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Biomedical subjects

Oliver Hartley

Publications and source records attributed to Oliver Hartley.

At least 19 recordsLinked to original sources
PubMed2006

The biological effectiveness of antiproton irradiation.

BACKGROUND AND PURPOSE: Antiprotons travel through tissue in a manner similar to that for protons until they reach the end of their range where they annihilate and deposit additional energy. This makes them potentially interesting for radiotherapy. The aim of this study was to conduct the first ever measurements of the biological effectiveness of antiprotons. MATERIALS AND METHODS: V79 cells were suspended in a semi-solid matrix and irradiated with 46.7MeV antiprotons, 48MeV protons, or (60)Co gamma-rays. Clonogenic survival was determined as a function of depth along the particle beams. Dose and particle fluence response relationships were constructed from data in the plateau and Bragg peak regions of the beams and used to assess the biological effectiveness. RESULTS: Due to uncertainties in antiproton dosimetry we defined a new term, called the biologically effective dose ratio (BEDR), which compares the response in a minimally spread out Bragg peak (SOBP) to that in the plateau as a function of particle fluence. This value was approximately 3.75 times larger for antiprotons than for protons. This increase arises due to the increased dose deposited in the Bragg peak by annihilation and because this dose has a higher relative biological effectiveness (RBE). CONCLUSION: We have produced the first measurements of the biological consequences of antiproton irradiation. These data substantiate theoretical predictions of the biological effects of antiproton annihilation within the Bragg peak, and suggest antiprotons warrant further investigation.

Animals↗
PubMed2006

N-terminal proteolytic processing by cathepsin G converts RANTES/CCL5 and related analogs into a truncated 4-68 variant.

N-terminal proteolytic processing modulates the biological activity and receptor specificity of RANTES/CCL5. Previously, we showed that an unidentified protease associated with monocytes and neutrophils digests RANTES into a variant lacking three N-terminal residues (4-68 RANTES). This variant binds CCR5 but exhibits lower chemotactic and antiviral activities than unprocessed RANTES. In this study, we characterize cathepsin G as the enzyme responsible for this processing. Cell-mediated production of the 4-68 variant was abrogated by Eglin C, a leukocyte elastase and cathepsin G inhibitor, but not by the elastase inhibitor elastatinal. Further, anti-cathepsin G antibodies abrogated RANTES digestion in neutrophil cultures. In accordance, reagent cathepsin G specifically digested recombinant RANTES into the 4-68 variant. AOP-RANTES and Met-RANTES were also converted into the 4-68 variant upon exposure to cathepsin G or neutrophils, while PSC-RANTES was resistant to such cleavage. Similarly, macaque cervicovaginal lavage samples digested Met-RANTES and AOP-RANTES, but not PSC-RANTES, into the 4-68 variant and this processing was also inhibited by anti-cathepsin G antibodies. These findings suggest that cathepsin G mediates a novel pathway for regulating RANTES activity and may be relevant to the role of RANTES and its analogs in preventing HIV infection.

Antibodies↗
PubMed2006

Analysis of HIV-1-X4 fusion with immature dendritic cells identifies a specific restriction that is independent of CXCR4 levels.

Immature dendritic cells (iDCs) are likely to be among the first targets of HIV infection during sexual transmission. We analyzed whether the relatively inefficient viral replication in iDCs could be attributed to specific restrictions during the viral life cycle. Using iDCs from a panel of donors, we set out to compare their capacity to support infection and propagation of X4- and R5-tropic viruses. We also performed quantitative flow cytometry to determine levels of relevant cell-surface CD4 and HIV-1 co-receptors. Although iDCs express comparable levels of functional CXC chemokine receptor 4 (CXCR4) and CC chemokine receptor 5 (CCR5) at the cell surface, they are 100- to 1,000-fold less susceptible to infection by X4- versus R5-tropic HIV-1 strains. Increasing surface expression of CXCR4 by transduction with lentiviral vectors did not lead to increased replication of the X4-tropic strains. Fusion of HIV-X4 with iDCs was markedly less efficient compared to that of HIV-R5. We conclude that an env-specific block early in the viral cycle operates in iDCs. This restriction may play a role in the exclusion of X4-tropic strains during HIV-1 transmission.

