Greater Killing Research Articles

Potent Antibacterial and Antibiofilm Activities of a Synthetic Remineralizing Preparation of Nano-Hydroxyapatite Against Cariogenic Streptococcus mutans Using an Ex-vivo Animal Model

AimThe aim to this study is to evaluate the biocompatibility and antibiofilm actions of two nano-hydroxy apatite (NHA).MethodologyNano-hydroxy apatites are biomaterials use in direct contact with living tissues. Therefore, they should be tested for their safety beside assessment of their minimum inhibitory (MIC) and minimum bactericidal concentration (MBC) using broth microdilution method. One hundred and twenty extracted bovine incisors were collected and cleaned to ensure the absence of any defects. Enamel blocks with different size (2 × 2 × 3 mm) and (5 × 5 × 2 mm) were prepared from their labial surfaces using an isomet saw. Enamel blocks are used for detecting the suitable concentration will be used in the following experiments using energy dispersive X-ray analysis (EDX). The remaining enamel blocks divided into 5 equal groups to detect inhibitory effect against bacterial adhesion to the initial enamel caries like lesions using viable count technique beside the antibiofilm activity against mature biofilm of Streptococcus mutans (S. mutans) using confocal laser microscopy. The remaining enamel blocks were used as a representing data for detecting surface topography for each group by using the scanning electron microscopy (SEM).ResultThe data showed safety of NHA suspensions. Additionally, only NHA suspension of large nanoparticle size (NHA-LPS) had MIC of 1.25 mg/ml against S. mutans. Also, have the higher percentages of Ca and P in the enamel blocks. Furthermore, the lowest level of bacterial adhesion was recorded in (group III) treated by NHA-LPS which was non-significantly different with the positive control group V. Biofilm thickness in group IV treated with NHA-small particle size (SPS) recorded high biofilm thickness followed by group III. Interestingly, group III showed greater killing effect against mature biofilm which is slightly higher than the positive control group V. In group III, surface topography revealed very smooth enamel surface with closed pores. Accordingly, NHA-LPS suspension had antiadhesive, antibacterial, and antibiofilm effect against cariogenic S. mutans representing a promising possibility to be recommended for safe effective remineralization.

In Vitro Time-Kill of Common Ocular Pathogens with Besifloxacin Alone and in Combination with Benzalkonium Chloride.

Background: Besifloxacin ophthalmic suspension 0.6% (w/v%) contains benzalkonium chloride (BAK) as a preservative. We evaluated the in vitro time-kill activity of besifloxacin, alone and in combination with BAK, against common bacteria implicated in ophthalmic infections. Methods: The activity of besifloxacin (100 µg/mL), BAK (10, 15, 20, and 100 µg/mL), and combinations of besifloxacin and BAK were evaluated against isolates of Staphylococcus epidermidis (n = 4), Staphylococcus aureus (n = 3), Haemophilus influenzae (n = 2), and Pseudomonas aeruginosa (n = 2) in time-kill experiments of 180 min duration. With the exception of one S. aureus isolate, all of the staphylococcal isolates were methicillin- and/or ciprofloxacin-resistant; one P. aeruginosa isolate was ciprofloxacin-resistant. The reductions in the viable colony counts (log10 CFU/mL) were plotted against time, and the differences among the time–kill curves were evaluated using an analysis of variance. Areas-under-the-killing-curve (AUKCs) were also computed. Results: Besifloxacin alone demonstrated ≥3-log killing of P. aeruginosa (<5 min) and H. influenzae (<120 min), and approached 3-log kills of S. aureus. BAK alone demonstrated concentration-dependent killing of S. epidermidis, S. aureus and H. influenzae, and at 100 µg/mL produced ≥3-log kills in <5 min against these species. The addition of BAK (10, 15, and 20 µg/mL) to besifloxacin increased the rate of killing compared to besifloxacin alone, with earlier 3-log kills of all species except P. aeruginosa and a variable impact on S. aureus. The greatest reductions in AUKC were observed among H. influenzae (8-fold) and S. epidermidis (≥5-fold). Similar results were found when the isolates were evaluated individually by their resistance phenotype. Conclusions: In addition to confirming the activity of 100 µg/mL BAK as a preservative in the bottle, these data suggest that BAK may help besifloxacin to achieve faster time-kills on-eye in the immediate timeframe post-instillation before extensive dilution against bacterial species implicated in ophthalmic infections, including drug-resistant S. epidermidis. Greater killing activity may help prevent resistance development and/or help treat resistant organisms.

