Stephen E. Christmas

Allele frequency net: a database and online repository for immune gene frequencies in worldwide populations

The allele frequency net database (http://www.allelefrequencies.net) is an online repository that contains information on the frequencies of immune genes and their corresponding alleles in different populations. The extensive variability observed in genes and alleles related to the immune system response and its significance in transplantation, disease association studies and diversity in populations led to the development of this electronic resource. At present, the system contains data from 1133 populations in 608 813 individuals on the frequency of genes from different polymorphic regions such as human leukocyte antigens, killer-cell immunoglobulin-like receptors, major histocompatibility complex Class I chain-related genes and a number of cytokine gene polymorphisms. The project was designed to create a central source for the storage of frequency data and provide individuals with a set of bioinformatics tools to analyze the occurrence of these variants in worldwide populations. The resource has been used in a wide variety of contexts, including clinical applications (histocompatibility, immunology, epidemiology and pharmacogenetics) and population genetics. Demographic information, frequency data and searching tools can be freely accessed through the website.

Inflammatory mediators in acute pancreatitis

Inflammatory mediators play a key role in acute pancreatitis and the resultant multiple organ dysfunction syndrome, which is the primary cause of death in this condition. Recent studies have confirmed the critical role played by inflammatory mediators such as TNF‐α, IL‐1β, IL‐6, IL‐8, PAF, IL‐10, C5a, ICAM‐1, and substance P. The systemic effects of acute pancreatitis have many similarities to those of other conditions such as septicaemia, severe burns, and trauma. The delay between the onset of inflammation in the pancreas and the development of the systemic response makes acute pancreatitis an ideal experimental and clinical model with which to study the role of inflammatory mediators and to test novel therapies. Elucidation of the key mediators involved in the pathogenesis of acute pancreatitis will facilitate the development of clinically effective anti‐inflammatory therapy. Copyright

Expression of the chemokines MCP-1/JE and cytokine-induced neutrophil chemoattractant in early acute pancreatitis

Introduction Inflammatory mediators play a critical role in acute pancreatitis. The precise role played by members of the chemokine family remains unclear. Aims To investigate the expression of the CC chemokine monocyte chemotactic protein (MCP)-1/JE and the CXC chemokine cytokine-induced neutrophil chemoattractant (CINC) in early acute pancreatitis. Methodology Pancreatitis was induced in rats, either by intraperitoneal injection of cerulein or by infusion of 5% sodium taurocholate into the pancreatic duct. Expression of MCP-1/JE and CINC in pancreas and plasma was determined by immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), Northern analysis, and quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). Results Following induction of acute pancreatitis, MCP-1/JE and CINC immunoreactivity was seen in acinar cells. Infiltrating neutrophils were strongly immunolabeled with an anti-MCP-1/JE antibody, whereas macrophages reacted strongly with an antibody to CINC. Northern analysis and quantitative real-time RT-PCR demonstrated upregulation of MCP-1/JE and CINC mRNA levels in pancreatic tissue. Plasma MCP-1 levels were significantly increased after 6 hours in the cerulein hyperstimulation model (2,444 ± 93 &mgr;g/mL versus control, 1,853 ± 262 &mgr;g/mL;p < 0.05). Plasma CINC levels were significantly increased after 6 hours in the cerulein hyperstimulation model (1,680 ± 134 &mgr;g/mL versus control, 725 ± 128 &mgr;g/mL;p < 0.005) and after 3 hours in the bile salt infusion model (6,663 ± 1,405 &mgr;g/mL versus control, 2,339 ± 800 &mgr;g/mL;p < 0.05). Conclusion CINC and MCP-1/JE may be early mediators of the inflammatory response in acute pancreatitis.

b3a2 BCR-ABL fusion peptides as targets for cytotoxic T cells in chronic myeloid leukaemia

