A fluorescent probe for monitoring PTP-PEST enzymatic activity.

Phosphatase non-receptor type 12 (PTPN12 or PTP-PEST) is a critical regulator of cell migration, acting as a tumor suppressor in cancer. Decreases in PTP-PEST expression correlate with aggressive phenotypes in hepatocellular carcinoma (HCC). Despite the importance of PTP-PEST in cellular signaling, methods to directly monitor its enzymatic activity are lacking. Herein, we report the design, synthesis, and optimization of a probe to directly monitor PTP-PEST enzymatic activity via a fluorescent readout. This activity sensor, termed pPEST1tide, is capable of detecting as little as 0.2 nM recombinant PTP-PEST. In addition, we demonstrate that this probe can selectively report on PTP-PEST activity using a panel of potential off-target enzymes. In the long-term, this activity probe could be utilized to identify small molecule modulators of PTP-PEST activity as well as provide a prognostic readout for HCC.

An Evolutionary Strategy for Identification of Higher Order, Green Fluorescent Host-Guest Pairs Compatible with Living Systems.

Engineered miniprotein host-small-molecule guest pairs could be utilized to design new processes within cells as well as investigate fundamental aspects of cell signaling mechanisms. However, the development of host-guest pairs capable of functioning in living systems has proven challenging. Moreover, few examples of host-guest pairs with stoichiometries other than 2:1 exist, significantly hindering the ability to study the influence of oligomerization state on signaling fidelity. Herein, we present an approach to identify host-guest systems for relatively small green fluorescent guests by incorporation into cyclic peptides. The optimal host-guest pair produced a 10-fold increase in green fluorescence signal upon binding. Biophysical characterization clearly demonstrated higher order supramolecular assembly, which could be visualized on the surface of living yeast cells using a turn-on fluorescence readout. This work further defines evolutionary design principles to afford host-guest pairs with stoichiometries other than 2:1 and enables the identification of spectrally orthogonal host-guest pairs.

Design and synthesis of fluorescent activity probes for protein phosphatases.

Protein phosphatases act in concert with protein kinases to regulate and maintain the phosphoproteome. However, the catalog of chemical tools to directly monitor the enzymatic activity of phosphatases has lagged behind their kinase counterparts. In this chapter, we provide protocols for repurposing the phosphorylation-sensitive sulfonamido-oxine fluorophore known as Sox to afford direct activity probes for phosphatases. With validated activity probes in-hand, inhibitor screens can be conducted with recombinant enzyme and the role of phosphatases in cell signaling can be investigated in unfractionated cell lysates.

Interrogating Protein Phosphatases with Chemical Activity Probes.

Protein phosphatases, while long overlooked, have recently become appreciated as drivers of both normal- and disease-associated signaling events. As a result, the spotlight is now turning torwards this enzyme family and efforts geared towards the development of modern chemical tools for studying these enzymes are well underway. This Minireview focuses on the evolution of chemical activity probes, both optical and covalent, for the study of protein phosphatases. Small-molecule probes, global monitoring of phosphatase activity through the use of covalent modifiers, and targeted fluorescence-based activity probes are discussed. We conclude with an overview of open questions in the field and highlight the potential impact of chemical tools for studying protein phosphatases.

Design and evaluation of a real-time activity probe for focal adhesion kinase.

Focal adhesion kinase (FAK) has been identified as a potential therapeutic target for the treatment of metastatic cancers. Herein we describe the design, synthesis and optimization of a direct activity sensor for FAK and its application to screening FAK inhibitors. We find that the position of the sensing moiety, a phosphorylation-sensitive sulfonamido-oxine fluorophore, can dramatically influence the performance of peptide sensors for FAK. Real-time fluorescence activity assays using an optimized sensor construct, termed FAKtide-S2, are highly reproducible (Z' = 0.91) and are capable of detecting as little as 1 nM recombinant FAK. Utilizing this robust assay format, we define conditions for the screening of FAK inhibitors and demonstrate the utility of this platform using a set of well-characterized small molecule kinase inhibitors. Additionally, we provide the selectivity profile of FAKtide-S2 among a panel of closely related enzymes, identifying conditions for selectively monitoring FAK activity in the presence of off-target enzymes. In the long term, the chemosensor platform described in this work can be used to identify novel FAK inhibitor scaffolds and potentially assess the efficacy of FAK inhibitors in disease models.

Oral tolerance to cancer can be abrogated by T regulatory cell inhibition.