674 The effective killing of the difficult-to-treat melanomas with the combinations of MCL1 inhibitors S63845/MIK665 plus Navitoclax

Despite the advancement of melanoma care, a subset of melanoma patients do not respond or relapse with current targeted or immuno-therapies. Therefore, it is necessary to explore other biological processes can be therapeutic. The BCL-2 family of proteins contributes to a cancer cell’s resistance to treatment, and BH3 mimetics drugs that target BCL-2 are effective in hematological cancers. We used genetic knockdown and BH3 mimetics therapeutics to target BCL-2’s anti-apoptotic defenses, and identified MCL1 and BCLXL as crucial melanoma pro-survival members. We then examined the effects of combining BH3 mimetics that target MCL1 and BCLXL in vivo and in vitro. We prioritized clinical-trial-ready compounds, such as ABT263 (Navitoclax) and S63845/S64315 (MIK655). We used commercial cell lines, as well as cells derived from patients with difficult-to-treat melanomas. The melanoma panel included the subtypes acral, mucosal, superficial spreading, and nodular, as well as diverse mutations, such as wild type for BRAF, NRAS and NF1. In vitro, the combined inhibition of MCL1 and BCLXL resulted in greater killing than either single agent (p < 0.05) in multiple assays. Genetic knock-down and knock-out studies showed that the combination-induced cell death was not dependent on pro-apoptotic BCL2 family members BID, BIM, or NOXA. Moreover, in mouse xenograft model, the combination inhibited tumor growth of multiple melanomas (p <0.01), had tolerable toxicity, and reduced sphere forming capacity (p < 0.05) in a downstream in vitro assay. In summary, this study suggests that dual targeting of MCL1 and BCLXL can be an alternative treatment for melanoma patients who have exhausted current treatment options.

β-1,3-glucan induces natural killer cell memory-like immunity

Abstract Natural killer (NK) cells are considered innate lymphocytes that are able to kill pathogenic microorganisms, including bacteria, parasites and fungi as well as virus-infected cells and tumor cells without prior stimulation. Recent studies suggest that they possess a memory-like property to previously encountered pathogens (e.g., cytomegalovirus). We showed that NK cells directly kill Cryptococcus and Candida, which are fatal opportunistic fungi often infecting immunocompromised individuals. We determined that NKp30 is the activating pattern recognition receptor that mediates NK cell killing of these fungi, and that β-1,3-glucan is the ligand for NKp30. Interestingly, we found that soluble β-1,3-glucan enhanced NK cell killing of C. neoformans and C. albicans. Since humans and animals are exposed to β-1,3-glucan through inhalation of components of the cell wall of microbes or consumption of dietary grains, we asked whether isolated β-1,3-glucan plays a role in shaping NK cell immunity. To determine whether β-1,3-glucan induces a training or memory effect, YT cells, an NK cell line, were treated for 24 hours with laminarin or Saccharomyces β-1,3-glucan and cultured for various times. After an initial period of enhanced C. neoformans killing, killing was reduced to the untreated level on the second week after the initial treatment. However, when cryptococcal killing was assessed on the fourth week, a much greater killing was observed compared to the enhanced killing. This suggests that a recall immune response was induced. We conclude that β-1,3-glucan may induce NK cell memory- like immunity.

Measuring Antimicrobial Efficacy against Biofilms: a Meta-analysis.

Through a statistical meta-analysis of published data on antimicrobial efficacy against biofilms formed by two common bacterial species, it was concluded that the particular experimental method used is the most important factor determining the outcome of the test. An expected dose-response relationship (greater killing with higher doses or longer treatment times) was observed for data sets derived from a single method but was not observed when data from multiple studies using diverse methods were pooled. Method-specific properties such as the surface area/volume ratio, areal biofilm cell density, and microbial species were shown to influence quantitative measurements of biofilm killing. A better appreciation of the method characteristics that affect antibiofilm efficacy tests could aid decision-making related to investment in research and development and regulatory approvals for biofilm control strategies. The following recommendations are offered to those working in research and development related to biofilm control: (i) report the log reduction, surface area/volume ratio, and biofilm areal cell density; (ii) include data for a benchmark agent, making sure that this agent performs competitively at the dose tested; (iii) measure the dose-response relationship, i.e., make measurements at multiple treatment concentrations or dose durations; and (iv) use a standardized method in addition to research methods.

Microarry Analysis Identifies Activation Of Multiple Signalling Pathways In Primary Prostate Epithelial Cells By Low Temperature Plasma

