Vimentin antibody production in transplant patients and immunomodulatory effects of vimentin in-vitro.

We investigated the presence of antibodies to vimentin in 150 patients awaiting transplant, (50 kidney, 50 liver and 50 thoracic) and in 51 previously transplanted kidney patients whose grafts had failed. Patients with primary end stage thoracic or kidney disease did not have increased levels of vimentin antibodies, but those with primary liver failure and those with kidney graft failure did. Those with kidney graft failure were more likely to form vimentin antibodies if the patient was HLA-DQ2 positive (p=<0.001). Further to this, we observed antibody mediated rejection in five HLA-DQ2 positive re-transplant patients where no other antibodies were identified. We investigated the effects of vimentin protein on cytokine production in phytohaemagglutinin stimulated and unstimulated peripheral blood mononuclear cells. When exposed to vimentin at low levels there was increased production of IL-10. When cultures were stimulated, there was a decrease in IL-10, IL-2 and IFN-gamma production and a large increase in IL-4 production (p=0.028) compared to the controls. These results suggest that under normal conditions exposure to vimentin can lead to regulation of the immune response. However, if the immune system is active, exposure to vimentin can enhance Th-2 immunity.

Variants in the genes encoding TNF-α, IL-10, and GSTP1 influence the effect of α-tocopherol on inflammatory cell responses in healthy men.

BACKGROUND: Despite evidence of antioxidant effects of vitamin E in vitro and in animal studies, large, randomized clinical trials have not substantiated a benefit of vitamin E in reducing inflammation in humans. An individual's genetic background may affect the response to α-tocopherol supplementation, but this has rarely been investigated. OBJECTIVE: The aim of this study was to explore the role of genetic polymorphisms on changes in LPS-stimulated inflammatory cytokine production from peripheral blood mononuclear cells (PBMCs) after α-tocopherol supplementation. DESIGN: A total of 160 healthy, middle-aged male volunteers (mean age: 52.7 y) were given dietary supplements of either 75 IU (low dose; n = 57) or 600 IU (high dose; n = 103) α-tocopherol/d for 6 wk. The production of TNF-α and IL-1ß, -6, and -10 by PBMCs after LPS stimulation was measured at baseline and after 6 wk. Polymorphisms in 15 genes involved in inflammation or responses to oxidative stress were characterized in the subjects. RESULTS: The ability of α-tocopherol to affect TNF-α production by LPS-stimulated PBMCs was influenced by the TNFA -238 polymorphism (P = 0.016). The ability of α-tocopherol to affect IL-6 production was influenced by the GSTP1 313 polymorphism (P = 0.019). The ability of α-tocopherol to affect IL-1ß production was influenced by the IL10 -592 and -1082 polymorphisms (P = 0.025 and P = 0.016, respectively). CONCLUSIONS: In healthy control subjects, the effect of α-tocopherol supplementation on the production of inflammatory cytokines appears to be dependent on an individual's genotype. These genotype-specific differences may help explain some of the discordant results in studies that used vitamin E.

Cancer cachexia is associated with the IL10 -1082 gene promoter polymorphism in patients with gastroesophageal malignancy.

BACKGROUND: The genetic predisposition of the host to local or systemic inflammation may contribute to the effect of cancer cachexia. OBJECTIVE: We investigated the relation between cytokine polymorphisms (IL1B -511, IL6 -174, IL10 -1082, TNFA -308, and LTA +252) and markers of nutritional status among patients with gastroesophageal cancer to determine whether any such association was reflected by cytokine concentrations in the tumor or plasma compartments. DESIGN: Patients (n = 203) with a diagnosis of gastroesophageal cancer underwent nutritional assessment (body mass index, anthropometric measures, dysphagia scoring, and estimation of dietary intake). Single nucleotide polymorphism genotyping was performed by TaqMan allelic discrimination genotyping. Serum cytokine and C-reactive protein concentrations were determined by enzyme-linked immunosorbent assay. Tumor tissue cytokine protein concentrations (n = 56) were determined by using the Cytometric Bead Array System. RESULTS: IL10 GG and IL6 CC polymorphisms were associated with elevated serum C-reactive protein concentrations, and the IL6 CC genotype was also associated with elevated tumor tissue cytokine concentrations. At diagnosis, the IL10 GG, but not the IL6, genotype was linked with increased total weight loss: 4.9% for AA, 7.1% for AG, and 12.0% for GG (P = 0.007). Serum C-reactive protein concentrations correlated with increased weight loss (r = 0.24, P < 0.001). Compared with other genotypes, the IL10 GG genotype retained an independent association in determining the extent of weight loss on multivariate analysis (95% CI: 0.52, 3.43; P = 0.008). Possession of the GG allele was associated with a 2.3 times increased risk of developing cachexia (95% CI: 1.2, 4.3; P = 0.014). CONCLUSION: These data suggest that the IL10 genotype of the host can influence the development of cachexia among patients with gastroesophageal malignancy.

