R. Adrian Robins

Direct ex vivo comparison of the breadth and specificity of the T cells in the liver and peripheral blood of patients with chronic HCV infection

The role of intrahepatic lymphocytes in the control of hepatitis C virus (HCV) infection and the pathology associated with it is not understood; most studies of the immunology of this infection use peripheral blood lymphocyte populations. To address this further, we examined in detail the IHL from HCV‐infected patients and controls, focusing on the antigen‐specific CD8+ T lymphocyte component. Individual T cells from needle liver biopsies and peripheral blood were isolated from patients with chronic HCV infection and examined directly ex vivo. We used RT‐PCR spectratyping to compare the breadth of the T cell receptor usage in the liver in comparison with the peripheral blood, and applied MHC class I tetramer technology to investigate the numbers of HCV‐specific CD8+ cells in the two compartments. T cell receptor usage in the liver of HCV‐infected patients was broad, comparable with that in the peripheral blood of the same patients. A much higher proportion of liver CD8+ cells expressed receptors specific for HCV antigens compared with paired peripheral blood CD8+ cells. A greater proportion of the liver tetramer‐positive cells expressed the activation marker CD69, compared with those in the periphery or other CD8+ cells in the liver. In the course of chronic HCV infection, HCV‐specific CD8 cells, which have been recently activated, appear to accumulate specifically in the livers of infected patients but are present in much lower numbers in the peripheral circulation. Further studies are needed to determine the function of these cells and their role in protection and immunopathology.

INTERLEUKIN‐8 AND INDUCIBLE NITRIC OXIDE SYNTHASE mRNA LEVELS IN INFLAMMATORY BOWEL DISEASE AT FIRST PRESENTATION

Interleukin‐8 (IL‐8) and nitric oxide (NO) may be important mediators in the pathogenesis of chronic idiopathic inflammatory bowel disease (CIIBD), but their roles in disease activity in ulcerative colitis (UC) and Crohns disease (CD) are uncertain. The aim of this study was to measure mRNA for IL‐8 and inducible NO synthase (iNOS) in small mucosal biopsies from untreated patients at first presentation and to relate these measurements to the histological levels of polymorph infiltration graded on a ten‐point scale. For this purpose, a sensitive enzyme‐linked oligonucleotide chemiluminescent assay (ELOCA) was developed to quantitate reverse transcription‐polymerase chain reaction (RT‐PCR) products amplified from RNA from paired biopsy samples. The levels of IL‐8 and iNOS mRNAs were calculated as ratios of the RT‐PCR products to glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) RT‐PCR product. In UC patients, median values of IL‐8/GAPDH and iNOS/GAPDH were significantly elevated compared with controls and CD. However, in both UC and CD, the IL‐8/GAPDH and iNOS/GAPDH ratios correlated significantly with polymorph infiltration. ELOCA enabled quantitation of multiple mRNAs in small mucosal biopsies from untreated patients with CIIBD and supported a role for IL‐8 and iNOS in acute inflammation in both UC and CD.

105Ad7 cancer vaccine stimulates anti‐tumour helper and cytotoxic T‐cell responses in colorectal cancer patients but repeated immunisations are required to maintain these responses

105AD7 is a human anti‐idiotypic antibody that recognises the binding site of the anti‐tumour antibody 791T/36 and can thereby mimic the CD55 antigen. The molecular basis of 105AD7 mimicry has been identified with 3 CDR regions of 105AD7 showing similarity to 3 regions of CD55. These regions have been analysed for potential T‐cell epitopes, and sequences that are predicted to bind to HLA/A1,3,24 and to HLA/DR1,3,7 have been identified within the CDRH3 region of 105AD7. These epitopes should be stimulating CD8 and CD4 responses, respectively. This prediction was tested on 12 colorectal cancer patients receiving 105AD7 therapy. There was good concordance in 10 of 11 patients between accumulation of CD8RO cells or tumour killing and expression of HLA/A1,3,24. The only patient who failed to respond had a non‐permissive class II haplotype and failed to show a helper response. Again 10 of 11 patients showing accumulation of CD4RO cells, in vitro blastogenesis responses, enhanced IL‐2 or enhanced NK activity expressed one or more of the HLA/DR1,3,7 haplotypes. Although there was a consistent accumulation of CD45RO cells following 14 of 18 immunisations, only one patient showed a sustained memory response. Our results suggest that 105AD7 can stimulate CD4 and CD8 responses in patients with the appropriate haplotype. However, it may be necessary to continue to immunise, since few patients produce a sustained memory response. Int. J. Cancer 85:87–92, 2000.

