Bacteriophages: biosensing tools for multi-drug resistant pathogens.

Pathogen detection is of utmost importance in many sectors, such as in the food industry, environmental quality control, clinical diagnostics, bio-defence and counter-terrorism. Failure to appropriately, and specifically, detect pathogenic bacteria can lead to serious consequences, and may ultimately be lethal. Public safety, new legislation, recent outbreaks in food contamination, and the ever-increasing prevalence of multidrug-resistant infections have fostered a worldwide research effort targeting novel biosensing strategies. This review concerns phage-based analytical and biosensing methods targeted towards theranostic applications. We discuss and review phage-based assays, notably phage amplification, reporter phage, phage lysis, and bioluminescence assays for the detection of bacterial species, as well as phage-based biosensors, including optical (comprising SPR sensors and fiber optic assays), electrochemical (comprising amperometric, potentiometric, and impedimetric sensors), acoustic wave and magnetoelastic sensors.

Identifying former injecting drug users infected with hepatitis C: an evaluation of a general practice-based case-finding intervention.

BACKGROUND: In Scotland, a general practice-based case-finding initiative, to diagnose and refer hepatitis C virus (HCV) chronically infected former injecting drug users (IDUs), was evaluated. METHODS: Testing was offered in eight Glasgow general practices in areas of high deprivation and high HCV and IDU prevalence to attendees aged 30-54 years with a history of IDU. Test uptake and diagnosis rates were compared with those in eight demographically similar control practices. RESULTS: Of 422 eligible intervention practice attendees, 218 (52%) were offered an HCV test and, of these, 121 (56%) accepted. Poor venous access in 13 individuals prevented testing. Of 105 tested, 70% (74/105) were antibody positive of which 58% (43/74) were RNA positive by PCR. Of 43 chronically infected individuals identified in intervention practices, 22 (51%) had attended specialist care within 30 months of the study, while 9 (21%) had defaulted. In control practices, 8 (22%) of 36 individuals tested were antibody positive. Test uptake and case yield were approximately 3 and 10 times higher in intervention compared with control practices, respectively. CONCLUSIONS: Targeted case-finding in primary care demonstrated higher test uptake and diagnosis rates; however, to optimize diagnosis and referral of chronically infected individuals, alternative means of testing (e.g. dried blood spots) and retention in specialist care (e.g. outreach services) must be explored.

Evaluation of a general practice based hepatitis C virus screening intervention.

BACKGROUND: In 2003 an estimated 37,500 of Scotland's population was chronically infected with HCV; 44% were undiagnosed former injecting drug users (IDU)--a priority group for antiviral therapy. AIM: To evaluate a hepatitis C virus (HCV) screening intervention. DESIGN: Outcome measures among two similar General Practice populations in an area of high HCV and drug use prevalence, one of which was exposed to an HCV screening intervention, were compared. METHODS: Thirty to fifty four year old attendees of the intervention practice were opportunistically offered testing and counselling, where clinically appropriate, (November 2003-April 2004). OUTCOMES: HCV test uptake, case detection, referral and treatment administration rates. RESULTS: Of 584 eligible attendees, 421 (72%) were offered and 117 (28%) accepted testing in the intervention practice; no testing was undertaken in the comparison practice. Prevalences of HCV antibody were 13% (15/117), 75% (3/4) and 91% (10/11) among all tested persons, current IDUs and former IDUs respectively. For 4/15 (27%) evidence of binge drinking following the receipt of their positive result, was available. Of the 11 referred to specialist care because they were HCV RNA positive, nine attended at least one appointment. Two received treatment: one had achieved a sustained viral response as of February 2008. CONCLUSION: While non targeted HCV screening in the general practice setting can detect infected former IDU, the low diagnostic yield among non IDUs limited the effectiveness of the intervention. A more targeted approach for identifying former IDUs is recommended. Additionally, the low uptake of treatment among chronically infected persons four years after diagnosis demonstrates the difficulties in clinically managing such individuals. Strategies, including support for those with a history of problem alcohol use, to improve treatment uptake are required.

Novel alternatives to antibiotics: bacteriophages, bacterial cell wall hydrolases, and antimicrobial peptides.