Cell Membrane↗
PubMed2006

Microbicides and other topical strategies to prevent vaginal transmission of HIV.

The HIV epidemic is, by many criteria, the worst outbreak of infectious disease in history. The rate of new infections is now approximately 5 million per year, mainly in the developing world, and is increasing. Women are now substantially more at risk of infection with HIV than men. With no cure or effective vaccine in sight, a huge effort is required to develop topical agents (often called microbicides) that, applied to the vaginal mucosa, would prevent infection of these high-risk individuals. We discuss the targets for topical agents that have been identified by studies of the biology of HIV infection and provide an overview of the progress towards the development of a usable agent.

Administration, Topical↗
PubMed2005

Identification of peptide ligands to the chemokine receptor CCR5 and their maturation by gene shuffling.

The determination of protein-protein interactions and their role in diverse pathophysiological processes is a promising approach to the identification of molecules of therapeutic potential. This paper describes the identification of peptidic CCR5 receptor ligands as potential drug leads against HIV-1 infection using in vitro evolution based on phage display. A phage-displayed peptide library was used to select for anti-CCR5 peptide. Further in vitro evolution of the peptide by exon shuffling was performed to identify peptides with optimized characteristics for CCR5 receptor. This peptide inhibited HIV coreceptor activity in a cell fusion assay with an IC50 of 5 microM. It did not exhibit either agonistic or antagonistic activity on CCR5 in the concentration range used. To our knowledge, this is a first report that describes the identification of peptide ligands specific to the CCR5 receptor from a phage-displayed library and the maturation of the selected peptide sequence by gene shuffling.

Animals↗
PubMed2005

Chemokine receptors in the central nervous system: role in brain inflammation and neurodegenerative diseases.

Chemokines were originally described as chemotactic cytokines involved in leukocyte trafficking. Research over the last decade, however, has shown that chemokine receptors are not restricted to leukocytes. In the brain, chemokine receptors are not only found in microglia (a brain macrophage), but also in astrocytes, oligodendrocytes and neurons. In this review, we describe the spatial and cellular distribution of chemokine receptors in the brain, distinguishing between constitutively and inducibly expressed receptors. We then discuss possible physiological functions, including neuronal migration, cell proliferation and synaptic activity. Evidence is emerging that chemokine receptors are also involved in neuronal death and hence neurodegenerative diseases. Chemokines may induce neuronal death either indirectly (e.g. through activation of microglia killing mechanisms) or directly through activation of neuronal chemokine receptors. Disease processes in which chemokines and their receptors are likely to be involved include multiple sclerosis (MS), Alzheimer's disease (AD), HIV-associated dementia (HAD) and cerebral ischemic disease. The study of chemokines and their receptors in the central nervous system (CNS) is not only relevant for the understanding of brain physiology and pathophysiology, but may also lead to the development of targeted treatments for neurodegenerative diseases.

Animals↗
PubMed2005

V3: HIV's switch-hitter.

The third variable region, V3, of the gp120 surface envelope glycoprotein is an approximately 35-residue-long, frequently glycosylated, highly variable, disulfide-bonded structure that has a major influence on HIV-1 tropism. Thus the sequence of V3, directly or indirectly, can determine which coreceptor (CCR5 or CXCR4) is used to trigger the fusion potential of the Env complex, and hence which cells the virus can infect. V3 also influences HIV-1's sensitivity to, and ability to escape from, entry inhibitors that are being developed as antiviral drugs. For some strains, V3 is a prominent target for HIV-1 neutralizing antibodies (NAbs); indeed, for many years it was considered to be the "principal neutralization determinant" (PND). Some efforts to use V3 as a vaccine target continue to this day, despite disappointing progress over more than a decade. Recent findings on the structure, function, antigenicity, and immunogenicity of V3 cast new doubts on the value of this vaccine approach. Here, we review recent advances in the understanding of V3 as a determinant of viral tropism, and discuss how this new knowledge may inform the development of HIV-1 drugs and vaccines.

AIDS Vaccines↗
PubMed2005

Immunogenicity of HIV type 1 gp120 CD4 binding site phage mimotopes.

