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Biomedical subjects

A G Dalgleish

Publications and source records attributed to A G Dalgleish.

At least 19 recordsLinked to original sources
PubMed1998

CC-3052: a water-soluble analog of thalidomide and potent inhibitor of activation-induced TNF-alpha production.

The immunomodulatory drug thalidomide has been shown to be clinically useful in a number of situations due to its ability to inhibit TNF-alpha synthesis. However, its use is restricted by potentially serious side effects, including teratogenicity and neuorotoxicity; furthermore, insolubility may present problems in terms of systemic bioavailability. Recently, structural modifications of thalidomide have been designed enabling greatly enhanced anti-TNF-alpha activity in LPS-treated mice. In contrast to thalidomide (LPS-induced TNF-alpha IC50 approximately 200 microM in DMSO) and other analogs tested, one of these compounds, CC-3052 (IC50 approximately 1 microM in water), is water soluble. Furthermore, this analog exhibits increased stability in human plasma (t(1/2) approximately 17.5 vs 1.5 h for thalidomide) and appears to be nontoxic, nonmutagenic, and nonteratogenic. At pharmacologically active levels, cellular proliferation and LPS-induced IL-6 mRNA and IL-12p40 mRNA (as well as IL-1beta and IL-6 protein levels) in whole blood cultures were not affected; apparent inhibition of NK activity by CC-3052 was reversed upon addition of exogenous rTNF-alpha. In addition, IL-10 mRNA and protein levels were increased. These properties are consistent with results indicating inhibition of phosphodiesterase type IV activity by CC-3052. Furthermore, CC-3052 did not increase the degradation rate of macrophage TNF-alpha transcripts nor inhibit LPS-induced primary macrophage NF-kappaB activation. Taken together, the potency of selective TNF-alpha inhibition, water solubility, and increased plasma stability make CC-3052 an excellent candidate for further development and clinical evaluation for the treatment of TNF-alpha-mediated disease.

Adjuvants, Immunologic↗
PubMed1998

Repertoire breadth of human CD4+ T cells specific for HIV gp120 and p66 (primary antigens) or for PPD and tetanus toxoid (secondary antigens)

Antigen derived peptides bound on MHC class II molecules on presenting cells stimulate specific CD4 lymphocytes that are in a naive state if antigen is given for the first time, or in a memory state if antigen has been previously encountered. In order to compare clonal heterogeneity of the human CD4+ T helper repertoire in primary vs. recall responses, we have generated T cell lines in vitro by repeated stimulation of peripheral lymphocytes with primary or with recall antigens. Clonal heterogeneity was broad in the case of recall response to tetanus toxoid or PPD, with a high frequency of specific precursors (> 100 cells/10(6) lymphocytes). In contrast, T cell lines responsive to primary antigens (HIV gp120 or HIV p66) were oligoclonal as defined by TCR V beta gene usage and by spectratyping, and the precursor frequency was low (< 2 cells/10(6) lymphocytes). Primary T cell lines generated from blood samples drawn at different times from the same donor showed that clones with identical TCR CDR3 region coding sequences were expanded, suggesting that in these individuals a large progeny derived from one single precursor is present, even though a previous encounter with the antigen was not documented. Assuming an even in vivo distribution of such cells, the presence of one precursor every 10(6) CD4 lymphocytes (within the CD4 T repertoire that comprises roughly 10(11) CD4 T cells) indicates that approximately 10(5) identical T cells from the same clonal precursor account for the primary response against the model antigens we have studied.

Amino Acid Sequence↗
PubMed1998

Low CD4 counts rather than superantigenic-like effects account for differences in expressed T-cell receptor (TCR) repertoires between HIV-1 seropositive long-term non-progressors and individuals with progressive disease.

Analysis of HIV-infected individuals who have stable CD4 counts many years after seroconversion may provide an insight as to how the host's immune system can successfully control HIV infection. In this study we analysed the T-cell receptor (TCR) Vbeta repertoire in 13 HIV+ individuals, seven of whom were classed as long-term non-progressors (LTNP), using a technique which couples anchor PCR (AnPCR) amplification of beta-chain cDNA to differential probe hybridization with non-radioactively labelled Vbeta family specific oligonucleotide probes. There were no significant differences in the expressed TCR repertoires between the LTNP group and the other HIV-infected individuals. However, there was a statistically significant inverse correlation between CD4 count and the number of Vbeta family-specific perturbations in the recent seroconverters (SC) and those with progressive infection (PI), consistent with other shared features of clinical disease progression (Th1/Th2 switch and high frequency of viral isolation). We conclude that long-term clinical non-progression in HIV-1 infection is not associated with the loss or expansion of a particular Vbeta family; instead, low CD4 count in the PI and SC individuals was correlated with increased number of Vbeta family-specific perturbations relative to the LTNP group and that it is hence unlikely that HIV encodes a superantigen.