Extensive research has been conducted on the development of three groups of naturally occurring antimicrobials as novel alternatives to antibiotics: bacteriophages (phages), bacterial cell wall hydrolases (BCWH), and antimicrobial peptides (AMP). Phage therapies are highly efficient, highly specific, and relatively cost-effective. However, precautions have to be taken in the selection of phage candidates for therapeutic applications as some phages may encode toxins and others may, when integrated into host bacterial genome and converted to prophages in a lysogenic cycle, lead to bacterial immunity and altered virulence. BCWH are divided into three groups: lysozymes, autolysins, and virolysins. Among them, virolysins are the most promising candidates as they are highly specific and have the capability to rapidly lyse antibiotic-resistant bacteria on a generally species-specific basis. Finally, AMP are a family of natural proteins produced by eukaryotic and prokaryotic organisms or encoded by phages. AMP are of vast diversity in term of size, structure, mode of action, and specificity and have a high potential for clinical therapeutic applications.

A study of human leukocyte D locus related antigens in Graves' disease.

An association between Graves' disease and the human leukocyte antigen (HLA) system has previously been reported. The disease was more strongly associated with the HLA D locus antigen Dw3 than with HLA B8. Products of the HLA D locus are determined by the interaction of test cells with standard typing lymphocytes, a technically difficult procedure. Recently, it has been possible to type serologically for D locus related (DRw) specificities on peripheral bone marrow-derived (B) lymphocytes. Blood B lymphocytes from 50 unrelated controls and 41 patients with Graves' disease were typed for seven HLA DRw specificities. 28 patients with Graves' disease (68%) were positive for DRw3, in contrast to 14 controls (28%); whereas only 21 patients (50%) were HLA B8 positive, compared with 13 (26%) controls. Thus, positivity for DRw3 afforded a relative risk for Graves' disease of 5.5, whereas that for HLA B8 amounted to 3.0. Additionally, a family with multiple cases of Graves' disease in which the disease was previously shown to be inherited with the haplotype, was linked to DRw2, which suggests that the susceptibility to the disease was inherited in association with that antigen. Two HLA B/glyoxalase recombination events were observed in this family; in both instances HLA DRw followed HLA B. This study thus demonstrates that the disease susceptibility gene for Graves' disease is in strong linkage disequilibrium with DRw3; however, it may be associated with other DRw specificities and inherited within family units in association with them.

Hypersensitizing effect of pluronic L61 on cytotoxic activity, transport, and subcellular distribution of doxorubicin in multiple drug-resistant cells.

The present study demonstrated that poly(oxypropylene) and poly(oxyethylene) block copolymer pluronic L61 (L61)-hypersensitized multidrug-resistant CHRC5 Chinese hamster ovary cells and MCF-7/ADR human breast carcinoma cells to the cytotoxic action of doxorubicin (Dox). CHRC5 and MCF-7/ADR cells manifested 290- and 700-fold increases, respectively, in their sensitivity to Dox/L61 formulation compared with free Dox. Their sensitive counterparts Aux-B1 and MCF-7 displayed only marginal or no increase at all in their response to Dox/L61. The study of the drug transport performed by flow cytometry showed that L61 enhanced the drug uptake and reduced the P-glycoprotein-mediated drug efflux. Visualization of Dox subcellular distribution in CHRC5 cells by fluorescent microscopy revealed that Dox was sequestered in cytoplasmic vesicles, whereas incubation of the cells with Dox/L61 altered the drug compartmentalization by releasing the drug from these vesicles and shifting it to the nucleus. These findings suggested that the hypersensitive response of multidrug-resistant cells to the action of Dox/L61 was caused by an increase in the drug accumulation and changes in its subcellular distribution.

In vitro expression of MMP-2 and MMP-9 in glioma cells following exposure to inflammatory mediators.