Cytokine gene polymorphisms, cancer susceptibility, and prognosis.

IL-10 is a multifunctional cytokine with both immunosuppressive and antiangiogenic functions and may have both tumor-promoting and -inhibiting properties. A large number of polymorphisms (primarily single-nucleotide polymorphisms) have been identified in the IL10 gene promoter. Convincing evidence that certain of these polymorphisms are associated with differential expression of IL-10 in vitro and in some cases in vivo was obtained, and a number of studies investigated associations between IL10 polymorphisms and cancer susceptibility and prognosis. The results from 22 studies in 13 different malignancies are reviewed. In 17 of these studies, positive associations between IL10 genotype or haplotype and disease susceptibility, progression, or both were reported. In some of these cancers genotypes associated with low IL-10 expression were a risk factor for disease or disease progression, whereas in others genotypes associated with high IL-10 expression were a risk factor. Published findings in breast cancer are as yet conflicting. Most but not all of the studies reviewed are based on small sample sizes and a limited number of IL10 polymorphisms. However, the preliminary data indicate that larger studies are required in a number of cancers to confirm initial results, extend studies to include more detailed genotype and haplotype analysis, and combine genotype and gene expression studies in the same subjects. Such studies will contribute significantly to our understanding of the biological role of IL-10 in cancer development.

Interleukin-10 polymorphisms, cancer susceptibility and prognosis.

Interleukin-10 (IL-10) is a multifunctional cytokine with both immunosuppressive and anti-angiogenic functions and may have both tumour-promoting and -inhibiting properties. A large number of polymorphisms (primarily single nucleotide polymorphisms (SNPs)) have been identified in the IL-10 gene promoter. Convincing evidence that certain of these polymorphisms are associated with differential expression of IL-10 in vitro and in some cases in vivo has been obtained and a number of studies have investigated associations between IL-10 polymorphisms and cancer susceptibility/prognosis. The results from 22 studies in 13 different malignancies are reviewed. In 17 of these studies, positive associations between IL-10 genotype or haplotype and disease susceptibility and/or progression were reported. In some of these cancers genotypes associated with low IL-10 expression were a risk factor for disease or disease progression, while in others genotypes associated with high IL-10 expression were a risk factor. Published findings in breast cancer are as yet conflicting. Most, but not all of the studies reviewed are based on small sample sizes and a limited number of IL-10 polymorphisms. However, the preliminary data obtained thus far indicate that larger studies are required in a number of cancers, in order to confirm initial results, extend studies to include more detailed genotype/haplotype analysis and combine genotype and gene expression studies in the same subjects. Such studies will contribute significantly to our understanding of the biological role of IL-10 in cancer development.

Pulmonary cachexia, systemic inflammatory profile, and the interleukin 1beta -511 single nucleotide polymorphism.