Adult↗
PubMed1998

Abnormal intracellular IL-2 and interferon-gamma (IFN-gamma) production as HIV-1-assocated markers of immune dysfunction.

We used three-colour cytometry to analyse intracellular cytokine production in activated whole blood cultures derived from patients with HIV-1 infection. We assessed mitogen-induced IL-2, IL-4 and IFN-gamma production from T cells as possible markers of immune dysfunction. The percentages of T cells staining for IL-2 were significantly reduced in stimulated cultures from HIV+ individuals relative to normal controls (P<0.0001); this reduction was observed in both the CD4+ and the CD8+ subsets. IL-2 production was significantly reduced in CD4+ T cells from HIV+ individuals clinically classified as symptomatics compared with HIV+ asymptomatics (P<0.001); in addition, production of IL-2 inversely correlated with viral load (r2=0.832). On the other hand, HIV+ individuals showed significantly more T cells staining positive for IFN-gamma (P<0.0001); subset analysis identified these T cells as CD8+. Increased IFN-gamma production in the CD8+ T cell subset of HIV+ individuals correlated neither with clinical status nor with plasma viral load. IL-4 staining in activated T cells was low (<5%) and no differences were observed between HIV+ and control groups. Three-colour FACS analysis of whole blood provides a sensitive, rapid and relatively easy means to detect cytokine profiles within T cell subpopulations. Only small volumes of blood are required (0.5 ml), since there is no need for cell isolation, making it more practical than ELISA or reverse transcriptase-polymerase chain reaction (RT-PCR) for the analysis of immune function in HIV+ individuals. This technique could therefore play a role in mapping the dynamics and extent of immune recovery in AIDS patients undergoing triple combination therapy.

Adult↗
PubMed1998

Tumour necrosis factor (TNF) and TNF-related molecules in HIV-1+ individuals: relationship with in vitro Th1/Th2-type response.

We examined the secretion and expression by peripheral blood mononuclear cells (PBMC) of TNF-alpha and TNF-related molecules with regard to Th1/Th2-type cytokine production. In 76 HIV+ patients at different disease stages and in 25 controls we measured cytokine (TNF-alpha/beta, interferon-gamma (IFN-gamma), IL-2, IL-4, IL-10), and activation marker secretion (sCD4, sCD8, sCD30) in phytohaemagglutinin (PHA)-stimulated and unstimulated PBMC cultures by ELISA, and membrane-bound TNF-alpha and CD30 expression by flow cytometry. We found an expansion of the TNF system in HIV+ individuals, that positively correlated with TNF-alpha, IFN-gamma and sCD8, probably representing activation of the cytotoxic compartment. In advanced disease these correlations disappeared, and TNF-alpha and TNF-related molecules positively correlated with IL-10. Our results are in line with the hypothesis that an expanded TNF system is immunopathological in conjunction with Th2-type immunity in the advanced stage of disease and with the inexorable progression to disease seen when both IL-10 and TNF-alpha are elevated.

Adult↗
PubMed1998

Immunotherapy of advanced prostate cancer: a phase I/II trial using Mycobacterium vaccae (SRL172).

OBJECTIVE: To assess whether a new heat-killed Mycobacterium vaccae preparation (SRL172), which enhances cell-mediated immunity and has been proposed for use as an immunotherapeutic agent against cancer, is safe in patients with advanced hormone-refractory prostate cancer, can stimulate desirable cytokine changes in these patients and modulate the progression of the disease. PATIENTS AND METHODS: Ten patients were given SRL172 intradermally at regular intervals. The serum prostate specific antigen (PSA) level was used as a surrogate marker of response. The proportion of peripheral blood mononuclear cells (PBMC) secreting interleukin 2 (IL2), interferon gamma (IFNgamma) and interleukin 4 (IL4) was measured by flow cytometry (FACS) before and after vaccination to assess whether the treatment induced a Th2 (predominantly humoral) to Th1 (predominantly cell-mediated) switch. RESULTS: There were no significant adverse events. In five patients the serum PSA declined during the trial and in two of these there was no concomitant change of therapy apart from vaccination with SRL172. Before vaccination with SRL172 patients had a low proportion of PBMC producing IFNgamma and IL2 (all 10) and a higher proportion secreting IL4 (all three tested), suggesting a predominantly Th2 cytokine profile. After vaccination the proportion of IL4 secreting PBMC fell in all three patients tested. The proportion of IL2 secreting PBMC increased in three patients whose PSA fell. The proportion of IFNgamma-secreting cells remained depressed in nine of 10 patients. CONCLUSION: Two patients with advanced hormone-refractory prostate cancer had a PSA response to the vaccination with SRL172. The proportion of PBMC secreting IL2 is a potential marker of response to immunotherapy.