Progression of glioma is associated with local degenerative processes which are attributed to the activity of gelatinases. As glioma cells are candidate for secretion of these enzymes, we have studied in vitro the potential of cytokines (interleukin-1alpha (IL-1), tumor necrosis factor-alpha (TNFalpha) and transforming growth factor-beta (TGFbeta2)) to regulate the expression of gelatinase A and B (Gels A and B, respectively) in two glioma cells of human (A172) and rat origin (C6). We showed that IL-1 and TNFalpha both induced gene expression and protein secretion of Gel B in both cell lines, as revealed by RT-PCR and gelatin zymography, respectively. In C6 cells, TNFalpha had no effect on Gel A constitutive expression while IL-1 increased its production, but only at high doses. We have also demonstrated that TGFbeta2 inhibited both IL-1- or TNFalpha-induced gene expression and Gel B production in a dose-dependent manner but had no effect on Gel A secretion. The effect of TGFbeta2 on Gel B secretion was reversed by phorbol myristate acetate (PMA). Taken together, these data suggest that IL-1, TNFalpha and TGFbeta2 tightly regulate Gel B secretion in glioma cells, an enzyme which is believed to play an important role in the local invasion of brain tissue by tumor cells.

Synergistic and selective stimulation of gelatinase B production in macrophages by lipopolysaccharide, trans-retinoic acid and CGP 41251, a protein kinase C regulator.

The production of gelatinase B by macrophages is relevant in the immunological and migratory functions of macrophages. CGP 41251, an inhibitor of protein kinase C (PKC), was found to stimulate the expression of gelatinase B in macrophages, as shown by the study of two different monocytic/macrophagic cell lines, mouse RAW 264.7 and human THP-1 cells. When human monocytes and rat peritoneal macrophages were treated with CGP 41251, insignificant increases of 10 and 25% were obtained. This can possibly be due to the presence of contaminating cells in these two enriched populations, since the CGP 41251 treatment of non-macrophagic cell lines inhibited their PMA-induced gelatinase B production. Taken together, these results suggest that the stimulatory effect of CGP 41251 is specific to cells of the monocytic lineage. Using RAW 264.7 cells as a model, the effect of CGP 41251 is additive to that obtained using lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA), as revealed by gelatin zymography and Northern blot analysis. The stimulatory effect of CGP 41251 on gelatinase B production in RAW 264.7 was: (a) inhibited by calphostin C (as is the LPS-induced response), indicating a PKC-dependence; (b) inhibited by dexamethasone (as opposed to the LPS-induced response); and (c) enhanced by addition of trans-retinoic acid (RA). In fact, RA can induce gelatinase B production, either alone or in synergy with LPS and/or CGP 41251, since the combination of the three agents gives the highest gelatinase B response, at both the protein and the mRNA levels. This represents an important observation considering the RA is now being tested as an anti-cancer agent and proposed for prevention studies.

Epitope-specific antibody response to HT-1080 fibrosarcoma cells by mimotope immunization.

Mouse monoclonal antibody (mAb) BCD-F9, which recognizes an unknown antigen found on the surface of many tumor cells, was used to screen a phage display library expressing random peptide decamers. The phage that was selected encoded the unique sequence GRRPGGWWMR, representing the peptide capable of binding to the BCD-F9 mAb. The peptide was synthesized and found to specifically inhibit the binding of mAb to HT-1080 fibrosarcoma cells. Alanine mutagenesis of the sequence encoding this peptide indicated that three residues, PXXWW, were critical for its binding to the BCD-F9 mAb. Polyclonal antibodies generated by immunization of rabbits with the synthetic peptide GRRPGGWWMR (anti-mimotope antiserum or AM-F9) bound specifically to HT-1080 cells and inhibited the binding of the BCD-F9 mAb to these cells. Using an experimental animal model in which CD-1 nude mice are inoculated i.v. with HT-1080 cells, develop lung metastasis, and die within 30 days, we have shown that AM-F9 could significantly prolong the life span of these animals. Our results suggest that a peptide mimotope can potentially be used as a novel immunotherapy to induce a beneficial antitumor response.

Inhibition of tumour growth and metastasis of human fibrosarcoma cells HT-1080 by monoclonal antibody BCD-F9.