Oral administration of tumour cells induces an immune hypo-responsiveness known as oral tolerance. We have previously shown that oral tolerance to a cancer is tumour antigen specific, non-cross-reactive and confers a tumour growth advantage. We investigated the utilisation of regulatory T cell (Treg) depletion on oral tolerance to a cancer and its ability to control tumour growth. Balb/C mice were gavage fed homogenised tumour tissue--JBS fibrosarcoma (to induce oral tolerance to a cancer), or PBS as control. Growth of subcutaneous JBS tumours were measured; splenic tissue excised and flow cytometry used to quantify and compare systemic Tregs and T effector (Teff) cell populations. Prior to and/or following tumour feeding, mice were intraperitoneally administered anti-CD25, to inactivate systemic Tregs, or given isotype antibody as a control. Mice which were orally tolerised prior to subcutaneous tumour induction, displayed significantly higher systemic Treg levels (14% vs 6%) and faster tumour growth rates than controls (p<0.05). Complete regression of tumours were only seen after Treg inactivation and occurred in all groups--this was not inhibited by tumour feeding. The cure rates for Treg inactivation were 60% during tolerisation, 75% during tumour growth and 100% during inactivation for both tolerisation and tumour growth. Depletion of Tregs gave rise to an increased number of Teff cells. Treg depletion post-tolerisation and post-tumour induction led to the complete regression of all tumours on tumour bearing mice. Oral administration of tumour tissue, confers a tumour growth advantage and is accompanied by an increase in systemic Treg levels. The administration of anti-CD25 Ab decreased Treg numbers and caused an increase in Teffs. Most notably Treg cell inhibition overcame established oral tolerance with consequent tumor regression, especially relevant to foregut cancers where oral tolerance is likely to be induced by the shedding of tumour tissue into the gut.

Control and augmentation of long-term plasmid transgene expression in vivo in murine muscle tissue and ex vivo in patient mesenchymal tissue.

PURPOSE: In vivo gene therapy directed at tissues of mesenchymal origin could potentially augment healing. We aimed to assess the duration and magnitude of transene expression in vivo in mice and ex vivo in human tissues. METHODS: Using bioluminescence imaging, plasmid and adenoviral vector-based transgene expression in murine quadriceps in vivo was examined. Temporal control was assessed using a doxycycline-inducible system. An ex vivo model was developed and optimised using murine tissue, and applied in ex vivo human tissue. RESULTS: In vivo plasmid-based transgene expression did not silence in murine muscle, unlike in liver. Although maximum luciferase expression was higher in muscle with adenoviral delivery compared with plasmid, expression reduced over time. The inducible promoter cassette successfully regulated gene expression with maximum levels a factor of 11 greater than baseline. Expression was re-induced to a similar level on a temporal basis. Luciferase expression was readily detected ex vivo in human muscle and tendon. CONCLUSIONS: Plasmid constructs resulted in long-term in vivo gene expression in skeletal muscle, in a controllable fashion utilising an inducible promoter in combination with oral agents. Successful plasmid gene transfection in human ex vivo mesenchymal tissue was demonstrated for the first time.

Induction of effective antitumor response after mucosal bacterial vector mediated DNA vaccination with endogenous prostate cancer specific antigen.

PURPOSE: The induction of systemic immune responses against antigenic targets that are over expressed by cancer cells represents a powerful therapeutic strategy to target metastatic cancer. We generated specific antitumor immune responses in a murine model of prostate cancer by oral administration of an attenuated strain of Salmonella typhimurium containing a plasmid coding for murine prostate stem cell antigen. MATERIALS AND METHODS: Trafficking of S. typhimurium SL7207 in the initial 10 hours after gavage feeding was determined using a bacterial lux expressing strain and live bioluminescence imaging. For vaccination trials male C57 BL/6 mice were gavage fed SL7207/murine prostate stem cell antigen expressing plasmid or controls twice at 2-week intervals. One week after the last feeding the mice were challenged subcutaneously with TRAMPC1 murine prostate carcinoma cells. Tumor dynamics and animal survival were recorded. RESULTS: Clearance of bacterial vector from animals was complete 9 hours after feeding. Delivery of vector transformed with a firefly luciferase reporter plasmid resulted in maximal eukaryotic reporter gene expression in splenocytes 48 hours after feeding. Induction of tumor protective immunity was achieved by feeding the mice murine prostate stem cell antigen expressing plasmid bearing bacteria and greater than 50% of immunized mice remained tumor free. No significant toxicity was observed. Induction of T-helper type 1 immune responses was determined by measuring interferon-γ produced by splenocytes from vaccinated mice. When adoptively transferred to naive animals, splenocytes from vaccinated mice prevented tumor growth in 66% of challenged animals. CONCLUSIONS: Endogenous prostate cancer antigen gene delivery using a bacterial vector resulted in breaking immune tolerance to murine prostate stem cell antigen and significant retardation of tumor growth.