BACKGROUND: Cachexia is common in chronic obstructive pulmonary disease (COPD) and is thought to be linked to an enhanced systemic inflammatory response. OBJECTIVE: We investigated differences in the systemic inflammatory profile and polymorphisms in related inflammatory genes in COPD patients. DESIGN: A cross-sectional study was performed in 99 patients with COPD (Global Initiative for Chronic Obstructive Lung Disease stages II-IV), who were stratified by cachexia based on fat-free mass index (FFMI; in kg/m2: <16 for men and <15 for women) and compared with healthy control subjects (HCs). Body composition was determined by bioelectrical impedance analysis. Plasma concentrations and gene polymorphisms of interleukin 1beta (IL-1beta -511), IL-6 (IL-6 -174), and the tumor necrosis factor system (TNF-alpha -308 and lymphotoxin-alpha +252) were determined. Plasma C-reactive protein, leptin, and urinary pseudouridine (as a marker of cellular protein breakdown) were measured. RESULTS: Fat mass, leptin, and pseudouridine were significantly different (P < 0.001) between noncachectic patients (NCPs) and cachectic patients (CPs: n = 35); the systemic inflammatory cytokine profile was not. NCPs had a body compositional shift toward a lower fat-free mass and a higher fat mass compared with HCs. CPs and NCPs had a greater systemic inflammatory response (P < 0.05) than did HCs, as reflected in C-reactive protein, soluble TNF-R75, and IL-6 concentrations. The overall distribution of the IL-1beta -511 polymorphism was significantly different between the groups (P < 0.05). CONCLUSIONS: In COPD patients, who are characterized by an elevated systemic inflammatory response, cachexia is not discriminatory for the extent of increase in inflammatory status. This study, however, indicates a potential influence of genetic predisposition on the cachexia process.

Quantitative detection of changes in cytokine gene expression in peripheral blood mononuclear cells correlates with and precedes acute rejection in renal transplant recipients.

Immunological monitoring of transplant recipients is an attractive concept. Cytokines provide an obvious focus for research, as they are central to the human immune response. This study aimed to identify cytokines whose sequential gene expression differentiated rejectors from non-rejectors immediately following renal transplantation. Forty-five renal transplant recipients (15 rejectors) and 13 living donors were recruited. Total RNA was extracted from the peripheral blood mononuclear cells and reverse transcribed. Cytokine gene expression levels of IL-4, IL-10, TNF-alpha and TGF-beta1 were measured using TaqMan. IL-10 expression increased significantly following donor surgery. IL-4 and TNF-alpha patterns clearly differentiated between rejectors and non-rejectors. In the rejectors significant increases occurred more than 48 h before clinical graft dysfunction. Negative predictive values were 76% and 80% for IL-4 and TNF-alpha, respectively. This study has identified two cytokines (IL-4 and TNF-alpha) whose gene expression patterns differentiate between rejecting and non-rejecting renal transplant recipients making immunological monitoring possible.

Workshop on cancer biometrics: identifying biomarkers and surrogates of cancer in patients: a meeting held at the Masur Auditorium, National Institutes of Health.

The current excitement about molecular targeted therapies has driven much of the recent dialog in cancer diagnosis and treatment. Particularly in the biologic therapy of cancer, identifiable antigenic T-cell targets restricted by MHC molecules and the related novel stress molecules such as MICA/B and Letal allow a degree of precision previously unknown in cancer therapy. We have previously held workshops on immunologic monitoring and angiogenesis monitoring. This workshop was designed to discuss the state of the art in identification of biomarkers and surrogates of tumor in patients with cancer, with particular emphasis on assays within the blood and tumor. We distinguish this from immunologic monitoring in the sense that it is primarily a measure of the tumor burden as opposed to the immune response to it. Recommendations for intensive investigation and targeted funding to enable such strategies were developed in seven areas: genomic analysis; detection of molecular markers in peripheral blood and lymph node by tumor capture and RT-PCR; serum, plasma, and tumor proteomics; immune polymorphisms; high content screening using flow and imaging cytometry; immunohistochemistry and tissue microarrays; and assessment of immune infiltrate and necrosis in tumors. Concrete recommendations for current application and enabling further development in cancer biometrics are summarized. This will allow a more informed, rapid, and accurate assessment of novel cancer therapies.

Genotypic effect of the -565C>T polymorphism in the ABCA1 gene promoter on ABCA1 expression and severity of atherosclerosis.