Curcumin modulation of IFN-β and IL-12 signalling and cytokine induction in human T cells

Curcumin is a polyphenol derived from the dietary spice turmeric. It possesses diverse anti‐inflammatory and anti‐cancer properties. Curcumin has been shown to exhibit an inhibitory effect on the production of inflammatory cytokines by human monocytes and has inhibited the animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE) in association with a decrease in interleukin 12 (IL‐12) production and signal transducer and activator of transcription 4 (STAT4) activation. The type I interferon (IFN) IFN‐has the ability to suppress IL‐12. Both IL‐12 and IFN‐α/β signal through the activation by phosphorylation of STAT4. Our aim was to investigate the effects of curcumin on the ability of T cells to respond to IL‐12 or IFN‐α/β. We report that curcumin decreases IL‐12‐induced STAT4 phosphorylation, IFN‐γ production, and IL‐12 Rβ1 and β2 expression. IFN‐β‐induced STAT4 phosphorylation, IL‐10 production and IFN receptor (IFNAR) subunits 1 and 2 expression were enhanced by curcumin. Curcumin increased IFN‐α‐induced IL‐10 and IFNAR1 expression. Prior exposure to curcumin decreased IFN‐α‐induced IFNAR2 expression and did not modify the level of IFN‐α‐induced pSTAT4 generation. Thus, the effect of curcumin on STAT4 activation in T cells is dependent upon the stimulus to which the T cells have been exposed.

Regulation in chronic obstructive pulmonary disease: the role of regulatory T-cells and Th17 cells

COPD (chronic obstructive pulmonary disease) is an inflammatory disorder of the airways, which is associated with irreversible airway obstruction. The pathological hallmarks of COPD are destruction of the lung parenchyma (pulmonary emphysema), inflammation of the central airways (chronic bronchitis) and inflammation of the peripheral airways (respiratory bronchiolitis). Tobacco smoking is established as the main aetiological factor for COPD. A maladaptive modulation of inflammatory responses to inhalation of noxious particles and gases is generally accepted as being a key central pathogenic process; however, the precise regulatory mechanisms of the disease are poorly understood. Two cell types are known to be important in immune regulation, namely regulatory T-cells and the newly identified Th17 (T-helper 17) cells. Both types of cells are subsets of CD4 T-lymphocytes and modulate the immune response through secretion of cytokines, for example IL (interleukin)-10 and IL-17 respectively. The present review will begin by describing the current understanding of inflammatory cell involvement in the disease process, and then focus on the possible role of subsets of regulatory and helper T-cells in COPD.

Cell cycle- and age-dependent activation of Sod1p drives the formation of stress resistant cell subpopulations within clonal yeast cultures.

Phenotypic heterogeneity describes non‐genetic variation that exists between individual cells within isogenic populations. The basis for such heterogeneity is not well understood, but it is evident in a wide range of cellular functions and phenotypes and may be fundamental to the fitness of microorganisms. Here we use a suite of novel assays applied to yeast, to provide an explanation for the classic example of heterogeneous resistance to stress (copper). Cell cycle stage and replicative cell age, but not mitochondrial content, were found to be principal parameters underpinning differential Cu resistance: cell cycle‐synchronized cells had relatively uniform Cu resistances, and replicative cell‐age profiles differed markedly in sorted Cu‐resistant and Cu‐sensitive subpopulations. From a range of potential Cu‐sensitive mutants, cup1Δ cells lacking Cu‐metallothionein, and particularly sod1Δ cells lacking Cu, Zn‐superoxide dismutase, exhibited diminished heterogeneity. Furthermore, age‐dependent Cu resistance was largely abolished in cup1Δ and sod1Δ cells, whereas cell cycle‐dependent Cu resistance was suppressed in sod1Δ cells. Sod1p activity oscillated ∼fivefold during the cell cycle, with peak activity coinciding with peak Cu‐resistance. Thus, phenotypic heterogeneity in copper resistance is not stochastic but is driven by the progression of individual cells through the cell cycle and ageing, and is primarily dependent on only Sod1p, out of several gene products that can influence the averaged phenotype. We propose that such heterogeneity provides an important insurance mechanism for organisms; creating subpopulations that are pre‐equipped for varied activities as needs may arise (e.g. when faced with stress), but without the permanent metabolic costs of constitutive expression.

Effects of glucocorticoids on STAT4 activation in human T cells are stimulus-dependent.

Glucocorticoids affect the immune system by a number of mechanisms, including modulation of cytokine production in lymphocytes. Glucocorticoids suppress T helper cell type 1 immune responses by decreasing the ability of T cells to respond to interleukin (IL)‐12, a major inducer of interferon (IFN)‐γ. IFN‐β increases the expression of the anti‐inflammatory cytokine IL‐10 and suppresses IL‐12. Signaling pathways through IFN‐β and the IL‐12 receptor (IL‐12R) involve activation by phosphorylation of signal transducer and activator of transcription 4 (STAT4). Our aim was to investigate the effects of dexamethasone on STAT4 activation by IFN‐β and IL‐12 in human T cell blasts. We report that dexamethasone decreases IL‐12‐induced STAT4 phosphorylation and IFN‐γ production and enhances IFN‐β‐induced STAT4 activation and IL‐10 production. These effects are associated with a down‐regulation of IL‐12Rβ1 expression but an up‐regulation of IFN‐βR. These results indicate that the effect of glucocorticoids on the STAT4 signaling pathway depends on the stimulus activating that pathway.