Sonoporation mediated immunogene therapy of solid tumors.

Development of gene-based therapies for the treatment of inherited and acquired diseases, including cancer, has seen renewed interest in the use of nonviral vectors coupled to physical delivery modalities. Low-frequency ultrasound (US), with a well-established record in a clinical setting, has the potential to deliver DNA efficiently, accurately and safely. Optimal in vivo parameters for US-mediated delivery of naked plasmid DNA were established using the firefly luciferase reporter gene construct. Optimized parameters were used to administer a therapeutic gene construct, coding for granulocyte-macrophage colony-stimulating factor (GM-CSF) and B7-1 costimulatory molecule, to growing murine fibrosarcoma tumors. Tumor progression and animal survival was monitored throughout the study and the efficacy of the US-mediated gene therapy determined and compared with an electroporation-based approach. Optimal parameters for US-mediated delivery of plasmid DNA to tumors were deduced to be 1.0 W/cm(2) at 20% duty cycle for 5 min (60 J/cm(2)). In vivo US-mediated gene therapy resulted in a 55% cure rate in tumor-bearing animals. The immunological response invoked was cell mediated, conferring resistance against re-challenge and resistance to tumor challenge after transfer of splenocytes to naïve animals. US treatment was noninjurious to treated tissue, whereas therapeutic efficacy was comparable to an electroporation-based approach. US-mediated delivery of an immune-gene construct to growing tumors was therapeutically effective. Sonoporation has the potential to be a major factor in the development of nonviral gene delivery approaches.

Optimised electroporation mediated DNA vaccination for treatment of prostate cancer.

BACKGROUND: Immunological therapies enhance the ability of the immune system to recognise and destroy cancer cells via selective killing mechanisms. DNA vaccines have potential to activate the immune system against specific antigens, with accompanying potent immunological adjuvant effects from unmethylated CpG motifs as on prokaryotic DNA. We investigated an electroporation driven plasmid DNA vaccination strategy in animal models for treatment of prostate cancer. METHODS: Plasmid expressing human PSA gene (phPSA) was delivered in vivo by intra-muscular electroporation, to induce effective anti-tumour immune responses against prostate antigen expressing tumours. Groups of male C57 BL/6 mice received intra-muscular injections of phPSA plasmid. For phPSA delivery, quadriceps muscle was injected with 50 microg plasmid. After 80 seconds, square-wave pulses were administered in sequence using a custom designed pulse generator and a custom-designed applicator with 2 needles placed through the skin central to the muscle. To determine an optimum treatment regimen, three different vaccination schedules were investigated. In a separate experiment, the immune potential of the phPSA vaccine was further enhanced with co- administration of synthetic CpG rich oligonucleotides. One week after last vaccination, the mice were challenged subcutaneously with TRAMPC1/hPSA (prostate cancer cell line stably expressing human PSA) and tumour growth was monitored. Serum from animals was examined by ELISA for anti-hPSA antibodies and for IFN gamma. Histological assessment of the tumours was also carried out. In vivo and in vitro cytotoxicity assays were performed with splenocytes from treated mice. RESULTS: The phPSA vaccine therapy significantly delayed the appearance of tumours and resulted in prolonged survival of the animals. Four-dose vaccination regimen provided optimal immunological effects. Co - administration of the synthetic CpG with phPSA increased anti-tumour responses, preventing tumour occurrence in 54% of treated animals. Vaccination with phPSA resulted in anti-hPSA Abs production and a significant production of IFN gamma was observed in immunised animals (p < 0.05). Immune responses were tumour specific and were transferable in adoptive T cell transfer experiments. CONCLUSIONS: This phPSA plasmid electroporation vaccination strategy can effectively activate tumour specific immune responses. Optimisation of the approach indicated that a four-dose regimen provided highest tumour protection. In vivo electroporation mediated vaccination is a safe and effective modality for the treatment of prostate cancer and has a potential to be used as a neo-adjuvant or adjuvant therapy.