The conserved domain of the CD4 binding site (CD4bs) on the human immunodeficiency virus type 1 (HIV- 1) envelope represents a potential target for vaccine development. Here we describe selection of peptide mimotopes by panning a phage peptide library on the HIV-1 CD4bs-specific, broadly neutralizing anti-HIV-1 monoclonal antibody, IgG(1) b12. We identified an initial consensus sequence for IgG1 b12 binding (M/VThetaSD, where Theta represents an aromatic amino acid). A molecular evolution approach, using second- and third-generation libraries, led us to identify a refined consensus sequence (GLLVWSDEL). The resulting IgG1 b12 phage mimotopes compete with gp160 for the IgG1 b12 antigen-binding site, but the phage coat protein (pIII) may play an important structural role, since both free peptides and KLH-conjugated peptides have no detectable binding activity. Mice immunized with IgG1 b12 phage mimotopes elicited a weak but persistent humoral response directed against the HIV-1 envelope. An antibody fragment was isolated from the antibody repertoires of these animals. It is noteworthy that while it has a relatively low affinity for HIV-1 gp160, the antibody targets an epitope that overlaps with that of IgG1 b12. Our data therefore suggest that engineered IgG1 b12 mimotopes share immunogenic features with the CD4bs. However, these peptidic structures will require further improvement in order to generate broad specificity neutralizing antibodies like IgG1 b12.

Amino Acid Sequence↗
PubMed2005

Engineering chemokines to develop optimized HIV inhibitors.

Since the discovery that to enter target cells HIV uses receptors for the class of proteins known as chemokines, attempts have been made to generate anti-HIV molecules based on the chemokine ligands. A significant level of knowledge of the structure-activity relationships of chemokines has been amassed since the beginning of the 1990s. This, together with work that has elucidated the mechanisms underlying the inhibitory activity of chemokines, has guided not only the rational design of anti-HIV chemokine analogues, but also strategies by which chemokine variants with potent anti-HIV activity can be isolated from large libraries by phage display. This review summarizes the current knowledge about the structure-activity relationships and receptor biology of chemokines that is relevant to the development of analogues with anti-HIV activity. We present specific examples of engineered chemokine analogues with potent anti-HIV activity and describe the challenges that will need to be faced if these molecules are to be further developed for clinical applications. Finally, we discuss how these challenges might be met through further engineering of the molecules.

Anti-HIV Agents↗
PubMed2004

Synthesis of N alpha-(1-phenyl-2-mercaptoethyl) amino acids, new building blocks for ligation and cyclization at non-cysteine sites: scope and limitations in peptide synthesis.

A new and convenient method for the synthesis and incorporation of N(alpha)-(1-phenyl-2-mercaptoethyl)-derivatized amino acids applicable to chemical ligation at non-cysteine sites is presented. N(alpha)-Auxiliary derivatives of glycine and alanine were easily prepared using reductive amination approaches. Several strategies for the incorporation of these derivatives into peptide chains were investigated: coupling without protection, with acid-labile protection, with base-labile protection, and via a novel protection strategy using the thiazolidine derivative. All amino acid derivatives were successfully coupled to various peptide resins, and with the exception of those incorporating Boc-protected derivatives, all resins yielded the desired peptide fragments. However, the coupling of the two alanine derivative diastereomers generated some epimerization. Finally, N-terminal auxiliary glycine and alanine peptides were cyclized, and the corresponding native circular peptides were obtained upon successful removal of the auxiliary.

Amino Acids↗
PubMed2004

Medicinal chemistry applied to a synthetic protein: development of highly potent HIV entry inhibitors.

We have used total chemical synthesis to perform high-resolution dissection of the pharmacophore of a potent anti-HIV protein, the aminooxypentane oxime of [glyoxylyl1]RANTES(2-68), known as AOP-RANTES, of which we designed and made 37 analogs. All involved incorporation of one or more rationally chosen nonnatural noncoded structures, for which we found a clear comparative advantage over coded ones. We investigated structure-activity relationships in the pharmacophore by screening the analogs for their ability to block the HIV entry process and produced a derivative, PSC-RANTES [N-nonanoyl, des-Ser1[L-thioproline2, L-cyclohexylglycine3]-RANTES(2-68)], which is 50 times more potent than AOP-RANTES. This promising group of compounds might be optimized yet further as potential prophylactic and therapeutic anti-HIV agents. The remarkable potency of our RANTES analogs probably involves the unusual mechanism of intracellular sequestration of CC-chemokine receptor 5 (CCR5), and it has been suggested that this arises from enhanced affinity for the receptor. We found that inhibitory potency and capacity to induce CCR5 down-modulation do appear to be correlated, but that unexpectedly, inhibitory potency and affinity for CCR5 do not. We believe this study represents the proof of principle for the use of a medicinal chemistry approach, above all one showing the advantage of noncoded structures, to the optimization of the pharmacological properties of a protein. Medicinal chemistry of small molecules is the foundation of modern pharmaceutical practice, and we believe we have shown that techniques have now reached the point at which the approach could also be applied to the many macromolecular drugs now in common use.