OBJECTIVE: Loss-of-function mutations of the ATP-binding cassette transporter A1 (ABCA1) gene cause Tangier disease, a rare genetic disorder with accumulation of lipid-laden macrophages and increased risk of atherosclerosis. Common variants of this gene may be a genetic factor for atherosclerosis in the general population. This study was performed to test the reported association between the -565C>T polymorphism and atherosclerosis severity and to investigate whether this variant per se had an effect on promoter activity of the ABCA1 gene. METHODS AND RESULTS: A cohort of patients with coronary atherosclerosis were genotyped for the -565C>T polymorphism. Logistic regression analyses showed that homozygotes of the -565T allele had greatest mean number of diseased coronary arteries, particular in nonsmokers. Real-time reverse-transcriptase polymerase chain reaction showed that in atherosclerotic plaques removed from patients undergoing endarteretomy, ABCA1 expression levels were lowest in those who had the T/T genotype and highest in those of the C/C genotype. Transfection and reporter assays demonstrated that in cultured macrophages, the -565T allelic promoter had a lower activity in driving gene expression than the -565C allelic promoter. Electrophoretic mobility shift assays displayed differential binding of nuclear proteins to the 2 alleles. CONCLUSIONS: These results indicate that the -565C>T polymorphism has an allele-specific effect on ABCA1 gene expression and provide further evidence of a genotypic effect on coronary atherosclerosis severity. The study showed that the ABCA1 gene -565C>T polymorphism was associated with severity of coronary atherosclerosis in a cohort of patients from Southern England and that this sequence variant per se had an effect on promoter activity of the ABCA1 gene. The data support the notion that common ABCA1 gene variants can contribute to interindividual variability in atherosclerosis susceptibility and severity.

EGF +61 gene polymorphism and susceptibility to and prognostic markers in cutaneous malignant melanoma.

CMM is the most serious cutaneous malignancy and is increasing in frequency among most Caucasian populations, where the most important risk factor is exposure to UV light. Relatively little is known of the genetic factors that mediate susceptibility to and prognosis in sporadic CMM, although a number of genes have been implicated. A striking association between EGF polymorphism and Breslow thickness of invasive CMM has been reported. We have sought confirmation of this finding in an independent study of 159 patients and 310 controls using TaqMan fluorescence-based genotyping for EGF +61. In our study group, there were no significant differences in EGF genotype frequencies between patients and controls nor was EGF genotype associated with tumour growth phase, stage or mitotic count. However, correlation between EGF genotype and Breslow thickness showed a modestly significant increase in frequency of the EGF (G/G) genotype among tumours >3.5 mm thick (30.0% vs. 9.8%, p = 0.03). In summary, in our group, the EGF +61 polymorphism was not a risk factor for CMM susceptibility, but this polymorphism may play a role in disease progression.

House keeping genes and gene expression analysis in transplant recipients: a note of caution.

BACKGROUND: House keeping genes are often used as a means of standardising results obtained in gene expression investigations. This study was performed to investigate whether beta-actin, beta2-microglobulin (two genes frequently quoted as house keeping genes) and/or transferrin receptor would be suitable house keeping genes for use in gene expression analysis of renal transplant recipients. METHODS: Sequential expression of all three genes was measured in the peripheral blood mononuclear cells of 13 living donors and 45 renal transplant recipients, pre-operatively and then daily for up to 2 weeks. Fifteen of the recipients experienced an episode of biopsy proven acute rejection. Gene expression measurement was performed using quantitative real time 'TaqMan' PCR technology. RESULTS: Gene expression of all three genes was unchanged in the living donor cohort. However, in the transplant recipients there were significant increases in expression following transplantation in the non-rejectors, and preceding the diagnosis of acute rejection. In the latter group, levels returned to pre-transplant values after the commencement of anti-rejection therapy. CONCLUSIONS: Beta-actin, beta2-microglobulin and transferrin receptor gene expression, although not influenced by surgery, is influenced by transplantation, acute rejection and anti-rejection therapy making these genes unsuitable as house keeping genes following renal transplantation. These findings may cast doubt on the results of some studies that used these genes for the purposes of standardisation when looking at cDNA measurement. We suggest that any group wishing to use a house keeping gene ensure that its expression is independent of study parameters prior to the start of the study.

Comparative evaluation of 'TaqMan' RT-PCR and RT-PCR ELISA for immunological monitoring of renal transplant recipients.