BCD-F9 is a murine IgG(2a) monoclonal antibody (mAb) that recognises a conformational epitope found on the surface of many human tumour cells. The aim of this study was to investigate the ability of BCD-F9 to recognise a variety of neoplastic cell lines and to test BCD-F9 in vivo for anticancer activity in subcutaneous (s.c.) and metastatic tumour models. Intravenous (i.v.) administration of BCD-F9 in CD-1 nude mice xenografted s.c. with human HT-1080 cells led to a significant inhibition of tumour growth. We demonstrated that BCD-F9 administrated i.v. significantly prolonged the life-span of CD-1 nude mice inoculated i.v. with the same tumour cell line that induces aggressive lung metastases in the untreated mice. We also investigated the antitumour activity of BCD-F9 in vitro in antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent complement-mediated cytotoxicity (ADCMC) assays. The effector cell subpopulations were obtained by macrophage stimulation (thioglycollate) or natural killer (NK) cell enrichment (negative selection). BCD-F9 was found to be effective in mediating tumour cell killing in vitro by ADCC and ADCMC mechanisms. These results suggest that the mAb BCD-F9 can have a potential use in immunotherapy for treatment of tumours of different origin.

Molecular analysis of episomal human papillomavirus type 16 DNA in a cervical carcinoma cell line.

Integration of human papillomavirus type 16 DNA sequences into host DNA is a frequent event in cervical carcinogenesis. However, recent studies showing that HPV16 is present exclusively in an episomal form in many primary cervical cancers suggest that HPV16 can transform target cells by mechanisms that do not require viral integration. We have established a cervical carcinoma cell line that harbors episomal copies of HPV16 DNA of approximately 10 kb. Restriction enzyme and two-dimensional gel analysis confirmed that HPV16 DNA was extrachromosomal with both monomeric and multimeric forms present. HPV16 was maintained as episomes with passage both in culture and after subcutaneous growth in nude mice. The 10 kb viral genome, consisting of a full-length copy of HPV16 and a partial duplication of the long control region and the L1 open reading frame, exhibited transforming activity comparable to prototype HPV16. This cell line should provide a useful model system for studying the biological significance of the physical state of the HPV16 genome in cervical carcinoma cells.

Immunogold and immunogold/silver staining in the ultrastructural localization of target molecules identified by monoclonal antibodies.

A new method for demonstrating the binding and internalization of target molecules identified by two different monoclonal antibodies (MAbs) is described. This double staining technique utilizes, in a pre-embedding procedure, an immunogold/silver staining and a MAb that recognized cell surface antigens and a post-embedding technique where only immunogold is used to identify intracytoplasmic antigens. We could demonstrate that immunogold and gold followed by silver enhancement are two highly sensitive and accurate techniques. Colloidal gold particles are versatile tracers at the electron microscopic level when used at two different particle sizes (5 and 20 nm in diameter) in a double labelling method for the simultaneous identification of antigenic sites on the same section; the combined use of immunogold and gold/silver staining for the simultaneous localization of breast cancer-associated antigens on the same thin section is more accurate. These techniques have also been used to visualize the internationalization of antigen-antibody complexes by incubating MAb-gold labelled tissue sections for 30 min at 37 degrees C. We conclude that the use of both colloidal gold and gold/silver constitutes a further improvement in immunoelectron microscopy techniques and can help visualize the relative pattern of reactivity of two MAbs on the same cells. Further applications of these techniques will have an important impact on the development and use of MAbs in oncology.

An in vitro model for cancer invasion: 1. Grading and quantitation of aggressiveness.

Invasion, destruction and replacement of normal tissues by cancer cells is the first critical step in the metastatic cascade and is the result of complex interactions between tumor and host factors. In an attempt to understand the complex mechanism of local invasion, we have developed a simple, reliable and generally applicable in vitro system using the confrontation of precultured heart fragments (PHF) with a constant known number of potentially invading (10(5)) cells. This grading system is based essentially on quantitative data standardized both for the portion of the explant invaded by the neoplastic cells and the type of invasion demonstrated. Using this system we could measure the ability of an aggressor cell to (a) adhere or attach to PHF (Grade 1), (b) invade the outer fibroblastic layers (Grade II) and/or the cardiac muscle cells (Grade III) and (c) destroy and completely replace the PHF (Grade IV). It was at once apparent from these experiments that, irrespective of their invasive capacity and/or their neoplastic state, both malignant (SKBR-2 III, BT-20, MCF-7, ZR-75-30 LoVo and YAC-1) and non-malignant (HBL-100) cells attach to the PHF. Only malignant cells, however, showed substantial local invasion of both the outer fibroblastic layers and/or the cardiac muscle cells. Most important for the actual problems of invasion in vivo, is the fact that malignant cells from different cell lines demonstrate a wide range in their invasion capacity: three different patterns of invasion were thus established; a highly invasive, a slowly invasive and a poorly invasive pattern. We also show that invasion and proliferation, as defined for the purposes of this study, are two different and independent properties of a given cell line. SKBR-2 III and YAC-1 are here shown to possess the most aggressive potential; they both invade the PHF very early and completely. The rapid proliferation of these aggressive cells and the destruction of the host tissue lead to the rapid disappearance of the myoblasts and their complete replacement by the invading cells. Non-malignant epithelial cells attached to but could not invade even the outer fibroblastic layers of the PHF.