Aged↗
PubMed1997

A double-blind placebo-controlled phase II trial of thalidomide in asymptomatic HIV-positive patients: clinical tolerance and effect on activation markers and cytokines.

A randomized double-blind, placebo-controlled study was performed to determine the safety, efficacy, and effect of thalidomide on a variety of immunological and biochemical parameters in asymptomatic human immunodeficiency virus (HIV)-positive patients. Nineteen male patients with elevated markers of immune activation and CD4 cell counts above 400/mm3 were randomized to either placebo or thalidomide at 100 mg/day for 24 weeks. However, only 3 (of 10) patients receiving thalidomide completed all 24 weeks compared to 6 (of 9) patients receiving placebo. This was mainly due to fatigue (somnolence is a recognized side effect), although this was also seen to a lesser extent in the placebo group and so may not be drug attributable. No significant changes in CD4/CD8 count, activation markers, TNF-alpha, or TNFR1 were observed. However, a nonsignificant trend toward inhibition of mitogen-induced TNF-alpha production was observed in the thalidomide arm. The lack of systemic effect and the lower tolerance of thalidomide (at this dose) in asymptomatic patients highlights the need for pharmacokinetic analysis to address possible absorption problems and the need for more potent and less toxic TNF-alpha inhibitors to be developed for use in this type of study.

Adult↗
PubMed1997

Optimisation of a peptide-based indirect ELISA for the detection of antibody in the serum of HIV-1 seropositive patients.

A modified peptide-based indirect ELISA technique for the detection of HIV-1 specific antibodies in the sera of HIV-1 seropositive individuals is described. We found that the reduction of non-specific binding of HIV-1 seropositive sera to the ELISA plate was essential for the reliable detection of serum antibodies in the peptide based indirect ELISA. Optimal results were obtained using Immulon microtitre plates, different concentrations of denatured. purified grade of casein in the blocking (1%) and washing (0.25%) solutions and by diluting HIV-1 seropositive sera 1 in 1600. These conditions reduced non-specific binding and improved assay sensitivity. We show that the inclusion of a control peptide is essential to reducing the incidence of false positive and false negative results. Taken together, the modifications described in this report improve reliability of the peptide-based indirect ELISA without compromising its sensitivity and have particular relevance for those wishing to apply the peptide-based indirect ELISA technique to serum samples which exhibit high levels of non-specific binding. To illustrate this, levels of antibody in the sera of HIV-1 seropositive and seronegative donors that are specific for peptides derived from a conserved region of HIV-1 gp120 sharing homology with the FAS apoptosis antigen were analysed using this technique.

Amino Acid Sequence↗
PubMed1997

Priming of human CD4+ antigen-specific T cells to undergo apoptosis by HIV-infected monocytes. A two-step mechanism involving the gp120 molecule.

The study of the pathology of HIV-1 infection in chimpanzees supports the idea of the crucial role of HIV-infected monocytes in the pathogenesis of AIDS, although viral mechanisms that lead to T cell dysfunction and deletion during HIV infection are still unclear. We show here that HIV-1-infected antigen-presenting monocytes (APCs) are able to prime in vitro non-HIV-infected antigen-specific CD4+ T cell lines or peripheral blood CD4+ T cells to undergo apoptosis after antigen-specific restimulation. The priming of T cells for apoptosis occurs in the absence of HIV replication in the T cells. Priming for apoptosis required two concomitant signals present on the same APC, an antigenic stimulus and a second signal provided by the HIV gp120 protein as demonstrated by the use as APCs of EBV-LCLs infected with different recombinant deleted proviruses or transfected with different HIV proteins. These results provide a mechanism for the priming for apoptosis of T cells in HIV-infected patients, implicating a role for HIV-infected APCs in the induction of T cell dysfunction and depletion in AIDS.

Antigen-Presenting Cells↗
PubMed1997

Lack of T cell response to cardiac myosin and a reduced response to PPD in patients with idiopathic dilated cardiomyopathy.