Animals↗
PubMed2004

Prevention of vaginal SHIV transmission in rhesus macaques through inhibition of CCR5.

Topical agents, such as microbicides, that can protect against human immunodeficiency virus (HIV) transmission are urgently needed. Using a chimeric simian/human immunodeficiency virus (SHIV SF162), which is tropic for the chemokine receptor CCR5, we report that topical application of high doses of PSC-RANTES, an amino terminus-modified analog of the chemokine RANTES, provided potent protection against vaginal challenge in rhesus macaques. These experimental findings have potentially important implications for understanding vaginal transmission of HIV and the design of strategies for its prevention.

Administration, Intravaginal↗
PubMed2004

PSC-RANTES blocks R5 human immunodeficiency virus infection of Langerhans cells isolated from individuals with a variety of CCR5 diplotypes.

Topical microbicides that effectively block interactions between CCR5(+) immature Langerhans cells (LC) residing within genital epithelia and R5 human immunodeficiency virus (HIV) may decrease sexual transmission of HIV. Here, we investigated the ability of synthetic RANTES analogues (AOP-, NNY-, and PSC-RANTES) to block R5 HIV infection of human immature LC by using a skin explant model. In initial experiments using activated peripheral blood mononuclear cells, each analogue compound demonstrated marked antiviral activity against two R5 HIV isolates. Next, we found that 20-min preincubation of skin explants with each RANTES analogue blocked R5 HIV infection of LC in a dose-dependent manner (1 to 100 nM) and that PSC-RANTES was the most potent of these compounds. Similarly, preincubation of LC with each analogue was able to block LC-mediated infection of cocultured CD4(+) T cells. Competition experiments between primary R5 and X4 HIV isolates showed blocking of R5 HIV by PSC-RANTES and no evidence of increased propagation of X4 HIV, data that are consistent with the specificity of PSC-RANTES for CCR5 and the CCR5(+) CXCR4(-) phenotype of immature LC. Finally, when CCR5 genetic polymorphism data were integrated with results from the in vitro LC infection studies, PSC-RANTES was found to be equally effective in inhibiting R5 HIV in LC isolated from individuals with CCR5 diplotypes known to be associated with low, intermediate, and high cell surface levels of CCR5. In summary, PSC-RANTES is a potent inhibitor of R5 HIV infection in immature LC, suggesting that it may be useful as a topical microbicide to block sexual transmission of HIV.

Anti-HIV Agents↗
PubMed2003

UCP3 protein regulation in human skeletal muscle fibre types I, IIa and IIx is dependent on exercise intensity.

It has been proposed that mitochondrial uncoupling protein 3 (UCP3) behaves as an uncoupler of oxidative phosphorylation. In a cross-sectional study, UCP3 protein levels were found to be lower in all fibre types of endurance-trained cyclists as compared to healthy controls. This decrease was greatest in the type I oxidative fibres, and it was hypothesised that this may be due to the preferential recruitment of these fibres during endurance training. To test this hypothesis, we compared the effects of 6 weeks of endurance (ETr) and sprint (STr) running training on UCP3 mRNA expression and fibre-type protein content using real-time PCR and immunofluorescence techniques, respectively. UCP3 mRNA and protein levels were downregulated similarly in ETr and STr (UCP3 mRNA: by 65 and 50%, respectively; protein: by 30 and 27%, respectively). ETr significantly reduced UCP3 protein content in type I, IIa and IIx muscle fibres by 54, 29 and 16%, respectively. STr significantly reduced UCP3 protein content in type I, IIa and IIx muscle fibres by 24, 31 and 26%, respectively. The fibre-type reductions in UCP3 due to ETr, but not STr, were significantly different from each other, with the effect being greater in type I than in type IIa, and in type IIa than in type IIx fibres. As a result, compared to STr, ETr reduced UCP3 expression significantly more in fibre type I and significantly less in fibre types IIx. This suggests that the more a fibre is recruited, the more it adapts to training by a decrease in its UCP3 expression. In addition, the more a fibre type depends on fatty acid beta oxidation and oxidative phosphorylation, the more it responds to ETr by a decrease in its UCP3 content.

