDY DESIGN AND METHODS: Identical whole blood or RBC units inoculated with Mycoplasma arthritidis or M. pneumoniae were incubated with PEN110 (inactivating agent) for 24 hours at 23 degrees C. Sham controls were treated with buffer under the same conditions. 4 degrees C controls were put on storage immediately after the spike. RESULTS: No viable microorganisms were detected in PEN110-treated units after 24 hours of incubation. Sham controls showed no changes to mycoplasma titers during the incubation. In 4 degrees C controls, minor decrease of mycoplasma titers was observed during the storage. CONCLUSION: The INACTINE process inactivates more than 107 mycoplasma CFU per mL in whole blood and RBCs. This study is the first demonstration of susceptibility of mycoplasmas to pathogen reduction. The data provide further support for the ability of INACTINE technology to address microbial safety issues that are not well characterized.

Prevention of Yersinia enterocolitica, Pseudomonas fluorescens, and Pseudomonas putida outgrowth in deliberately inoculated blood by a novel pathogen-reduction process.

BACKGROUND: Yersinia enterocolitica, Pseudomonas fluorescens, and P. putida are responsible for a significant amount of the bacterial sepsis cases attributed to RBC transfusions. INACTINE is a pathogen-reduction process for RBCs, which consists of incubation of RBCs with PEN110 (proprietary compound) followed by automated washing of the RBCs. INACTINE is an electrophilic agent, which inactivates a wide range of viruses and WBCs by disruption of nucleic acid replication. The present study investigated the effect of the PEN110 process on Y. enterocolitica, P. fluorescens, and P. putida. STUDY DESIGN AND METHODS: Identical units of reduced CPD/ADSOL additive solution (AS-1) or CP2D/Nutricel additive solution (AS-3) RBCs were inoculated with 10 to 100 CFU per mL of either Y. enterocolitica, P. fluorescens, or P. putida. The control units were put on storage immediately after the bacterial spike. The test units were subjected to the PEN110 process and then stored. Sham control units were processed the same way as test units without addition of PEN110. Bacterial titer in all units was monitored during the 6-week storage period. RESULTS: No bacteria were detected in any of the RBC units (n = 9 for each microorganism) prepared using the PEN110 process throughout 6 weeks of storage. Substantial bacterial growth occurred in all control and in a majority of sham control units (11 out of 15 experiments). The bacterial inactivation by the INACTINE process was found to be equally effective in CPD/AS-1 and CP2D/AS-3 RBC units. CONCLUSION: The INACTINE process effectively prevented the outgrowth of Y. enterocolitica, P. fluorescens, and P. putida deliberately inoculated into WBC-reduced CPD/AS-1 and CP2D/AS-3 RBCs. The results demonstrated the crucial bactericidal role of PEN110 in the INACTINE process.