Safety, acceptability and tolerability of uncoated and excipient-coated high density silicon micro-projection array patches in human subjects.

Most vaccinations are performed by intramuscular injection with a needle and syringe. However, this method is not ideal due to limitations, such as the risk of needle-stick injury, the requirement for trained personnel to give injections and the need to reconstitute lyophilized vaccines. Therefore, we tested an alternative delivery technology that overcomes the problems with needle and syringe.

Pyruvate:ferredoxin oxidoreductase and thioredoxin reductase are involved in 5-nitroimidazole activation while flavin metabolism is linked to 5-nitroimidazole resistance in Giardia lamblia.

Objectives: The mechanism of action of, and resistance to, metronidazole in the anaerobic (or micro-aerotolerant) protozoan parasite Giardia lamblia has long been associated with the reduction of ferredoxin (Fd) by the enzyme pyruvate:ferredoxin oxidoreductase (PFOR) and the subsequent activation of metronidazole by Fd to toxic radical species. Resistance to metronidazole has been associated with down-regulation of PFOR and Fd. The aim of this study was to determine whether the PFOR/Fd couple is the only pathway involved in metronidazole activation in Giardia.

Chromosome sequence maps of the Giardia lamblia assemblage A isolate WB.

Two genotypes, assemblages A and B, of the pathogenic gut protozoan parasite Giardia lamblia infect humans. Symptoms of infection range from asymptomatic to chronic diarrhea. Giardia chromosomes have long been characterized but not until the publication of the first Giardia genome sequence was chromosome mapping work, commenced nearly two decades ago, completed.

Sequence map of the 2 Mb Giardia lamblia assemblage A chromosome.

The gut protozoan parasite, Giardia lamblia (Assemblage A), has 5 major chromosomes, 1 of which is 2 Mb, as determined from gel separations of whole chromosomes. We originally published a physical map of this chromosome and, now, using the sequence data from 46 chromosome-specific probes, have produced a sequence map of the 2 Mb chromosome. Comparison of the probe sequences with the Giardia genome database (http://GiardiaDB.

Susceptibility in vitro of clinically metronidazole-resistant Trichomonas vaginalis to nitazoxanide, toyocamycin, and 2-fluoro-2'-deoxyadenosine.

This study investigates the susceptibility of a clinically metronidazole (Mz)-resistant isolate of Trichomonas vaginalis to alternative anti-trichomonal compounds. The microaerobic minimal inhibitory concentration (MIC) of the 5-nitroimidazole (NI) drug, Mz, against a typical Mz-susceptible isolate of T. vaginalis is around 3.

A new-generation 5-nitroimidazole can induce highly metronidazole-resistant Giardia lamblia in vitro.

The 5-nitroimidazole (NI) compound C17, with a side chain carrying a remote phenyl group in the 2-position of the imidazole ring, is at least 14-fold more active against the gut protozoan parasite Giardialamblia than the 5-NI drug metronidazole (MTR), with a side chain in the 1-position of the imidazole ring, which is the primary drug for the treatment of giardiasis. Over 10 months, lines resistant to C17 were induced in vitro and were at least 12-fold more resistant to C17 than the parent strains. However, these lines had ID(90) values (concentration of drug at which 10% of control parasite ATP levels are detected) for MTR of >200 microM, whilst lines induced to be highly resistant to MTR in vitro have maximum ID(90) values around 100 microM (MTR-susceptible isolates typically have an ID(90) of 5-12.

Sequence map of the 3-Mb Giardia duodenalis assemblage A chromosome.

The genome of the gut protozoan parasite Giardia duodenalis (assemblage A) has been sequenced and compiled as contigs and scaffolds (GiardiaDB- http://GiardiaDB.org ), but specific chromosome location of all scaffolds is unknown. To determine which scaffolds belong to the 3-Mb chromosome, a library of probes specific for this chromosome was constructed.

Epstein-Barr virus nuclear antigen 3A contains six nuclear-localization signals.

The Epstein-Barr nuclear antigen 3A (EBNA3A) is one of only six viral proteins essential for Epstein-Barr virus-induced transformation of primary human B cells in vitro. Viral proteins such as EBNA3A are able to interact with cellular proteins, manipulating various biochemical and signalling pathways to initiate and maintain the transformed state of infected cells. EBNA3A has been reported to have one nuclear-localization signal and is targeted to the nucleus during transformation, where it associates with components of the nuclear matrix.