International Society for Cell and Gene Therapy of Cancer 2009 Annual Meeting held in Cork, Ireland.

The International Society for Cell and Gene Therapy (ISCGT) of Cancer annual meeting was held from September 2 through September 4, 2009, in Cork, Ireland ( www.iscgt2009.com ). The conference was held in conjunction with the Irish Society for Gene and Cell Therapy third annual meeting, and brought together scientists and clinicians from around the world in a country developing its knowledge economy. Next year's ISCGT meeting will be held in Doha, the capital of Qatar ( www.iscgt.net ), from September 27 through September 29, 2010.

Prostate stem cell antigen DNA vaccination breaks tolerance to self-antigen and inhibits prostate cancer growth.

Prostate stem cell antigen (PSCA) is a cell surface antigen expressed in normal human prostate and over expressed in prostate cancer. Elevated levels of PSCA protein in prostate cancer correlate with increased tumor stage/grade, with androgen independence and have higher expression in bone metastases. In this study, the PSCA gene was isolated from the transgenic adenocarcinoma mouse prostate cell line (TRAMPC1), and a vaccine plasmid construct was generated. This plasmid PSCA (pmPSCA) was delivered by intramuscular electroporation (EP) and induced effective antitumor immune responses against subcutaneous TRAMPC1 tumors in male C57 BL/6 mice. The pmPSCA vaccination inhibited tumor growth, resulting in cure or prolongation in survival. Similarly, the vaccine inhibited metastases in PSCA expressing B16 F10 tumors. There was activation of Th-1 type immunity against PSCA, indicating the breaking of tolerance to a self-antigen. This immunity was tumor specific and was transferable by adoptive transfer of splenocytes. The mice remained healthy and there was no evidence of collateral autoimmune responses in normal tissues. EP-assisted delivery of the pmPSCA evoked strong specific responses and could, in neoadjuvant or adjuvant settings, provide a safe and effective immune control of prostate cancer, given that there is significant homology between human and mouse PSCA.

Tripartite meeting in gene and cell therapy, 2008: Irish Society for Gene and Cell Therapy, British Society for Gene Therapy, and International Society for Cell and Gene Therapy of Cancer.

The second annual meeting of the Irish Society for Gene and Cell Therapy was held in Cork, Ireland on May 15 and 16, 2008 (http://crr.ucc.ie/isgct/). The meeting was jointly organized with the British Society for Gene Therapy and the International Society for Cell and Gene Therapy of Cancer. Because of the location of the conference and the co-organization of this meeting with the British and International Gene Therapy societies, the meeting enjoyed a range of talks from some of the major leaders in the field. Particularly notable were the talented molecular and cell biologists from Ireland who have contributed cutting edge science to the field of gene therapy. Topics including cardiovascular disease, repair of single-gene disorders, and cancer gene therapy were discussed with presentations ranging from basic research to translation into the clinic. Here we describe some of the most exciting presentations and their potential impact on imminent clinical gene therapy trials.

Effective tumor treatment using optimized ultrasound-mediated delivery of bleomycin.

Bleomycin is a nonpermeant, hydrophilic macromolecule with a high intrinsic anticancer cytotoxicity. However, the cytotoxic potential of the drug is restricted by its low membrane permeability. Application of low-intensity ultrasound to growing tumors enhances intracellular delivery of bleomycin after IP or intratumoral administration, thereby potentiating its cytotoxicity. Optimization of ultrasound parameters for in-vivo bleomycin delivery was undertaken, and an effective antitumor effect was demonstrated in solid tumors of both murine and human cell lines. Cell death after treatment was shown to occur by an apoptotic mechanism. The results achieved in these experiments were equivalent to those achieved using electroporation to mediate delivery of bleomycin-electrochemotherapy. We found that, although temperature rises of up to 5 degrees C occur using the optimized ultrasound conditions, this effect is not responsible for the potentiated drug cytotoxicity. This technique could be used with focused ultrasound or with endoscopic ultrasound probes to develop a localized and effective anticancer treatment with little or no systemic toxicity. (E-mail: Geraldc@ccrc.ie).

A five-strain probiotic combination reduces pathogen shedding and alleviates disease signs in pigs challenged with Salmonella enterica Serovar Typhimurium.