We have previously reported the killing effects of Low Temperature Plasma (LTP) on primary prostate epithelial cells, of both normal and cancerous origin, via necrosis [1] following initial DNA damage. However it was evident that a population of cells was resistant to LTP treatment. To discern possible survival mechanisms in treated cells; the gene expression of 6 samples (4 cancers and 2 matched normal) was analysed 2 hours post-treatment, on a transcriptome wide level, by microarray. Initial analysis found alteration in the expression of 646 transcripts with significant activation of Notch, NF-kB, Nrf2 and AP-1 signalling in our primary cells. Pathway activation was subsequently validated by qRT-PCR in four different patient-derived prostate cultures to give a robust LTP-induced expression signature. Upstream effector activation was then confirmed by immunofluorescence and Western blot. We found as other groups have; [2, 3] accumulation of Nrf2 in response to LTP, alongside AP-1 activation [4, 5] – through phosphorylation of both JNK and Jun. The Notch signalling pathway is typically described in relation to stem cell maintenance and we observed LTP induced Notch activation through release of its intracellular domain (NICD) that then acts as a nuclear transcription factor. Tumours are innately heterogeneous and contain multiple cell populations, both intrinsic and recruited. We have established that our cultures contain three readily identifiable epithelial cell pools that can be separated to permit further analysis.[6] Preliminary investigation has found that the progenitor epithelial cells respond in greater measure than their more differentiated progeny, suggesting that they are more resistant to plasma. Similar observations of prostate stem cell resistance have been made with radiotherapy.[7] However, if these stem-like cells can be targeted – LTP may have a greater killing effect if utilized in prostate cancer treatments. Download : Download high-res image (151KB) Download : Download full-size image Figure – Pathway activation in primary prostate cancer epithelial culture; Nrf2 accumulation, AP-1 activation by JNK and Jun phosphorylation and Notch cleavage, with release of NICD by LTP. U – Untreated, T- Treated. Samples taken 0.5 and 2 hours following treatment with 3 minutes LTP.

Silencing of PKG1 expression enhances the efficacy of vemurafenib against melanoma cell

To explore whether targeting phosphoglycerate kinase 1 (PGK1) can enhance the sensitivity of BRAFV600E mutation melanoma cells to vemurafenib. The methods of cell biology, molecular biology and pharmacology(MTT assay, Western blot, FCM, Colongenic assay) were used in this study. ① Silencing of PGK1 expression increased the efficacy of vemurafenib in melanoma cells, as evidenced by greater killing in the tumor cells subjected to combined treatment of vemurafenib with siPGK1; ②The mechanism of enhanced sensitivity of melanoma cells to vemurafenib was associated with activation of apoptotic signaling pathway. Targeting of PGK1 may represent a novel strategy of sensitizing melanoma cells to vemurafinib.

Influence of rhlR and lasR on Polymyxin Pharmacodynamics in Pseudomonas aeruginosa and Implications for Quorum Sensing Inhibition with Azithromycin.

The impact of quorum sensing on polymyxin and azithromycin pharmacodynamics was assessed in Pseudomonas aeruginosa PAO1 and an isogenic rhlR/lasR double knockout. For polymyxin B, greater killing against the rhlR/lasR knockout than against PAO1 was observed at 108 CFU/ml (polymyxin B half-maximal effective concentration [EC50], 5.61 versus 12.5 mg/liter, respectively; P < 0.005). Polymyxin B combined with azithromycin (256 mg/liter) was synergistic against each strain, significantly reducing the respective polymyxin B EC50 compared to those with monotherapy (P < 0.005), and is a promising strategy by which to combat P. aeruginosa.

Cooperative Supression of MEIS2 Mediates Dexamethasone Enhancement of Lenalidomide Killing in Myeloma Cells