Nuclear localization of the Epstein-Barr virus EBNA3B protein.

The Epstein-Barr virus nuclear antigen (EBNA) 3B is a hydrophilic, proline-rich, charged protein that is thought to be involved in transcriptional regulation and is targeted exclusively to the cell nucleus, where it localizes to discrete subnuclear granules. Co-localization studies utilizing a fusion protein between enhanced green fluorescent protein (EGFP) and EBNA3B with FLAG-tagged EBNA3A and EBNA3C proteins demonstrated that EBNA3B co-localized with both EBNA3A and EBNA3C in the nuclei of cells when overexpressed. Computer analyses identified four potential nuclear-localization signals (NLSs) in the EBNA3B amino acid sequence.

The Epstein-Barr virus nuclear antigen-6 protein co-localizes with EBNA-3 and survival of motor neurons protein.

The Epstein-Barr virus nuclear antigen (EBNA)-6 protein is essential for Epstein-Barr virus (EBV)-induced immortalization of primary human B-lymphocytes in vitro. In this study, fusion proteins of EBNA-6 with green fluorescent protein (GFP) have been used to characterize its nuclear localization and organization within the nucleus. EBNA-6 associates with nuclear structures and in immunofluorescence demonstrate a punctate staining pattern.

Identification of the nuclear localization signals within the Epstein-Barr virus EBNA-6 protein.

Epstein-Barr virus nuclear antigen (EBNA)-6 is essential for EBV-induced immortalization of primary human B-lymphocytes in vitro. Previous studies have shown that EBNA-6 acts as a transcriptional regulator of viral and cellular genes; however at present, few functional domains of the 140 kDa EBNA-6 protein have been completely characterized. There are five computer-predicted nuclear localization signals (NLS), four monopartite and one bipartite, present in the EBNA-6 amino acid sequence.

The EBNA-3 gene family proteins disrupt the G2/M checkpoint.

The Epstein-Barr nuclear antigens (EBNA), EBNA-3, -4 and -6, have previously been shown to act as transcriptional regulators, however, this study identifies another function for these proteins, disruption of the G2/M checkpoint. Lymphoblastoid cell lines (LCLs) treated with a G2/M initiating drug azelaic bishydroxamine (ABHA) did not show a G2/M checkpoint response, but rather they display an increase in cell death, a characteristic of sensitivity to the cytotoxic effects of the drug. Cell cycle analysis demonstrated that the individual expression of EBNA-3, -4 or -6 are capable of disrupting the G2/M checkpoint response induced by ABHA resulting in increased toxicity, whereas EBNA-2, and -5 were not.

A histone deacetylase inhibitor, azelaic bishydroxamic acid, shows cytotoxicity on Epstein-Barr virus transformed B-cell lines: a potential therapy for posttransplant lymphoproliferative disease.

Background: Posttransplant lymphoproliferative disease (PTLD), driven by the presence of Epstein-Barr virus (EBV), is becoming an increasingly important clinical problem after solid organ transplantation. The use of immunosuppressive therapy leads to the inhibition of the cytotoxic T cells that normally control the EBV latently infected B cells. The prognosis for many patients with PTLD is poor, and the optimal treatment strategy is not well defined.

Characterization of the transcriptional repressor RBP in Epstein-Barr virus-transformed B cells.

RBP, a transcriptional repressor, is intricately involved in Epstein-Barr virus (EBV) transformation of human B cells. The EBV nuclear proteins EBNA-2, -3, -4 and -6 all utilize RBP to regulate the transcription of both cellular and viral genes. This study investigates the isoforms of the RBP protein in Burkitt's lymphoma (BL) cells and in EBV-transformed lymphoblastoid cell lines (LCLs).

Epstein-Barr virus-encoded RK-BARF0 protein expression.

The cellular localization of the Epstein-Barr virus-encoded RK-BARF0 protein was analyzed by fluorescence microscopy and immunoblotting. The recombinant RK-BARF0 protein was found to be tightly bound to nuclear structures, whereas 16- to 20-kDa RK-BARF0 derivatives, generated by differential splicing of the RK-BARF0 transcript, were present throughout the cell. Moreover, a previously generated anti-RK-BARF0 rabbit serum was found to cross-react with cellular proteins, showing that the previously identified 30- to 35-kDa membrane-associated proteins do not represent RK-BARF0.

A-type and B-type Epstein-Barr virus differ in their ability to spontaneously enter the lytic cycle.