Peptide sequences spanning the BCR–ABL protein junction potentially constitute novel leukaemia‐specific antigens. 9‐mer b3a2 fusion peptides have been reported to bind with high affinity to HLA‐A3, ‐A11 and ‐B8. We have studied the effect of b3a2 BCR–ABL junctional peptides on the cytotoxic T‐cell (CTL) response against normal and chronic myeloid leukaemia (CML) cells. Antigen‐presenting cells (APCs) were prepared from HLA‐A3‐ or ‐B8‐positive peripheral blood mononuclear cells (PBMCs) by incubation with phytohaemagglutinin (PHA) and interleukin (IL)‐2 for 7 d. These APCs were pulsed with the respective b3a2 junctional peptide in the presence of β2‐microglobulin and were then used to challenge autologous PBMCs at 7‐d intervals in the presence of IL‐2, IL‐6, IL‐7 and IL‐12. On subsequent exposure to target cells (either further pulsed normal APCs or unpulsed CML cells), specific HLA‐restricted CTL responses were observed against all HLA‐A3/‐B8 matched normal target cells tested, but not to targets that were HLA mismatched. Cytotoxicity was also induced against HLA‐A3/‐B8 unpulsed CML cells, but not against unmatched CML cells. These data indicate (i) that endogenous BCR–ABL junctional peptides may be presented by CML cells and (ii) that exogenous peptides are potential stimulators of autologous antileukaemic CTLs.

Treatment with Met‐RANTES reduces lung injury in caerulein‐induced pancreatitis

Severe acute pancreatitis leads to a systemic inflammatory response characterized by widespread leucocyte activation and, as a consequence, distant lung injury. In CC chemokines the first two cysteine residues are adjacent to each other. The aim of this study was to evaluate the effect of Met‐RANTES, a CC chemokine receptor antagonist, on pancreatic inflammation and lung injury in caerulein‐induced acute pancreatitis in mice.

Detection of interleukin-8 mRNA and protein in human colorectal carcinoma cells

Interleukin-8 (IL-8) is a member of the chemokine family of pro-inflammatory chemotactic cytokines and is secreted by some human colorectal carcinoma cell lines. We have used in situ hybridisation and immunohistochemistry to determine whether IL-8 mRNA and protein, respectively, are produced by human colorectal carcinoma cells in vivo. IL-8 mRNA was detected within the cytoplasm of tumour cells in all nine samples tested, including that of a tumour which had metastasised to a lymph node. Non-involved colonic mucosa within the same tissue blocks showed much weaker labelling. IL-8 protein was detected in 74% (23/31) of tumour samples and was mainly localised to the tumour cell cytoplasm. In 30% of cases, staining was heterogeneous, with between 1 and 30% of cells being positive. In some tumour cells, IL-8 showed a perinuclear distribution resembling that found by in situ hybridisation. Some infiltrating leucocytes, endothelial cells and fibroblast-like cells within the tumour sections were also positive for IL-8 mRNA and protein. The possibilities that colorectal tumours produce IL-8 to aid invasion and/or metastasis or as a tumour growth factor are discussed.

Associations between genes for killer immunoglobulin-like receptors and their ligands in patients with solid tumors

Killer immunoglobulin-like receptor (KIR) and human leukocyte antigen (HLA) genotypes were analyzed from panels of lung (non-small-cell lung cancer [NSCLC] and small-cell lung cancer [SCLC]), colon, and kidney cancer patients and compared with normal control subjects. No significant differences were noted between KIR gene frequencies in patients compared with normal subjects. When combinations of KIR genes and their HLA ligands were considered, there were significant decreases in frequencies of both KIR2DL2 and KIR2DL3 in homozygotes for their ligand HLA-C1, and an increase in the frequency of KIR3DL1 and its ligand HLA-Bw4 in kidney cancer patients compared with controls. Both associations were partly attributable to changes in ligand frequencies alone. NSCLC patients showed a significant increase in the frequency of KIR2DL1 and its ligand HLA-C2 and a corresponding decrease in frequency of KIR2DL3 and its ligand HLA-C1 in homozygotes. In NSCLC, the Ile80 form of HLA-Bw4 was decreased in KIR3DL1+ HLA-Bw4+ patients, whereas in SCLC the Ile80 form was increased and the Thr80 form decreased in KIR3DS1+ HLA-Bw4+ patients. These findings are consistent with increased co-expression of high-affinity inhibitory KIRs and their ligands, potentially resulting in decreased natural killer cell function, and hence with natural killer cells having a protective role in lung and kidney cancer but not colon cancer.