Dexamethasone (Dex) enhances the clinical response to most therapies in multiple myeloma, the immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide in particular. As a steroid, Dex may modulate the therapeutic response by acting on multiple cell types in vivo. Whether and how Dex enhances the tumor intrinsic clinical response to IMiDs is not well understood. To address this important question, we focus on the role of Dex in modulating the proximal events in IMiD signaling that lead to the killing of myeloma cells.Cereblon (CRBN), a substrate receptor of the CRL4CRBN E3 ligase, is required for IMiD's anti-myeloma activity. MEIS2 is a homeobox transcription factor of the mammalian myeloid ecotropic insertion site (MEIS) family, which plays a pivotal role in development and leukemogenesis. Recent crystal structure studies in conjunction with biochemical screen have identified MEIS2 as an endogenous substrate of CRBN. IMiDs apparently bind CRBN and block MEIS2 from binding to CRBN, thereby facilitating the recruitment of transcription factors IKZF1 and IKZF3 to CRL4CRBN for degradation. This then leads to loss of IRF4, a target of IKZF1/3, necessary for myeloma survival. This model posits that MEIS2 inhibits the function of CRBN in myeloma cells. However, as all the studies of MEIS2 were performed heterologous cell lines, evidence that supports a function of MEIS2 in myeloma cells is lacking.We now showed by RNA-seq and protein analysis that MEIS2 was absent in normal bone marrow plasma cells (BMPC), but highly expressed in freshly isolated primary bone marrow myeloma cells (BMMC)s at both RNA and protein levels. We further showed that MEIS2 was regulated by the cell cycle: reduced in G1 arrest and restored in S phase in human myeloma cell lines (HMCL)s as well as primary BMMCs. Depletion of MEIS2 markedly enhanced lenalidomide killing, and overexpression of MEI2 attenuated lenalidomide killing of HMCL MM1.S. These results provide the first evidence that MEIS2 is aberrantly expressed in primary myeloma cells and acts within myeloma cells to inhibit IMiD killing.To investigate if Dex enhances the myeloma intrinsic response to IMiDs, we first determined that lenalidomide and pomolidomide induced late G1 arrest within 24 hours, as evidenced by the repression of cyclin A, the increases in p21 and p27 proteins, and the reduction of myeloma cells in S phase by BrdU labeling. Kinetic studies further indicate that induction of G1 arrest precedes apoptosis, detectable at 72 hours in MM1.S and CAG cells. These data suggest that IMiDs preferentially kill myeloma cells arrested in G1. Dex killed CAG and MM1.S cells marginally at 10 nM and killing was not greater at 100 nM. However, concurrent addition of Dex enhanced lenalidomide killing, more prominently in CAG cells then in MM1.S cells. Addition of Dex at these concentrations to CAG cells after 48 hours of exposure to lenalidomide leads to synergistic and greater killing in 24 hours in a dose-dependent manner. These data indicate that Dex enhances lenalidomide killing, preferentially in G1 arrest.To investigate the mechanism by which Dex enhances lenalidomide killing, we found that Dex did not regulate MEIS2 or CRBN protein expression in CAG cells, in line with its inability to significantly kill myeloma cells. Sequential addition of lenalidomide and Dex led to a > 2-fold reduction in the MEIS2 protein, which corresponds to a > 2-fold reduction in the MEIS2/CRBN ratio and synergistic killing. Concurrent addition of Dex and lenalidomide also resulted in a 2-fold reduction of the MEIS2 protein and the MEIS2/CRBN ratio, but it required 72 hours of the presence of Dex instead of 24 hours.In summary, our study demonstrates, for the first time, that MEIS2 is expressed in primary myeloma cells and inhibits IMiDs killing. Dex enhances IMiD killing of myeloma cells through cooperative suppression of MEIS2 at proximal IMiD signaling. These findings suggest a novel mechanism by which Dex enhances the anti-myeloma activity of IMiD, which has important implication for myeloma therapy. DisclosuresRossi:Celgene: Consultancy, Speakers Bureau; Onyx: Research Funding, Speakers Bureau; Takeda: Speakers Bureau; Janssen: Speakers Bureau. Niesvizky:Celgene: Consultancy, Research Funding, Speakers Bureau; Onyx: Consultancy, Research Funding, Speakers Bureau; Takeda: Consultancy, Research Funding, Speakers Bureau.

In vitro pharmacokinetics/pharmacodynamics of the combination of avibactam and aztreonam against MDR organisms.

The combination of aztreonam and avibactam has been proposed for the treatment of infections caused by metallo-β-lactamase-producing Gram-negative organisms, given the stability of aztreonam against metallo-β-lactamases plus the broad coverage of avibactam against AmpC β-lactamases and ESBLs. This study aimed to evaluate the efficacy of the combination against four clinical isolates with defined but diverse β-lactamase profiles. The MICs of aztreonam were determined without and with avibactam (1, 2, 4, 8 and 16 mg/L). Using the MIC values, the static time-kill kinetic studies were designed to encompass aztreonam concentrations of 0.25, 0.5, 1, 2 and 4 times the MIC at the respective avibactam concentrations from 0 to 8 mg/L. Aztreonam and avibactam concentrations were determined by LC-MS/MS during the course of the time-kill kinetic studies to evaluate whether avibactam protects aztreonam from degradation. Three of the four isolates had aztreonam MICs ≥128 mg/L in monotherapy. Dramatically increasing susceptibility associated with a decrease in aztreonam MIC was observed with increasing avibactam concentration. Against all isolates, the combinations resulted in greater killing with a much lower dose requirement for aztreonam. The resulting changes in base-10 logarithm of cfu/mL at both the 10 h and 24 h references (versus 0 h) were synergistic. In contrast, a significantly higher concentration of aztreonam in the monotherapy was required to produce the same kill as that in the combination therapy, due to rapid aztreonam degradation in two isolates. The aztreonam/avibactam combination protects aztreonam from hydrolysis and provides synergy in antimicrobial activity against multiple β-lactamase-expressing strains with a wide MIC range.