Salmonella spp. infection is a major cause of gastroenteritis, with many thousands of cases reported in the European Union every year. The use of probiotics offers the potential to improve this situation. Here, we investigate the effects of oral treatment of pigs with a defined lactic acid bacteria culture mixture on both clinical and microbiological signs of Salmonella enterica serovar Typhimurium infection. Fifteen weaned pigs blocked by sex and weight were administered control milk or a mixture of five probiotic strains as either a milk fermentate or milk suspension for a total of 30 days. The mixture consisted of two strains of Lactobacillus murinus and one strain each of Lactobacillus salivarius subsp. salivarius, Lactobacillus pentosus, and Pediococcus pentosaceous. Following probiotic administration for 6 days, animals were challenged orally with serovar Typhimurium; the health of the animals and the microbiological composition of their feces were monitored for 23 days postinfection. Animals treated with probiotic showed reduced incidence, severity, and duration of diarrhea. These animals also gained weight at a greater rate than control pigs administered skim milk. Mean fecal numbers of Salmonella were significantly reduced in probiotic-treated animals at 15 days postinfection (P = 0.01). The administered probiotic bacteria improved both the clinical and microbiological outcome of Salmonella infection. These strains offer significant benefit for use in the food industry and may have potential in human applications.

Oral immune tolerance mediated by suppressor T cells may be responsible for the poorer prognosis of foregut cancers.

The poor prognosis of foregut cancers might, in part, be due to the immune tolerising effect of tumour antigens which are shed into the gastrointestinal tract and processed by the gut immune system. This would create a tumour specific tolerance without compromise of global immune functions. Experimental data shows that orally fed cancer tissue induces a non cross reactive attenuation of the cellular anti tumour host responses and confers a growth advantage specific to individual cancers. Although the cellular basis of such pro-tumourogenic responses has yet to be established, it is likely, based on studies of oral tolerance mechanisms, that recruitment of immune suppressive T cells (T(regs)) may be responsible. Abrogation of oral immune tolerance to the tumour by immune based therapy could represent a significant advance in the management of upper gastrointestinal cancers.

Relative ability of orally administered Lactobacillus murinus to predominate and persist in the porcine gastrointestinal tract.

Five porcine-derived Lactobacillus or Pediococcus isolates administered to pigs (n = 4), either singly or as a combination at approximately 10(10) CFU per day varied with respect to intestinal survival and persistence. Two Lactobacillus murinus strains survived best and were excreted at approximately 10(7) to 10(8) CFU/g of feces. In contrast, Pediococcus pentosaceus DPC6006 had the lowest fecal count at approximately 10(5) CFU/g and was excreted at a significantly lower level than both L. murinus strains. Fecal L. murinus DPC6003 counts were also significantly higher than both Lactobacillus salivarius DPC6005 and Lactobacillus pentosus DPC6004 ( approximately 10(6) CFU/g). The L. murinus strains persisted for at least 9 days postadministration in both the feces and the cecum. Animals fed a combination of all five strains excreted approximately 10(7) CFU of the administered strains/g, with L. murinus predominating, as determined by randomly amplified polymorphic DNA PCR. Postadministration, variation was observed between animals fed the strain combination, but in general, L. murinus DPC6002 and DPC6003 and L. pentosus DPC6004 predominated in the feces and the cecum while P. pentosaceus DPC6006 was detected only in the cecum. Fifteen days after the start of culture administration, mean fecal Enterobacteriaceae counts were significantly lower in some of the treatment groups. In addition, when mean preadministration counts were compared with those obtained after 21 days of culture administration, Enterobacteriaceae counts were reduced by approximately 87 to 98% in pigs fed L. salivarius DPC6005, P. pentosaceus DPC6006, L. pentosus DPC6004, and the culture mix. In conclusion, the porcine intestinal isolates have potential as probiotic feed additives for pigs, with differences in strain performance highlighting the advantages of using culture combinations.

Potential of using real-time PCR-based detection of spoilage yeast in fruit juice--a preliminary study.

A real-time PCR system was used to differentiate between the common spoilage yeasts, Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Candida krusei, Rhodotorula glutinis and Saccharomyces cerevisiae, based on melting peak Tm analysis of the 5.8S rDNA subunit and the adjacent ITS2 region of these yeasts. By using the real-time PCR system and by targeting the citrate synthase (cs 1) gene of C. krusei, it was possible to develop a sensitive detection system to both identify and quantitate the level of C. krusei growth in an artificially contaminated apple juice sample.