By sequentially monitoring cytokine gene expression (using RT-PCR ELISA technology) in peripheral blood cells of renal transplant recipients in the early post-operatively period we have shown that expression patterns correlate with clinical events, namely acute allograft rejection. This strategy may have the potential of predicting acute rejection prior to clinical detection. Unfortunately, the technique used was time consuming and only semi-quantitative and, therefore, not suitable for clinical application. In this study, we have sought to confirm the results of the early work using a real time quantitative RT-PCR technique ('TaqMan'), which may be applicable in the clinical laboratory. 'TaqMan' primers and probes were designed for Interleukin (IL)-4 and IL-10 using Primer Express software. Cytokine gene expression for both cytokines was re-measured in stored cDNA samples from 27 non-rejectors and 14 patients experiencing an episode of biopsy proven acute rejection. Compared to pre-transplant levels, IL-4 gene expression fell significantly on post-operative days 2 and 7 before returning to baseline values by day 14 in the non-rejectors. In the rejectors, the initial significant fall was again seen, but with an earlier return to pre-transplant levels at the time of rejection diagnosis. This was followed by a further significant fall in levels 48 h after the initiation of anti-rejection therapy. These different patterns for rejectors and non-rejectors were seen using both techniques. For IL-10, gene expression increased significantly following transplantation throughout the study period when compared to baseline values. This pattern was seen using both techniques. In the rejectors, there were different patterns seen depending on the technique used. When using RT-PCR ELISA, the initial rise was again seen followed by a return to baseline values at the time of rejection diagnosis followed by a further significant rise in gene expression after the start of anti-rejection treatment. The pattern resembled those of the non-rejectors when expression was measured using 'TaqMan'. This study has confirmed that sequential monitoring of cytokine gene expression, measured in peripheral blood mononuclear cells, detects significant changes that correlate with clinical events in renal transplant recipients, including acute rejection, although not all changes detected with RT-PCR ELISA were confirmed. Therefore, real time quantitative RT-PCR technology may be useful in monitoring the immunological status of these patients in the early post-operative period.

The role of hemochromatosis susceptibility gene mutations in protecting against iron deficiency in celiac disease.

BACKGROUND & AIMS: Celiac disease and hereditary hemochromatosis are common HLA-defined conditions in northwestern Europe. We sought to determine whether there is a genetic relationship between the 2 diseases and if hemochromatosis susceptibility gene (HFE) mutations are protective against iron deficiency in celiac disease. METHODS: Polymerase chain reaction amplification using sequence-specific primers capable of identifying the 2 HFE gene mutations (H63D and C282Y) and the HLA class I and II alleles was used to type 145 white patients with celiac disease and 187 matched controls. Hemoglobin and fasting serum iron levels in celiac patients were measured at diagnosis. RESULTS: HFE gene mutations, H63D or C282Y, were identified in 70 celiac patients (48.3%) and 61 controls (32.6%) (P = 0.004). The C282Y mutation was associated with HLA-A*03 and B*07 alleles in controls and with A*01, A*03, B*08, and DRB1*0301 alleles in celiac patients; the H63D mutation was associated with HLA-A*25 and DRB1*03 alleles in controls and A*29 and DRB1*03 alleles in celiac patients. At diagnosis, celiac patients with the C282Y mutation had higher mean hemoglobin and fasting serum iron levels compared with the HFE wild type (P = 0.0002 and 0.006, respectively). This was not observed with the H63D mutation. CONCLUSIONS: In celiac disease, HFE gene mutations are common and are in linkage disequilibrium with different HLA alleles compared with controls. A disease-specific haplotype that carries C282Y and DQB1*02 is suggested. We propose that HFE gene mutations provide a survival advantage by ameliorating the iron deficiency seen in celiac patients.

The ability of fish oil to suppress tumor necrosis factor alpha production by peripheral blood mononuclear cells in healthy men is associated with polymorphisms in genes that influence tumor necrosis factor alpha production.