Inhibitory effect of 131I-labeled epidermal growth factor receptor-targeted liposome nanoparticle on EGFR-overexpressing cancer cells in vitro

Objective To construct 131I labeled anti-EGFR immunoliposome nanoparticle (131I-Cetuaximab (C225)-BSA-PCL), and investigate its inhibitory effect on EGFR-overexpressing cancer cells in vitro. Methods Anti-EGFR liposome nanoparticle C225-BSA-PCL and non-targeted liposomes BSA-PCL were constructed. The products were observed with transmission electron microscopy and dynamic light scattering. The EGFR-targeted binding and cellular uptake in EGFR-overexpressing cancer cells were observed with flow cytometry and confocal microscopy. Anti-EGFR and non-targeted liposomes were labeled with 131I using the chloramine-T method. The targeted cell killing effects of 131I labeled liposomes were analyzed using MTT assay. The time-dependent cellular uptake analysis was used to evaluate the slow-release effects of the 131I labeled liposomes. The independent-samples t test was used for data analysis. Results The EGFR-targeted liposome C225-BSA-PCL and non-targeted liposome BSA-PCL were successfully constructed, and the effective diameters were approximately 130-180 nm. Flow cytometry and confocal microscopy revealed significant uptake of C225-BSA-PCL in EGFR-overexpressing tumor cells. BSA-PCL could also bind to cells with minimal and weak tumor retention. The EGFR-targeted radioactive liposome 131I-C225-BSA-PCL showed greater targeted cell killing effect than non-targeted liposome 131I-BSA-PCL, the IC50 values of 131I-C225-BSA-PCL and 131I-BSA-PCL were 0.03-1.32 and 0.25-12.19, respectively. The uptakes of 131I-C225-BSA-PCL was higher than that of 131I-BSA-PCL (t=3.03-16.86, all P<0.05) and reached the maximal level at 4 h after incubation. Conclusions The EGFR-targeted liposome C225-BSA-PCL demonstrated superior cellular binding and uptake on EGFR-overexpressing cancer cells compared with BSA-PCL. The EGFR-targeted radioactive liposome 131I-C225-BSA-PCL had favorable intracellular retention and excellent targeted cell killing effect, and could effectively suppress the growth of EGFR-overexpressing cancer cells. Key words: Liposomes; Nanotechnology; Receptors, epidemal growth factor-urogastrone; Iodine radioisotopes; Tumor cells, cultured

The cytotoxity of paclitaxel-nanoparticles with dual ligands combined with ultrasound irradiation on drug-resistant breast cancer cells

Objective To screening optimized ultrasound (US) condition for MCF-7 cells and MCF-7/Tax cells, and to investigate the effect of dual-ligands nanoparticles (NPs) combined with US on overcoming multidrug resistance (MDR). Methods To obtain optimized US condition, cell viability and cellular uptake of NPs treated with MCF-7 cells and MCF-7/Tax cells were detected under different ultrasonic parameters including ultrasound intensity, exposure duration and microbubble concentration. MTT assay was performed to observe the effect of NPs combined with US on cells. Results The optimal US conditions for MCF-7 cells was irradiation intensity 0.6 W/cm2, irradiation time 80 s, and the MBs concentration with volume ratio of microbubbles to medium was 1∶10; and for MCF-7/Tax cells, irradiation intensity 0.6 W/cm2, irradiation time 80 s, and the MBs concentration with volume ratio of microbubbles to medium was 1∶20. Compared with individual NPs, NPs assisted by US exhibited greater killing ability for MCF-7 (IC50 1.43 ng/ml) and MCF-7/Tax cells (IC50<800 ng/ml). Conclusions US effect could enhance drug sensitivity of the dual-functionized NPs for MCF-7/Tax cells, indicating that US irradiation had potential assisted modality to reverse tumor MDR. Key words: Ultrasonography; Microbubbles; Multidrug resistance; Dual-ligands nanoparticles; Breast neoplasms

An Acidic Microenvironment Increases NK Cell Killing of Cryptococcus neoformans and Cryptococcus gattii by Enhancing Perforin Degranulation

Cryptococcus gattii and Cryptococcus neoformans are encapsulated yeasts that can produce a solid tumor-like mass or cryptococcoma. Analogous to malignant tumors, the microenvironment deep within a cryptococcoma is acidic, which presents unique challenges to host defense. Analogous to malignant cells, NK cells kill Cryptococcus. Thus, as in tumor defense, NK cells must kill yeast cells across a gradient from physiologic pH to less than 6 in the center of the cryptococcoma. As acidic pH inhibits anti-tumor activities of NK cells, we sought to determine if there was a similar reduction in the anticryptococcal activity of NK cells. Surprisingly, we found that both primary human NK cells and the human NK cell line, YT, have preserved or even enhanced killing of Cryptococcus in acidic, compared to physiological, pH. Studies to explore the mechanism of enhanced killing revealed that acidic pH does not increase the effector to target ratio, binding of cytolytic cells to Cryptococcus, or the active perforin content in effector cells. By contrast, perforin degranulation was greater at acidic pH, and increased degranulation was preceded by enhanced ERK1/2 phosphorylation, which is essential for killing. Moreover, using a replication defective ras1 knockout strain of Cryptococcus increased degranulation occurred during more rapid replication of the organisms. Finally, NK cells were found intimately associated with C. gattii within the cryptococcoma of a fatal infection. These results suggest that NK cells have amplified signaling, degranulation, and greater killing at low pH and when the organisms are replicating quickly, which would help maintain microbicidal host defense despite an acidic microenvironment.