In this study replication of A-type and B-type Epstein-Barr virus (EBV) strains has been assessed. A-type and B-type type lymphoblastoid cell lines (LCLs) were established by infecting B lymphocytes, isolated from five EBV-seropositive donors, with different A-type and B-type virus isolates. The presence of viral capsid antigens (VCA) in these LCLs was determined by immunofluoresence assay and by immunoblotting.

Regulation of interleukin-1beta transcription by Epstein-Barr virus involves a number of latent proteins via their interaction with RBP.

Epstein-Barr virus (EBV) infects B cells, resulting in the outgrowth of immortalised lymphoblastoid cell lines (LCLs). Here, we demonstrate through the use of intracellular staining that interleukin-1beta (IL-1beta) is expressed in LCLs and investigate the influence of the individual latent proteins on the expression of IL-1beta. Using RT-PCR, IL-1beta was shown to be up-regulated in EBV-transformed LCLs as well as in group III Burkitt's lymphoma (BL) cell lines, compared with group I BL cell lines.

A functional link for major TCR expansions in healthy adults caused by persistent Epstein-Barr virus infection.

Dramatic clonal expansions of unknown functional significance have been documented in the T cell receptor (TCR) alpha beta peripheral blood repertoires of apparently healthy adults. In this study, we provide evidence that persistent infection with the ubiquitous Epstein-Barr virus (EBV) causes major distortions within the memory repertoire of healthy virus carriers. Using complementarity determining region 3 (CDR3) length analysis to measure repertoire diversity, dominant expansions that dramatically skewed the entire TCRBV6 blood repertoire towards oligoclonality were enriched in the CD8(+)CD45RO+CD45RA- subset of HLA B8(+) healthy virus carriers.

Interferon-resistant human melanoma cells are deficient in ISGF3 components, STAT1, STAT2, and p48-ISGF3gamma.

The mechanism of IFN resistance was examined in three long-term cell lines, SK-MEL-28, SK-MEL-3, and MM96, exhibiting significant variation in responsiveness to the antiproliferative and antiviral effects of type I IFNs. The JAK-STAT components involved in IFN signal transduction were analyzed in detail. After exposure to IFN, activation of the IFN type I receptor-linked tyrosine kinases, JAK-1 and TYK-2, was detected at similar levels in both IFN-sensitive and IFN-resistant cell types, indicating that IFN resistance did not result from a deficiency in signaling at the level of receptor-associated kinase activation.

Comparison of the biological properties of two anti-mucin-1 antibodies prepared for imaging and therapy.

A comparison was made between the murine anti-MUC1 antibody BC2 (which reacts with the peptide epitope APDTR) and the "humanised" antibody hCTMO1 from CellTech, which reacts with the MUC1 epitope RPAP. Preliminary studies demonstrated that hCTMO1 was a "good" antibody whereas BC2 was not. Various parameters were determined and conclusions reached.

Both A type and B type Epstein-Barr virus nuclear antigen 6 interact with RBP-2N.

Using the yeast two-hybrid system, Epstein-Barr virus nuclear antigen 6A (EBNA6A) was found to interact with the RBP-2N isoform of RBP-J kappa. The interaction of EBNA6A and EBNA6B with RBP-2N was compared and the results indicated that EBNA6B was less efficient at interacting with RBP-2N than was EBNA6A. Deletion mutation analysis of EBNA6A identified a region involved in the interaction with RBP-2N, while analysis of RBP-2N identified a domain which interacts with EBNA6A.

Epstein-Barr nuclear antigen-3 and -4 interact with RBP-2N, a major isoform of RBP-J kappa in B lymphocytes.

The Epstein-Barr nuclear antigen (EBNA)-3 and EBNA-4 proteins are thought to act as transcriptional transactivators. The yeast two-hybrid system and coimmunoprecipitation were used to demonstrate that EBNA-3 and -4 associate with the DNA-binding protein RBP-2N, an isoform of RBP-J kappa. A comparison between EBNA-3, EBNA-4, and EBNA-6 binding to RBP-2N indicated that EBNA-3 enhanced beta-galactosidase activity 4-fold more than EBNA-6 and 30-fold more than EBNA-4.

Antibody-targeted drugs for the therapy of cancer.

The advent of monoclonal antibodies has revitalised the concept of magic bullets and various agents (eg. drugs, toxins and isotopes) have been conjugated to monoclonal antibodies for selective delivery to tumours. Preclinical studies in mouse tumour models have been impressive and have lead to several clinical trials.