Immunolymphscintigraphy of axillary lymph node metastases in breast cancer patients using monoclonal antibodies: first clinical findings.

The results of preliminary investigations into the immunolymphscintigraphic (ILS) detection of axillary lymph node metastases in nine breast cancer patients by means of the 3C6F9 monoclonal antibody (MAs) are presented. The IgG2a monoclonal antibody detects a 37 KD antigen, consistently found on the surface of primary and metastatic breast tumors. Each patient received 1 mCi of I-123 (specific activity, 2 mCi per mg of antibody) as a subcutaneous injection between the 2nd and the 3rd finger of both hands, i.e., the healthy side serving as a control for the affected side. Clear images of lymph node metastases were visible 4 to 8 hours after injection of the antibody. Seven of the nine patients studied were positive by scanning and six showed positive lymph node involvement by histopathology (6/7; true positive = 86%). Two patients did not show any iodine uptake in the axilla and were subsequently found to be free of metastases (2/2; true negative = 100%). These data give an overall accuracy of ILS of 89% and demonstrate that 3C6F9 localizes preferentially in affected axillary lymph nodes compared to normal lymph nodes.

Combined use of 123I-labelled BCD-F9 and 4C4 monoclonal antibody with dissimilar specificity for breast cancer: implication for the detection limit of immunolymphoscintigraphy in the assessment of axillary lymph node metastases.

To determine whether the detection limit of immunolymphoscintigraphy (ILS), reported to be > or = 1 cm, can be improved by comparing imaging after administration of breast cancer-specific monoclonal antibody (MAb) BCD-F9 and breast cancer-nonspecific 4C4, 25 patients with suspected breast cancer were given injections of both 123I-labelled MAbs. The ILS was performed independently for both MAbs, the 4C4 scans serving as an ipsilateral negative control, and was used preoperatively to detect lymph node metastases. Twenty-one patients had breast cancer of whom 11 patients suffered from axillary involvement. Single interpretation of BCD-F9 scans gave true positive results in six of 11 and true negative results in 12 of 14 patients, whereas combined interpretation of BCD-F9 and 4C4 scans gave true positive results in nine of 11 and true negative results in 14 of 14 patients. On the basis of comparison of scintigrams of both MAbs, ILS allowed the detection of lymph node metastases 0.3-0.8 cm in diameter (n = 3). Immunohistochemistry of BCD-F9 and 4C4 MAbs of tumour-free and tumour-bearing lymph nodes correlated with ILS, with the exception of one patient. The study suggests that comparing scans obtained with BCD-F9 and 4C4 MAbs may improve the detection limit of ILS in the preoperative staging of axillae.

Clinical value of immunolymphscintigraphy in patients with breast cancer.

Thirty four patients suspected of suffering from breast cancer were investigated by immunolymphscintigraphy (ILS) a few days before a planned operation. Patients received 500 micrograms of the BCD-F9 monoclonal antibody or its F(ab')2 fragments containing 1 mCi of Iodin-123. Each preparation was given by a subcutaneous injection into the fingerwebs between the 2nd and 3rd fingers of both hands. Three static scintigrams were taken 4, 8 and 24 hours following injection of the radiolabelled antibodies. ILS results were always compared to histopathological findings and gave for the intact antibody a sensitivity of 83%, a specificity of 93% and an accuracy of 89%, the positive predictive value was 91% and the negative predictive value was 80%. On the other hand, when the F(ab')2 fragments were used the sensitivity of the ILS technique was 75%, the specificity was 100% while the accuracy was 86%, the positive predictive value was 100% and the negative predictive value was 75%.