BACKGROUND: Tumor necrosis factor alpha (TNF-alpha) mediates inflammation. High TNF-alpha production has adverse effects during disease. Polymorphisms in the TNF-alpha and lymphotoxin alpha genes influence TNF-alpha production. Fish oil suppresses TNF-alpha production and has variable antiinflammatory effects on disease. OBJECTIVE: We examined the relation between TNF-alpha and lymphotoxin alpha genotypes and the ability of dietary fish oil to suppress TNF-alpha production by peripheral blood mononuclear cells (PBMCs) in healthy men. DESIGN: Polymorphisms in the TNF-alpha (TNF*1 and TNF*2) and lymphotoxin alpha (TNFB*1 and TNFB*2) genes were determined in 111 healthy young men. TNF-alpha production by endotoxin-stimulated PBMCs was measured before and 12 wk after dietary supplementation with fish oil (6 g/d). RESULTS: Homozygosity for TNFB*2 was 2.5 times more frequent in the highest than in the lowest tertile of inherent TNF-alpha production. The percentage of subjects in whom fish oil suppressed TNF-alpha production was lowest (22%) in the lowest tertile and doubled with each ascending tertile. In the highest and lowest tertiles, mean TNF-alpha production decreased by 43% (P < 0.05) and increased by 160% (P < 0.05), respectively. In the lowest tertile of TNF-alpha production, only TNFB*1/TNFB*2 heterozygous subjects were responsive to the suppressive effect of fish oil. In the middle tertile, this genotype was 6 times more frequent than the other lymphotoxin alpha genotypes among responsive individuals. In the highest tertile, responsiveness to fish oil appeared unrelated to lymphotoxin alpha genotype. CONCLUSION: The ability of fish oil to decrease TNF-alpha production is influenced by inherent TNF-alpha production and by polymorphisms in the TNF-alpha and lymphotoxin alpha genes.

Influence of cytokine gene polymorphisms on the development of prostate cancer.

Polymorphisms in the promoter regions of cytokine genes may influence prostate cancer (PC) development via regulation of the antitumor immune response and/or pathways of tumor angiogenesis. PC patients (247) and 263 controls were genotyped for interleukin (IL)-1beta-511, IL-8-251, IL-10-1082, tumor necrosis factor-alpha-308, and vascular endothelial growth factor (VEGF)-1154 single nucleotide polymorphisms. Patient control comparisons revealed that IL-8 TT and VEGF AA genotypes were decreased in patients compared with controls [23.9 versus 32.3%; P = 0.04, odds ratio (OR) = 0.66, 95% confidence interval (CI) 0.44-0.99 and 6.3 versus 12.9%; P = 0.01, OR = 0.45, 95% CI 0.24-0.86, respectively], whereas the IL-10 AA genotype was significantly increased in patients compared with controls (31.6 versus 20.6%; P = 0.01, OR = 1.78, 95% CI 1.14-2.77). Stratification according to prognostic indicators showed association between IL-8 genotype and log prostate-specific antigen level (P = 0.05). These results suggest that single nucleotide polymorphisms associated with differential production of IL-8, IL-10, and VEGF are risk factors for PC, possibly acting via their influence on angiogenesis.

Gene polymorphisms, inflammatory diseases and cancer.

Genes whose products play a critical role in regulation of the immune response include the human leucocyte antigen (HLA) and cytokine families of genes. The HLA genes are the most polymorphic found in the human genome, and the bulk of this polymorphism results in functional differences in expressed HLA molecules, resulting in inter-individual differences in presentation of peptide antigens to T-cells. In addition, a considerable number of cytokine-associated gene polymorphisms have been identified, the bulk of which occur in the upstream promoter sequences of these genes, which in many cases results in differential in vitro expression of the respective pro- or anti-inflammatory gene product. Particular HLA polymorphisms result in well-defined associations with a large number of immunologically-mediated diseases, including some diseases with known dietary risk factors. For example, individuals of HLA-DQA1*0501, DQB1*0201 genotype have a greater than 200-fold increased risk of developing intolerance to dietary wheat gluten (coeliac disease), and additional HLA-related factors may influence the development of malignant lymphoma within pre-existing coeliac disease. Similarly, HLA-DRB1 alleles sharing a common sequence motif constitute the primary known genetic risk factor for rheumatoid arthritis. The influence of polymorphisms associated with differential cytokine expression on disease susceptibility is currently of much interest. Most attention has been focused on associations with susceptibility to benign immunologically-mediated diseases, including a number of gut diseases. However, recent work from our laboratory indicates that cytokine polymorphisms may influence susceptibility to and prognosis in a number of different cancers, including malignant melanoma skin cancer and solid tumours which may be influenced by diet, such as prostate cancer (collaboration with the CRC/BPG UK Familial Prostate Cancer study). In addition, preliminary work suggests that dietary modulation of expression levels of certain cytokines in healthy human subjects may be genotype dependent.