Regulation and transcript analysis of the ceroperon responsible for quorum sensing in Rhodobacter sphaeroides 2.4.1

Rhodobacter sphaeroides 2.4.1 is a Gram negative facultative photoheterotrophic bacterium that has been shown to have an N-acyl homoserine lactone-based quorum sensing system called cer for c&barbelow;ommunity e&barbelow;scape r&barbelow;esponse. The cer ORFs are cerR, the transcriptional regulator, cerI, the autoinducer synthase and cerA , whose function is unknown. The autoinducer molecule, 7,8- cis-N-(tetradecenoyl) homoserine lactone, has been characterized. The objective of this study was to identify an environmental stimulus that influences the regulation of cerRAI and, to characterize transcription of the cer operon. ^ A cerR::lacZ transcriptional fusion was made and β-Galactosidase assays were performed in R. sphaeroides 2.4.1 strains, wild type, AP3 (CerI−) and AP4 (CerR−). The cerR::lacZ β-Galactosidase assays were used as an initial survey of the mode of regulation of the Cer system. A cerA::lacZ translational fusion was created and was used to show that cerA can be translated. The presence of 7,8-cis-N-(tetradecenoyl) homoserine lactone was detected from R. sphaeroides strains wild type and AP4 (CerR−) using a lasR::lacZ translational fusion autoinducer bioassay. The cerR::lacZ transcriptional fusion in R. sphaeroides 2.4.1 wild type was tested under different environmental stimuli, such as various carbon sources, oxygen tensions, light intensities and culture media to determine if they influence transcription of the cer ORFs. Although lacZ assay data implicated high light intensity at 100 W/m2 to stimulate cer transcription, quantitative Northern RNA data of the cerR transcript showed that low light intensity at 3 W/m2 is at least one environmental stimulus that induces cer transcription. This finding was supported by DNA microarray analysis. Northern analysis of the cerRAI transcript provided evidence that the cer ORFs are co-transcribed, and that the cer operon contains two additional genes. Bioinformatics was used to identify genes that may be regulated by the Cer system by identifying putative lux box homologue sequences in the presumed promoter region of these genes. Genes that were identified were fliQ, celB and calsymin, all implicated in interacting with plants. Primer extension was used to help localize cis-elements in the promoter region. The cerR::lacZ transcriptional fusion was monitored in a subset of different global DNA binding transcriptional regulator mutant strains of R. sphaeroides 2.4.1. Those regulators involved in maintaining an anaerobic photosynthetic lifestyle appeared to have an effect. Collectively, the data imply that R. sphaeroides 2.4.1 activates the Cer system when grown anaerobic photosynthetically at low light intensity, 3 W/m2, and it may be involved in an interaction with plants. ^

Comparison of OG1RF and an isogenic fsrB deletion mutant by transcriptional analysis: the Fsr system of Enterococcus faecalis is more than the activator of gelatinase and serine protease.

The FsrABC system of Enterococcus faecalis controls the expression of gelatinase and a serine protease via a quorum-sensing mechanism, and recent studies suggest that the Fsr system may also regulate other genes important for virulence. To investigate the possibility that Fsr influences the expression of additional genes, we used transcriptional profiling, with microarrays based on the E. faecalis strain V583 sequence, to compare the E. faecalis strain OG1RF with its isogenic mutant, TX5266, an fsrB deletion mutant. We found that the presence of an intact fsrB influences expression of numerous genes throughout the growth phases tested, namely, late log to early stationary phase. In addition, the Fsr regulon is independent of the activity of the proteases, GelE and SprE, whose expression was confirmed to be activated at all three time points tested. While expression of some genes (i.e., ef1097 and ef0750 to -757, encoding hypothetical proteins) was activated in late log phase in OG1RF versus the fsrB deletion mutant, expression of ef1617 to -1634 (eut-pdu orthologues) was highly repressed by the presence of an intact Fsr at entry into stationary phase. This is the first time that Fsr has been characterized as a negative regulator. The newly recognized Fsr-regulated targets include other factors, besides gelatinase, described as important for biofilms (BopD), and genes predicted to encode the surface proteins EF0750 to -0757 and EF1097, along with proteins implicated in several metabolic pathways, indicating that the FsrABC system may be an important regulator in strain OG1RF, with both positive and negative effects.