Expression of Activity-Dependent Neuroprotective Protein in the Immune System: Possible Functions and Relevance to Multiple Sclerosis

Background: Activity-dependent neuroprotector (ADNP) is a neuroprotective molecule containing an 8-amino acid peptide, NAPVSIPQ (NAP), that is sufficient for its neuroprotective effects. Objective: To assess the expression of ADNP in the human immune system in normal subjects and multiple sclerosis patients. MaterialsandMethods: ADNP expression was assessed in peripheral blood mononuclear cells (PBMCs) from healthy donors and multiple sclerosis (MS) patients using staining with anti-ADNP (NAP) antibodies and markers for T cells, B cells, monocytes and natural killer cells. ADNP mRNA was determined in peripheral blood from MS patients (n = 24) and matched controls (n = 21). Expression of activation markers CD69 and CD154 and of IFN-γ was assessed by flow cytometry in stimulated PBMCs. Effects of NAP on immune cell proliferation was assessed by tritiated thymidine incorporation. Results: Monocytes, B cells and T cells, but not regulatory (CD4+CD25+) T cells expressed ADNP. NAP peptide decreased the expression of CD69, CD154 and IFN-γ in PBMC and caused suppressed anti-CD3-/anti-CD28-stimulated PBMC proliferation. ADNP mRNA was reduced in MS compared to control peripheral blood. Conclusion: ADNP is expressed in many immune system cells. ADNP mRNA is reduced in PBMCs in MS. The peptide NAP, which plays an important role in neuroprotection, has potential immunomodulatory properties.

HIV coreceptor and chemokine ligand gene expression in the male urethra and female cervix.

Objective:Isolates with a tropism for the coreceptor CCR5 are the predominant viral strain transmitted following heterosexual transmission. We have investigated coreceptor expression levels within male and female genital epithelia to assess whether selective transmission can be explained by elevated CCR5 expression within the genital epithelia per se. Design:Individuals attending a local genitourinary medicine unit were recruited, and samples of genital epithelia obtained using either a cytobrush (females) or urethral swab (males). Expression of coreceptor and cell marker mRNAs was then determined by reverse transcription (RT)–PCR. Methods:RNA was recovered from the epithelial cell samples then used as templates in competitive quantitative RT–PCR to measure mRNA expression of key chemokines, coreceptors and cell-type markers in the epithelial cell samples. Cell-surface coreceptor expression was also assessed in a sample of patients using fluorescent cell staining. Results:CXCR4 and CCR3 coreceptors were expressed at significantly higher levels than CCR5 within the female endo- and ectocervix and distal end of the male urethra. Increased levels of cell surface expressed CXCR4 compared to CCR5 was confirmed in samples obtained from the female genital tract by FACS analysis. Conclusions:The selective transmission of CCR5-tropic viral variants is unlikely to result simply from differential coreceptor abundance at the genital epithelia.

Colorectal Cancer Cells Induce Lymphocyte Apoptosis by an Endothelial Monocyte-Activating Polypeptide-II-Dependent Mechanism

Endothelial monocyte-activating polypeptide-II (EMAP-II) was first isolated from cell growth medium conditioned by tumor cells, and is closely related or identical with the p43 component of the mammalian multisynthase complex. In its secreted form, EMAP-II has multiple cytokine-like activities in vitro, inducing procoagulant activity on the surface of endothelial cells, increasing expression of E- and P-selectins and TNF-R1, and directing migration of monocytes and neutrophils. EMAP-II has also been shown to induce apoptosis in endothelial cells, leading to the suggestion that it is a proinflammatory polypeptide with antiangiogenic activity. The role of secreted EMAP-II in tumors remains poorly understood, and we hypothesized that EMAP-II may play a role in immune evasion by tumor cells. We investigated its effects on lymphocytes, using recombinant protein, or colorectal cancer cell lines, as a source of native EMAP-II. Recombinant EMAP-II inhibits DNA synthesis and cell division, and induces apoptosis in mitogen-activated lymphocytes in PBMC preparations, and in Jurkat T cells. Native EMAP-II, released by or expressed on the surface of colorectal carcinoma cells, also induces activation of caspase 8 and apoptosis of PBLs and Jurkat cells, which are partially blocked by addition of Abs against EMAP-II. Thus, activated lymphocytes, along with proliferating endothelial cells, are targets for the cytotoxic activity of EMAP-II. Membrane-bound and soluble EMAP-II appear to play multiple roles in the tumor microenvironment, one of which is to assist in immune evasion.