Abstract 3515: Maitake D-Fraction, a natural mushroom extract, synergizes with Interleukin-2 for increased lytic activity of peripheral blood mononuclear cells against various human tumor cell histologies

Abstract Maitake (Grifola frondosa) has been recognized by naturopathic physicians as a medicinal mushroom capable of immune support. Maitake D-Fraction (MDF), a component of the maitake mushroom isolated during hot-water extraction, is rich in α-Glucans and commonly prescribed as a dietary supplement. Because MDF is widely available and easily tolerated, it is highly desirable for the cancer patient looking for immune support via natural products. Previous research has suggested that a dietary MDF regimen can enhance immune function in humans. However, studies to investigate the capacity of MDF to increase tumor immunity are lacking. This study was designed to investigate whether human peripheral blood mononuclear cells (PBMC) treated with MDF develop increased cytolytic capacity against human tumor cells. Cytolysis mediated by MDF-activated PBMC was compared to that of naïve PBMC (media control) and PBMC that were treated with interleukin-2 (IL-2). Immune mediated tumor cytolysis was assessed used a standard Chromium-Release Assay and expressed as number of lytic units (LU)/10^7 effector cells. Target cells for the assays included the PANC1 pancreatic cancer cell line (PA) purchased from ATCC, and single cell suspensions created from peritoneal carcinomatosis (PC) and renal cell carcinoma (RC) tumors resected from patients. Against the PA cells, the LU observed were 16.9 (media control), 12.9 (1ug/ml MDF), 23.8 (10ug/ml MDF) and 28.9 (100ug/ml MDF). The corresponding LU values against PC cells were 7.6, –13.4, –14.2, and –12.1, respectively, and against RC cells 12.3, 2.7, 2.0, and 8.2, respectively. Predictably, PBMC stimulated with an optimally activating dose of IL2 (200U/ml) exhibited a marked increase in LU against all target cells. Nevertheless, when PBMC were stimulated concurrently with both IL-2 and MDF activation was increased further. Thus, LU against PA target cells were 73.0 for PBMC treated with IL-2 alone, and 110.6 for PBMC treated with IL-2 plus 100ug/ml MDF. The corresponding LU values against PC target cells were 37.1 (IL-2 alone), and 66.4 (IL-2 plus 100ug/ml MDF) and against RC target were 94.0 (IL-2 alone) and 110.7 (IL-2 plus 100ug/ml MDF). This study shows that MDF has the capacity to increase the lytic activity produced by an optimally activating dose of IL-2, eliciting greater killing of a variety of human cancer cells than what can be achieved with IL2 alone. It is reasonable to assume that MDF can elicit a similar effect at lower doses of IL-2. Given the toxicity of IL-2 observed in humans, the potential to elicit the desired immune stimulation by combining lower doses of IL-2 with MDF becomes highly desirable. Future clinical trials should be designed to investigate the ability of MDF to elicit optimal immune stimulation with more tolerable doses and schedules of IL-2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3515. doi:1538-7445.AM2012-3515

Synthesis and structure-activity study of quaternary ammonium functionalized β-cyclodextrin-carboxymethylcellulose polymers

Carboxymethylcellulose (CMC) and beta-cyclodextrin (beta-CD)-based polymers functionalized with two types of quaternary ammonium compounds (QACs), the alkaquat DMB-451 (N-alkyl (50% C14, 40% C12, 10% C10) dimethylbenzylammonium chloride) (DMD-451) named polymer DMB-451, and FMB 1210-8 (a blend of 32 w% N-alkyl (50% C14, 40% C12, 10% C10) dimethylbenzylammonium chloride and 48 w% of didecyldimethylammonium chloride) named polymer FMB 1210-8, were synthethized and characterized by Fourier transform infrared spectroscopy. The antimicrobial activities of these polymers against Eschericia coli were also evaluated at 25 degrees C in wastewater. The results have indicated that the polymer FMB 1210-8 possesses a high-affinity binding with bacterial cells that induces a rapid disinfection process. Moreover, in the same experimental conditions of disinfection (mixture of 1.0 g of polymer and 100 mL of wastewater), the polymer FMB 1210-8 has a higher antimicrobial efficiency (99.90%) than polymer DMB-451 (92.8%). This phenomenon might be associated to a stronger interaction with bacterial cells due to stronger binding affinity for E. coli cells and greater killing efficiency of the C10 alkyl chains QAC of polymer FMB 1210-8 to disrupt the bacterial cell membrane as compared to N-alkyl (50% C14, 40% C12, 10% C10) dimethylbenzylammonium chloride. Together, these results suggest that the polymer FMB 1210-8 could constitute a good disinfectant against Escherichia coli, which could be advantageously used in wastewater treatments due to the low toxicity of beta-CD and CMC, and moderated toxicity of FMB 1210-8 to human and environment.