Levels of expression of complement regulatory proteins CD46, CD55 and CD59 on resting and activated human peripheral blood leucocytes

The cell surface complement regulatory (CReg) proteins CD46, CD55 and CD59 are widely expressed on human lymphoid and non‐lymphoid cells. This study aimed to compare systematically levels of CReg expression by different leucocyte subsets and to determine whether levels were increased following activation in vitro. Levels of each CReg protein were similar on freshly isolated monocytes and all major lymphocyte subsets, except that CD4+ cells expressed significantly less CD46 than CD8+ cells (P < 0·05) while the reverse was observed for CD55 (P < 0·02). CD56+ cells, predominantly natural killer cells, expressed significantly lower levels of CD59 than T cells (P < 0·02). CD45RO+ cells had higher levels of surface CD46 and CD59, but lower levels of CD55, than CD45RO– cells (P < 0·02); CD25+ cells also expressed significantly less CD55 than CD25– cells (P < 0·002). Neutrophils expressed higher levels of CD59, but lower levels of CD55, than monocytes. Following activation with phytohaemagglutinin, CD46 was up‐regulated on all leucocyte subsets with the exception of CD56+ cells. Both CD55 and CD59 were also markedly up‐regulated on monocytes, and CD55 expression was greater on CD8+ than CD4+ cells following activation (P < 0·02). Lipopolysaccharide treatment did not significantly alter B‐cell expression of CReg proteins whereas CD55 and CD59, but not CD46, were significantly up‐regulated on monocytes (P < 0·02). These observations that CReg proteins are up‐regulated on certain activated leucocyte subsets indicate that levels would be increased following immune responses in vivo. This could enhance both protection against local complement activation at inflammatory sites and also the immunoregulatory properties of these leucocytes.

Expression of functional molecules by human CD3- decidual granular leucocyte clones.

Cell surface and cytoplasmic antigen expression by 35 CD3− decidual granular leucocyte (DGL) clones, derived from human endometrial tissue in the first trimester of pregnancy, has been compared with both that of fresh CD3− decidual leucocytes and that of CD3− peripheral blood natural killer (PBNK) cell clones (n = 12). The majority of DGL clones retained the antigenic phenotype of fresh cells, although CD103 (HML‐1) was expressed on 50% of DGL clones but only 17% of fresh DGL. Both cytoplasmic CD3ζ and CD3ε chains were detected in > 90% of DGL clones in the absence of cell surface CD3. Cytoplasmic CD3ζ was present in almost all fresh CD3− DGL, whereas CD3ε was not. Most DGL clones did not express surface Fcγ receptors I‐III (CD64, ‐32 and ‐16, respectively) and complement receptors (CR) types 1 and 2 (CD35 and 21, respectively), but 43% expressed CR3 (CD11b/18); in contrast, all PBNK clones were CR3+. The NK cell‐associated molecules Kp43 (CD94) and the p58 molecule recognized by the HP3E4 monoclonal antibody were both present on a higher proportion of CD3− PBNK (91% and 50%, respectively) than DGL clones (31% and 14%, respectively), despite expression of CD94 by > 90% of fresh CD56+ decidual leucocytes. Five of 35 CD3− DGL clones expressed cytoplasmic CD3ζ in the absence of expression of CD2, CD16 or the p58 molecule recognized by HP3E4. These variations between CD3− DGL and PBNK cell clones in expression of functional molecules may be related to previously reported differences in major histocompatibility complex‐non‐restricted cytotoxic activities between these two cell types.

Perforin-expressing lymphocytes in peripheral blood and decidua of human first-trimester pathological pregnancies

PROBLEM: We have shown previously that the decidua of first‐trimester human pregnancy is heavily infiltrated with perforin‐positive cells. The aim was to detect expression of perforin in both decidual lymphocytes (DL) and peripheral blood lymphocytes (PBL) in the first trimester of pathological pregnancies: Anembryonic pregnancy and missed abortion.