Adult↗
PubMed2003

Chemokine-induced cell death in CCR5-expressing neuroblastoma cells.

CCR5 is expressed in neurons but its function in this cellular context is hitherto poorly understood. We have generated CCR5-expressing SH-SY5Y neuroblastoma cells. CCR5 ligands induced cell death in these cells, but not in control neuroblastoma cells or in CCR5-expressing fibroblasts. CCR5-dependent killing of neuroblastoma cells occurred through apoptosis, since it was accompanied by caspase-3 activation and could be prevented by a caspase-3 inhibitor. Finally, cell killing by activated microglia was more rapid and extensive in CCR5-expressing neuroblastoma cells than in control cells. In summary, CCR5 may act as a death receptor in cells of neuronal lineage and therefore be involved in inflammatory neurodegeneration.

Animals↗
PubMed2003

Two mechanisms for human immunodeficiency virus type 1 inhibition by N-terminal modifications of RANTES.

C-C chemokine receptor 5 (CCR5) is the primary coreceptor for human immunodeficiency virus type 1 (HIV-1) infection. Native chemokines that bind to CCR5 inhibit HIV-1 infection, albeit weakly, but chemically modified chemokines inhibit infection more efficiently. We have investigated the inhibitory mechanism of three N-terminally modified RANTES variants (AOP-, NNY-, and PSC-RANTES) with the MT-2 human T-cell line stably expressing either native or mutated CCR5. The RANTES analogues showed the same rank order (PSC > NNY > AOP) in their capacity to induce prolonged CCR5 internalization, inhibit surface reexpression, and prevent HIV-1 infection on MT-2 cells expressing wild-type CCR5 or CCR5 with four C-terminal serine phosphorylation sites mutated to alanine. None of the RANTES analogues caused internalization of a C-terminal cytoplasmic domain deletion mutant of CCR5, and each derivative had equal potency in inhibiting HIV-1 infection of MT-2 cells expressing this mutant. We conclude that the C-terminal cytoplasmic residues of CCR5 are necessary for receptor sequestration by RANTES analogues but that the process and the relative activity of each derivative are not dependent upon phosphorylation of the C-terminal serine residues. Two mechanisms of antiviral activity are demonstrated: receptor blockade and receptor sequestration. Potency correlates with the ability to induce CCR5 sequestration but not with receptor binding, suggesting that sequestration may make the greater contribution to antiviral activity.

Anti-HIV Agents↗
PubMed2003

Human immunodeficiency virus type 1 entry inhibitors selected on living cells from a library of phage chemokines.

The chemokine receptors CCR5 and CXCR4 are promising non-virus-encoded targets for human immunodeficiency virus (HIV) therapy. We describe a selection procedure to isolate mutant forms of RANTES (CCL5) with antiviral activity considerably in excess of that of the native chemokine. The phage-displayed library of randomly mutated and N-terminally extended variants was screened by using live CCR5-expressing cells, and two of the selected mutants, P1 and P2, were further characterized. Both were significantly more potent HIV inhibitors than RANTES, with P2 being the most active (50% inhibitory concentration of 600 pM in a viral coat-mediated cell fusion assay, complete protection of target cells against primary HIV type 1 strains at a concentration of 10 nM). P2 resembles AOP-RANTES in that it is a superagonist of CCR5 and potently induces receptor sequestration. P1, while less potent than P2, has the advantage of significantly reduced signaling activity via CCR5 (30% of that of RANTES). Additionally, both P1 and P2 exhibit not only significantly increased affinity for CCR5 but also enhanced receptor selectivity, retaining only trace levels of signaling activity via CCR1 and CCR3. The phage chemokine approach that was successfully applied here could be adapted to other chemokine-chemokine receptor systems and used to further improve the first-generation mutants reported in this paper.

Animals↗