Activity of moxifloxacin against intracellular community-acquired methicillin-resistant Staphylococcus aureus: comparison with clindamycin, linezolid and co-trimoxazole and attempt at defining an intracellular susceptibility breakpoint

Co-trimoxazole, clindamycin and linezolid are used to treat community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infections, but little is known about intracellular activity. Moxifloxacin is active against intracellular methicillin-susceptible S. aureus (MSSA), but CA-MRSA has not been studied. We used 12 clinical CA-MRSA, 1 MSSA overexpressing norA and 2 hospital-acquired MRSA (moxifloxacin MICs: 0.03 to 4 mg/L). Activity was assessed in broth and after phagocytosis by THP-1 macrophages or keratinocytes {concentration-dependent experiments [24 h of incubation] to determine relative potencies [EC(50)], static concentrations [C(s)] and maximal relative efficacies [E(max) (change in log(10) cfu compared with initial inoculum)] and time-dependent experiments [0-72 h] at human C(max)}. Concentration-dependent experiments: in broth, EC(50) and C(s) were correlated with the MIC for all antibiotics, but moxifloxacin achieved significantly (P < 0.01) greater killing (more negative E(max)) than the comparators; and in THP-1 cells and keratinocytes, moxifloxacin acted more slowly but still reached a near bactericidal effect (2 to 3 log(10) cfu decrease) at 24 h with unchanged EC(50) and C(s) as long as its MIC was ≤0.125 mg/L (recursive partitioning analysis). Clindamycin and linezolid were static, and co-trimoxazole was unable to suppress the intracellular growth of CA-MRSA. At human C(max) in broth, moxifloxacin killed more rapidly and more extensively (≥5 log(10) cfu decrease at 10 h) than clindamycin (4 log(10) cfu at 48 h) or co-trimoxazole and linezolid (1-2 log(10) cfu at 72 h). Moxifloxacin is active against both extracellular and intracellular CA-MRSA if the MIC is low, and is more effective than clindamycin, co-trimoxazole and linezolid.

The antitumor effects of OSU-HDAC42, a novel histone deacetylase inhibitor, in HER-2-positive breast cancer

e14587 Background: Elevated HER-2/neu expression in primary breast tumors is associated with frequent relapse and poor prognosis. For this reason, HER-2/neu has been actively pursued as a target for novel therapeutic agents. Histone deacetylase inhibitors (HDACi), a new class of antitumor drugs, can downregulate HER2 through hyperacetylation of heat shock protein 90 (hsp90), an important ATP-dependent chaperone that mediates the stability and maturation of a variety of important oncogenic proteins, including HER2, Akt and ERα. In this study, we assess the effects of OSU-HDAC42, a novel orally bioavailable phenylbutyrate-based HDAC inhibitor, on hsp90 and HER2 downregulation. Methods: OSU-HDAC42 was tested for effects on the human breast cancer cell lines BT474, SKBR3, MDA-MB-231 and MCF-7. Assays of cell viability, cell cycle, apoptosis, HER2 expression, phosphorylation, and hsp90 acetylation were performed. Tumor growth was assessed in an orthotopic HER2+ mammary tumor model, using diets ± OSU-HDAC42. Results: SKBR3 (HER2+, ERα-) was the most susceptible to the antiproliferative effects of OSU-HDAC42 after 72 hours of treatment (IC50 = 0.025 μmol/L), followed by BT474 (HER2+, ERα+), MCF-7 (HER2-, ERα+) and MDA-MB-231 (HER2-, ERα-) cells with IC50 values calculated at 0.16, 0.19 and 0.2 μmol/L, respectively, which correlated with the level of inhibition of Hsp90 client protein expression (HER2, Akt). OSU-HDAC42 yields 43- or 65- fold greater cell killing than suberoylanilide hydroxamic acid (SAHA; vorinostat) or MS-275, respectively, and more potently suppresses the levels of Hsp90 client proteins (HER2, ERα and Akt) and induction of apoptosis in HER2+ breast cancer cells. In vivo administration of OSU-HDAC42 resulted in reductions of 76% and 82% in NT5 (HER2+,ER-) tumor mass and volume, respectively, concomitant with tubulin hyperacetylation, increased PARP cleavage, and decreased HER2 levels in OSU-HDAC42 treated mouse tumors. Conclusions: OSU-HDAC42 is a potent inhibitor of HER2+ breast cancer, mediated in part through hsp90 downregulation. As an orally bioavailable HDAC inhibitor with greater potency than other clinically available HDACi agents, OSU- HDAC42 warrants evaluation in clinical trials as a novel therapy for HER2+ breast cancer. [Table: see text]