REGULATION OF TOXIN SYNTHESIS BY CLOSTRIDIUM DIFFICILE

Clostridium difficile is the leading definable cause of nosocomial diarrhea worldwide due to its virulence, multi-drug resistance, spore-forming ability, and environmental persistence. The incidence of C. difficile infection (CDI) has been increasing exponentially in the last decade. Virulent strains of C. difficile produce either toxin A and/or toxin B, which are essential for the pathogenesis of this bacterium. Current methods for diagnosing CDI are mostly qualitative tests that detect the bacterium, the toxins, or the toxin genes. These methods do not differentiate virulent C. difficile strains that produce active toxins from non-virulent strains that do not produce toxins or produce inactive toxins. Based on the knowledge that C. difficile toxins A and B cleave a substrate that is stereochemically similar to the native substrate of the toxins, uridine diphosphoglucose, a quantitative, cost-efficient assay, the Cdifftox activity assay, was developed to measure C. difficile toxin activity. The concept behind the activity assay was modified to develop a novel, rapid, sensitive, and specific assay for C. difficile toxins in the form of a selective and differential agar plate culture medium, the Cdifftox Plate assay (CDPA). This assay combines in a single step the specific identification of C. difficile strains and the detection of active toxin(s). The CDPA was determined to be extremely accurate (99.8% effective) at detecting toxin-producing strains based on the analysis of 528 C. difficile isolates selected from 50 tissue culture cytotoxicity assay-positive clinical stool samples. This new assay advances and improves the culture methodology in that only C. difficile strains will grow on this medium and virulent strains producing active toxins can be differentiated from non-virulent strains. This new method reduces the time and effort required to isolate and confirm toxin-producing C. difficile strains and provides a clinical isolate for antibiotic susceptibility testing and strain typing. The Cdifftox activity assay was used to screen for inhibitors of toxin activity. Physiological levels of the common human conjugated bile salt, taurocholate, was found to inhibit toxin A and B in vitro activities. When co-incubated ex vivo with purified toxin B, taurocholate protected Caco-2 colonic epithelial cells from the damaging effects of the toxin. Furthermore, using a caspase-3 detection assay, taurocholate reduced the extent of toxin B-induced Caco-2 cell apoptosis. These results suggest that bile salts can be effective in protecting the gut epithelium from C. difficile toxin damage, thus, the delivery of physiologic amounts of taurocholate to the colon, where it is normally in low concentration, could be useful in CDI treatment. These findings may help to explain why bile rich small intestine is spared damage in CDI, while the bile salt poor colon is vulnerable in CDI. Toxin synthesis in C. difficile occurs during the stationary phase, but little is known about the regulation of these toxins. It was hypothesized that C. difficile toxin synthesis is regulated by a quorum sensing mechanism. Two lines of evidence supported this hypothesis. First, a small (KDa), diffusible, heat-stable toxin-inducing activity accumulates in the medium of high-density C. difficile cells. This conditioned medium when incubated with low-density log-phase cells causes them to produce toxin early (2-4 hrs instead of 12-16 hrs) and at elevated levels when compared with cells grown in fresh medium. These data suggested that C. difficile cells extracellularly release an inducing molecule during growth that is able to activate toxin synthesis prematurely and demonstrates for the first time that toxin synthesis in C. difficile is regulated by quorum signaling. Second, this toxin-inducing activity was partially purified from high-density stationary-phase culture supernatant fluid by HPLC and confirmed to induce early toxin synthesis, even in C. difficile virulent strains that over-produce the toxins. Mass spectrometry analysis of the purified toxin-inducing fraction from HPLC revealed a cyclic compound with a mass of 655.8 Da. It is anticipated that identification of this toxin-inducing compound will advance our understanding of the mechanism involved in the quorum-dependent regulation of C. difficile toxin synthesis. This finding should lead to the development of even more sensitive tests to diagnose CDI and may lead to the discovery of promising novel therapeutic targets that could be harnessed for the treatment C. difficile infections.