Idiopathic dilated cardiomyopathy (IDCM) is the main cause of cardiac transplantation in young adults in the 20-40 years age group in the Western world. Recent evidence supports a possible role for autoimmune pathogenesis in IDCM and it has been suggested that T cells could mediate the disease. Cardiac myosin is one of the putative autoantigens recognized by antibodies from patients with IDCM, but T cell responses to cardiac myosin have not previously been assessed. Proliferation to cardiac myosin by peripheral blood mononuclear cells (PBMC) from patients, their relatives and controls was assessed in a lymphoproliferation assay specifically designed to measure low frequency T cell precursor responses. The study group consisted of 23 patients with IDCM and 29 relatives. The control groups consisted of 10 patients with heart failure secondary to ischaemic heart disease (IHD) and 22 healthy laboratory controls. A response to myosin was observed in 16.7% of the subjects studied. However, these responses were all of low precursor frequency and no dose response for antigen-specific proliferation could be observed. More importantly, there was no correlation between myosin-specific T cell responses and IDCM, as only one IDCM patient and four IDCM relatives (three out of the four with left ventricular enlargement (LVE)) were among the 14 subjects whose PBMC exhibited a proliferative response. However, proliferation of PBMC to purified protein derivative of Mycobacterium bovis (PPD) was significantly suppressed in IDCM patients when compared with the laboratory control group (P<0.05). PPD response data suggested that the PPD suppression correlated with disease progression. The results of our present study indicate an absence, or lack, of cardiac myosin-specific peripheral blood T cells in IDCM patients, along with the possibility of underlying impaired cell mediated immunity, reflected in the suppressed responses to PPD. Future studies looking at T cell immune mechanisms in IDCM should concentrate on the analysis of T cells from the heart itself, or look at other potential cardiac antigens from normal and diseased heart tissue.

Adolescent↗
PubMed1997

Why: gene therapy?

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Clinical Medicine↗
PubMed1996

The use of 7-amino actinomycin D in identifying apoptosis: simplicity of use and broad spectrum of application compared with other techniques.

The detection and quantitation of apoptotic cells is becoming increasingly important in the investigation of the role of apoptosis in cellular proliferation and differentiation. The pathogenesis of hematologic disorders such as aplastic anemia and the development of neoplasia are believed to involve dysregulation of apoptosis. To quantitate accurately the proportion of apoptosis cells within different cell types of a heterogeneous cell population such as blood or bone marrow, a method is required that combines the analysis of large numbers of cells with concurrent immunophenotyping of cell surface antigens. In this study, we have evaluated such a method using the fluorescent DNA binding agent, 7-amino actinomycin D (7AAD), to stain three diverse human cell lines, induced to undergo apoptosis by three different stimuli. Flow cytometric analysis defines three populations on the basis of 7AAD fluorescence and forward light scatter. We have shown by cell sorting and subsequent morphological assessment and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling that the populations defined by 7AAD represent live, apoptotic, and late-apoptotic/dead cells. This method is quick, simple, reproducible, and cheap and will be a valuable tool in the investigation of the role of apoptosis in normal physiology and in disease states.

Apoptosis↗
PubMed1996

Cytokines and soluble receptor changes in the transition from primary to early chronic HIV type 1 infection.

We studied determinants of chronic inflammation and/or immune activation in plasma from patients in the transition from primary to early chronic HIV-1 infection. The following parameters were estimated in seven patients over time: plasma concentrations of soluble CD8 (sCD8), tumor necrosis factor alpha (TNF-alpha), soluble TNF receptor type II (sTNFRII), interleukin 6 (IL-6), soluble IL-6 receptor (sIL6R), IL-10, transforming growth factor beta1 (TGF-beta1), along with CD4- and CD8-positive T cell counts, p24 antigenemia, and clinical evaluation. Results showed that concentrations of sCD8, TNF-alpha, and sTNFRII, and peripheral CD8-positive lymphocyte counts, were significantly increased in patients, compared to HIV-negative controls, and showed a trend toward normal values over time. Levels of IL6, sIL6R, IL-10, and TGF-beta1 did not differ from those of controls and did not change over time. Heterogeneity was observed among the patients in terms of CD4-positive T cell depletion, levels of sCD8, concentrations of TNF-alpha/sTNFRII, and clinical outcome. These data indicate that in the transition phase from primary acute to chronic and asymptomatic infection the host immune activation in response to the virus is highly heterogeneous and that the sustained rise in TNF-alpha and its receptor may represent an important therapeutic target in early disease. The persistence of a state of chronic inflammation and/or immune activation could influence the progression of disease independently from CD4-positive T cell counts.

Adult↗