Affinity purification of a high molecular weight human breast cancer-associated antigen identified by the BCD-B4 monoclonal antibody.

This study reports the purification and characterization of a high molecular weight human breast cancer-associated antigen identified by a previously described (1,2) murine monoclonal antibody, BCD-B4. Immunohistochemical analysis indicated that BCD-B4 recognizes an antigen expressed in an altered form on the human breast carcinoma cell line, BT-20, compared to the non-malignant human mammary epithelial cell line, HBL-100. Chemical treatments and enzymatic digestions suggested that the recognized moiety was a protein. The antigenic determinant was resistant to neuraminidase and periodate treatments but was sensitive to trypsin and proteinase K. The antigen was purified by affinity chromatography and its molecular weight, determined by SDS-PAGE analysis under non-reducing conditions, was proven to be 250 Kd. Under reducing conditions, the molecule dissociated into two polypeptides of 125 and 45 Kd, respectively. Both subunits could be isolated from normal HBL-100 and neoplastic BT-20 cellular protein extracts by affinity chromatography. The higher molecular weight subunit showed; however, qualitative and quantitative differences between the two cell lines: it was expressed in greater quantity on BT-20 cells and its molecular weight was 15 Kd higher. Both subunits could also be identified by immunoblots of BT-20 cells.

Ornithine decarboxylase activity in tissues from rats exposed to 60 Hz magnetic fields, including harmonic and transient field characteristics.

Ornithine decarboxylase (ODC) activity is used widely as a biomarker for tumor promotion in animal model systems. Several previous studies have reported increases in ODC activity in tissues of rats exposed to 60 Hz magnetic fields. The goals of this study were to confirm these findings and to determine whether ODC activity is increased in tissues of animals exposed to magnetic fields containing complex metrics. Three experiments were conducted in male F344 rats. Each study included a sham control group and a group exposed to pure continuous 60 Hz fields (0.2 mT). Additional groups included animals exposed to randomly time-varying 60 Hz fields (range of 0.02 to 0.2 mT); intermittent 60 Hz fields (2 mT) with on-off cycles ranging from 5 s to 5 min; pure continuous 180 Hz fields (2 mT); 60 Hz fields with a superimposed 3rd harmonic (total field strength, 2 mT); 60 Hz fields with superimposed third, fifth, and seventh harmonics (total field strength, 2 mT); 60 Hz fields (2 mT) with superimposed transients; and randomly time-varying 60 Hz fields (range of 0.02 to 0.2 mT) with superimposed transients. After 4 weeks of exposure (18.5 h/day), eight animals per group were euthanized within 1 h of magnetic field deactivation. Homogenates of liver, kidneys, spleen, and brain were prepared from each animal, quick-frozen, and shipped for analysis by four independent laboratories. No consistent pattern of differences in the ODC activity among experimental groups was found either within a laboratory or among laboratories. The results do not support the hypothesis that exposure to extremely low frequency magnetic fields stimulates ODC activity.

CMOS Conductometric System for Growth Monitoring and Sensing of Bacteria.

We present the design and implementation of a prototype complementary metal-oxide semiconductor (CMOS) conductometric integrated circuit (IC) for colony growth monitoring and specific sensing of Escherichia coli (E. coli) bacteria. The detection of E. coli is done by employing T4 bacteriophages as receptor organisms. The conductometric system operates by measuring the resistance of the test sample between the electrodes of a two-electrode electrochemical system (reference electrode and working electrode). The CMOS IC is fabricated in a TSMC 0.35-µm process and uses a current-to-frequency (I to F) conversion circuit to convert the test sample resistance into a digital output modulated in frequency. Pulsewidth control (one-shot circuit) is implemented on-chip to control the pulsewidth of the output digital signal. The novelty in the current work lies in the ability of the CMOS sensor system to monitor very low initial concentrations of bacteria (4×10(2) to 4×10(4) colony forming unit (CFU)/mL). The CMOS system is also used to record the interaction between E. coli and its specific receptor T4 bacteriophage. The prototype CMOS IC consumes an average power of 1.85 mW with a 3.3-V dc power supply.