Mechanism of anti-tumor activity of adenovirus type 5 E1A in ovarian cancer

The adenovirus type 5 E1A gene was originally developed as a gene therapy to inhibit tumorigenicity of HER-2-overexpressing cells by transcriptional downregulation of HER-2. Our goal is to improve the overall efficacy of E1A gene therapy. To achieve this goal, we have conducted two preclinical experiments. ^ First, we hypothesized that Bcl-2 overexpressing ovarian cancer is resistant to E1A gene therapy. This hypothesis is based on that the 19 kDa protein product of the adenoviral E1B gene which is homologous to Bcl-2 inhibits E1A-induced apoptosis. Treating high Bcl-2-xpressing cells with E1A in combination with an antisense oligonucleotide to Bcl-2 (Bcl-2-ASO) resulted in a significant decrease in cell viability due to an increased rate of apoptosis relative to cells treated with E1A alone. In an ovarian cancer xenograft model, mice implanted with low HER-2, high Bcl-2 cells, treated with E1A plus Bcl-2-ASO led to prolonged survival. Bcl-2 thus may serve as a predictive molecular marker enabling us to select patients with ovarian cancer who will benefit significantly from E1A gene therapy. ^ Second, we elucidated the molecular mechanism governing the anti-tumor effect of E1A in ovarian cancer to identify a more potent tumor suppressor gene. We identified PEA-15 (phospho-protein enriched in astrocytes) upregulated in E1A transfected low HER-2-expressing OVCAR-3 ovarian cancer cell, which showed decreased cell proliferation. PEA-15 moved ERK from the nucleus to the cytoplasm and inhibited ERK-dependent transcription and proliferation. Using small interfering RNA to knock down PEA-15 expression in OVCAR-3 cells made to constitutively express E1A resulted in accumulation of phosphoERK in the nucleus, an increase in Elk-1 activity, DNA synthesis, and anchorage-independent growth. PEA-15 also independently suppressed colony formation in some breast and ovarian cancer cell lines in which E1A is known to have anti-tumor activity. We conclude that the anti-tumor activity of E1A depends on PEA-15. ^ In summary, (1) Bcl-2 may serve as a predictive molecular marker of E1A gene therapy, allowing us to select patients and improve efficacy of E1A gene therapy. (2) PEA-15 was identified as a component of the molecular mechanism governing the anti-tumor activity of E1A in ovarian cancer, (3) PEA-15 may be developed as a novel therapeutic gene. ^

Safety, pharmacokinetics, and antiretroviral activity of multiple doses of ibalizumab (formerly TNX-355), an anti-CD4 monoclonal antibody in HIV-1 infected adults