Galleria mellonella as a Model System To Study Acinetobacter baumannii Pathogenesis and Therapeutics

Nonmammalian model systems of infection such as Galleria mellonella (caterpillars of the greater wax moth) have significant logistical and ethical advantages over mammalian models. In this study, we utilize G. mellonella caterpillars to study host-pathogen interactions with the gram-negative organism Acinetobacter baumannii and determine the utility of this infection model to study antibacterial efficacy. After infecting G. mellonella caterpillars with a reference A. baumannii strain, we observed that the rate of G. mellonella killing was dependent on the infection inoculum and the incubation temperature postinfection, with greater killing at 37 degrees C than at 30 degrees C (P = 0.01). A. baumannii strains caused greater killing than the less-pathogenic species Acinetobacter baylyi and Acinetobacter lwoffii (P < 0.001). Community-acquired A. baumannii caused greater killing than a reference hospital-acquired strain (P < 0.01). Reduced levels of production of the quorum-sensing molecule 3-hydroxy-C(12)-homoserine lactone caused no change in A. baumannii virulence against G. mellonella. Treatment of a lethal A. baumannii infection with antibiotics that had in vitro activity against the infecting A. baumannii strain significantly prolonged the survival of G. mellonella caterpillars compared with treatment with antibiotics to which the bacteria were resistant. G. mellonella is a relatively simple, nonmammalian model system that can be used to facilitate the in vivo study of host-pathogen interactions in A. baumannii and the efficacy of antibacterial agents.

Pseudomonas aeruginosa Serious Infections: Mono or Combination Antimicrobial Therapy?

P. aeruginosa is a serious cause of infection with reported rates of mortality being up to 61%. Several studies evidenced a correlation between hospital mortality due to P. aeruginosa bloodstream infections and an inappropriate antimicrobial treatment. Increasing resistance in P. aeruginosa isolates complicates the selection of adequate empirical therapy in severe infections and P. aeruginosa is often indistinguishable from other gram-negative bacterial infections. For these reasons, present guidelines for the treatment of suspected P. aeruginosa bacteraemia recommend the rapid introduction of empirical antimicrobial therapy that includes at least one antipseudomonal agent until having microbiological results. Current consensus favours the use of empirical combination, balancing the potential for greater toxicity against the lower emergence of antimicrobial resistance and the greater killing that might be achieved by combination therapies acting synergistically. Advantages and disadvantages of combination therapy towards monotherapy for P. aeruginosa severe infections, current antibiotics used for P. aeruginosa severe infections and main studies published on this issue are reviewed.

Apoptosis Mechanisms Related to the Increased Sensitivity of Jurkat T‐cells vs A431 Epidermoid Cells to Photodynamic Therapy with the Phthalocyanine Pc 4†‡

To examine the clinical applicability of Pc 4, a promising second-generation photosensitizer, for the photodynamic treatment of lymphocyte-mediated skin diseases, we studied the A431 and Jurkat cell lines, commonly used as surrogates for human keratinocyte-derived carcinomas and lymphocytes, respectively. As revealed by ethyl acetate extraction and absorption spectrophotometry, uptake of Pc 4 into the two cell lines was linear with Pc 4 concentration and similar on a per cell basis but greater in Jurkat cells on a per mass basis. Flow cytometry showed that uptake was linear at low doses; variations in the dose-response for uptake measured by fluorescence supported differential aggregation of Pc 4 in the two cell types. As detected by confocal microscopy, Pc 4 localized to mitochondria and endoplasmic reticulum in both cell lines. Jurkat cells were much more sensitive to the lethal effects of phthalocyanine photodynamic therapy (Pc 4-PDT) than were A431 cells, as measured by a tetrazolium dye reduction assay, and more readily underwent morphological apoptosis. In a search for molecular factors to explain the greater photosensitivity of Jurkat cells, the fate of important Bcl-2 family members was monitored. Jurkat cells were more sensitive to the induction of immediate photodamage to Bcl-2, but the difference was insufficient to account fully for their greater sensitivity. The antiapoptotic protein Mcl-1 was extensively cleaved in a dose- and caspase-dependent manner in Jurkat, but not in A431, cells exposed to Pc 4-PDT. Thus, the greater killing by Pc 4-PDT in Jurkat compared with A431 cells correlated with greater Bcl-2 photodamage and more strongly to the more extensive Mcl-1 degradation. Pc 4-PDT may offer therapeutic advantages in targeting inflammatory cells over normal keratinocytes in the treatment of T-cell-mediated skin diseases, such as cutaneous lymphomas, dermatitis, lichenoid tissue reactions and psoriasis, and it will be instructive to evaluate the role of Bcl-2 family proteins, especially Mcl-1, in the therapeutic response.