Context: Despite tremendous strides in HIV treatment over the past decade, resistance remains a major problem. A growing number of patients develop resistance and require new therapies to suppress viral replication. ^ Objective: To assess the safety of multiple administrations of the anti-CD4 receptor (anti-CD4) monoclonal antibody ibalizumab given as intravenous (IV) infusions, in three dosage regimens, in subjects infected with human immunodeficiency virus (HIV-1). ^ Design: Phase 1, multi-center, open-label, randomized clinical trial comparing the safety, pharmacokinetics and antiviral activity of three dosages of ibalizumab. ^ Setting: Six clinical trial sites in the United States. ^ Participants: A total of twenty-two HIV-positive patients on no anti-retroviral therapy or a stable failing regimen. ^ Intervention: Randomized to one of two treatment groups in Arms A and B followed by non-randomized enrollment in Arm C. Patients randomized to Arm A received 10 mg/kg of ibalizumab every 7 days, for a total of 10 doses; patients randomized to Arm B received a total of six doses of ibalizumab; a single loading dose of 10 mg/kg on Day 1 followed by five maintenance doses of 6 mg/kg every 14 days, starting at Week 1. Patients assigned to Arm C received 25 mg/kg of ibalizumab every 14 days for a total of 5 doses. All patients were followed for safety for an additional 7 to 8 weeks. ^ Main Outcome Measures: Clinical and laboratory assessments of safety and tolerability of multiple administrations of ibalizumab in HIV-infected patients. Secondary measures of efficacy include HIV-1 RNA (viral load) measurements. ^ Results: 21 patients were treatment-experienced and 1 was naïve to HIV therapy. Six patients were failing despite therapy and 15 were on no current HIV treatment. Mean baseline viral load (4.78 log 10; range 3.7-5.9) and CD4+ cell counts (332/μL; range 89-494) were similar across cohorts. Mean peak decreases in viral load from baseline of 0.99 log10(1.11 log10, and 0.96 log 10 occurred by Wk 2 in Cohorts A, B and C, respectively. Viral loads decreased by >1.0 log10 in 64%; 4 patients viral loads were suppressed to < 400 copies/mL. Viral loads returned towards baseline by Week 9 with reduced susceptibility to ibalizumab. CD4+ cell counts rose transiently and returned toward baseline. Maximum median elevations above BL in CD4+ cell counts for Cohorts A, B and C were +257, +198 and +103 cells/μL, respectively and occurred within 3 Wks in 16 of 22 subjects. The half-life of ibalizumab was 3-3.5 days and elimination was characteristic of capacity-limited kinetics. Administration of ibalizumab was well tolerated. Four serious adverse events were reported during the study. None of these events were related to study drug. Headache, nausea and cough were the most frequently reported treatment emergent adverse events and there were no laboratory abnormalities related to study drug. ^ Conclusions: Ibalizumab administered either weekly or bi-weekly was safe, well tolerated, and demonstrated antiviral activity. Further studies with ibalizumab in combination with standard antiretroviral treatments are warranted.^

GMZ27 is a new organic arsenic derivative with anti-leukemic activity, via the induction of reactive oxygen species through NADPH oxidase and mitochondrial pathway that lead to leukemia cell apoptosis

Arsenic trioxide (ATO) is an inorganic arsenic derivative that is very effective against relapsed acute promyelocytic leukemia. It is being investigated as therapy for other cancers, but the risk/benefit ratio is questionable due to significant side effects. In contrast, organic arsenic derivatives (OAD) are known to be much less toxic than ATO. Based on high activity, we selected GMZ27 (dipropil-s-glycerol arsenic) for further study and have confirmed its potent activity against human acute leukemia cell lines. This anti-leukemic activity is significantly higher than that of ATO. Both in vivo and in vitro tests have shown that GMZ27 is significantly less toxic to normal bone marrow mononuclear cells and normal mice. Therefore, further study of the biological activity of GMZ27 was undertaken. ^ GMZ27, in contrast to ATO, can only marginally induce maturation of leukemic cells. GMZ27 has no effect on cell cycle. The anti-leukemic activity of GMZ27 against acute myeolocytic leukemia cells is not dependent upon degradation of PML-RARα fusion protein. GMZ27 causes dissipation of mitochondrial transmembrane potential, cleavage of caspase 9, caspase 3 activation. Further studies indicated that GMZ27 induces intracellular reactive oxygen species (ROS) production, and modification of intracellular ROS levels had profound effect on its potential to inhibit proliferation of leukemic cells. Therefore ROS production plays a major role in the anti-leukemic activity of GMZ27. ^ To identify how GMZ27 induces ROS, our studies focused on mitochondria and NADPH oxidase. The results indicated that the source of ROS generation induced by GMZ27 is dose dependent. At the low dose (0.3 uM) GMZ27 induces NADPH oxidase activity that leads to late ROS production, while at the high dose (2.0 uM) mitochondria function is disrupted and early ROS production is induced leading to dramatic cell apoptosis. Therefore, late, ROS production can be detected in mitochondria are depleted Rho-0 cells. Our work not only delineates a major biologic pathway for the anti-leukemic activity of GMZ27, but also discusses possible ways of enhancing the effect by the co-application of NADPH oxidase activator. Further study of this interaction may lead to achieving better therapeutic index.^