[Proinsulin: physiology, measurement, and interest in clinical biology]. / La pro-insuline : physiologie, dosage et intérêt en biologie clinique.

The proinsulin molecule results from the cleavage of pre-pro-insulin, produced in pancreatic beta cells. Its subsequent -cleavage allows the release of insulin, the key hormone of glycemia regulation and C-peptide in equimolar proportions. During fasting trial, insulinoma diagnosis relies on inadequately high insulin and C-peptide serum levels concomitant with an hypoglycemia. In this context, proinsulin assay can be interesting in the cases of discrepancy between the two parameters. In diabetes, endoplasmic reticulum stress and beta cells inflammation, lead to the secretion of misfolded proinsulin molecules. Thus, in type 2 diabetes, proinsulin/insulin ratio increases with the degree of insulin resistance. In type 1 diabetes, proinsulin/C-peptide ratio could predict the onset of diabetes in relatives. In our practice, serum pro-insulin determined using an Elisa immunoassay (Millipore®) during fasting trial can be complementary to C-peptide and insulin assays in relation to glycemia to label an hypoglycemia. In case of glucose intolerance and diabetes, proinsulin could thus be measured.

Biochemical Screening for Fetal Trisomy 21: Pathophysiology of Maternal Serum Markers and Involvement of the Placenta.

It is now well established that maternal serum markers are often abnormal in fetal trisomy 21. Their determination is recommended for prenatal screening and pregnancy follow-up. However, mechanisms leading to abnormal maternal serum levels of such markers are still debated. Our objective was to help clinicians and scientists unravel the pathophysiology of these markers via a review of the main studies published in this field, both in vivo and in vitro, focusing on the six most widely used markers (hCG, its free subunit hCGß, PAPP-A, AFP, uE3, and inhibin A) as well as cell-free feto-placental DNA. Analysis of the literature shows that mechanisms underlying each marker's regulation are multiple and not necessarily directly linked with the supernumerary chromosome 21. The crucial involvement of the placenta is also highlighted, which could be defective in one or several of its functions (turnover and apoptosis, endocrine production, and feto-maternal exchanges and transfer). These defects were neither constant nor specific for trisomy 21, and might be more or less pronounced, reflecting a high variability in placental immaturity and alteration. This explains why maternal serum markers can lack both specificity and sensitivity, and are thus restricted to screening.

Pattern of C-peptide response to oral glucose tolerance test: Interest and cut-off values.

OBJECTIVE: The oral glucose tolerance test (OGTT) classifies subjects as normal, glucose intolerant or diabetic depending on glycemia at 120 min (T120) post-test. Five insulin profiles associated with different incidences of diabetes over 10 years' follow-up were previously described following OGTT. However, insulin measurement is sensitive to hemolysis, and can be replaced by C-peptide assay on hemolyzed samples. However, little is known about patterns of C-peptide response to OGTT. DESIGN AND METHODS: In total, 128 patients were included, to establish preliminary baseline C-peptide values and to evaluate C-peptide response to OGTT in comparison to insulin response, using the Liaison XL immunoanalyzer. RESULTS: Hundred patients had a normal glycemic response, 19 were classified as glucose intolerant and 9 as diabetic. In normal subjects, median C-peptide values (nmol/L, with 5-95 percentiles) were 0.53 (0.23-1.37) at baseline, peaking at 2.36 (0.94-1.83) at T60, and decreasing to 2.09 (1.13-4.36) at T120. The C-peptide response pattern was similar but flatter than the insulin pattern because of different catabolism pathways. Nevertheless, C-peptide and insulin response profiles were discordant in only 9.4% of cases. Profile 3 (C-peptide peaking at T60) was the most prevalent in normal patients whereas profile 4 (peak at 120 min and lower level at T30 than at T60) was the most prevalent in glucose intolerant and diabetic patients. CONCLUSIONS: In OGTT, C-peptide could replace insulin determination on hemolyzed blood samples to predict the risk of type 2 diabetes.

Amniotic Aaquaporins (AQP) in Normal and Pathological Pregnancies: Interest in Polyhydramnios.

Polyhydramnios is a common feature diagnosed by ultrasound in the second half of pregnancy. Biochemical analysis of amniotic fluid can be useful when suspecting Bartter syndrome or digestive atresia but in most of cases, no etiology of polyhydramnios is found because of the complex regulation of amniotic fluid. Aquaporins (AQP) are transmembrane channel proteins contributing to water transfers. Some of them are expressed in fetal membranes and placenta. Their expression has been shown to be disrupted in some pathological conditions such as maternal diabetes, often associated with polyhydramnios. AQP-1, 3 and 8 levels in amniotic fluid were retrospectively measured in patients suffering from polyhydramnios (n=21) from 23 weeks of gestation (WG). They were compared to the levels observed in control subjects (n=96) and their relationship with maternal factors and neonatal issues was analyzed. AQP-1, 3, 8 levels were physiologically fluctuating, AQP-1 levels always being the lowest and AQP-3 the highest, with a significant decrease at the end of pregnancy. AQPs/AFP ratios increased about 8 folds during pregnancy, their kinetic profiles reflecting physiological dynamic evolution of amniotic fluid volume. In polyhydramnios, AQP-3 level tended to be decreased whereas AQP-8 level was decreased from mid-gestation whatever the etiology of polyhydramnios. No significant relationship was found between AQPs levels and either the fetal prematurity degree or macrosomia. No specific pattern was observed in idiopathic polyhydramnios, limiting the interest of AQPs dosage in amniotic fluid in the management of those complicated pregnancies.

Human catalase gene promoter haplotype and cardiometabolic improvement after bariatric surgery.

Although its powerful impact on most co-morbidities has been widely demonstrated, the metabolic outcomes of bariatric surgery (BS) show a great heterogeneity among patients. Haplotypes of one of the major antioxidant enzyme, catalase (CAT), are associated with hypertension, dyslipidemia, and diabetes. The haplotype referred to as CAT1 includes homozygous carriers of CATH1 [-844G,-89A,-20T], whereas CAT2 haplotype includes heterozygous carriers (CATH1/CATH2) and CATH2 homozygous [-844A,-89T,-20C]. The aim of our study was to evaluate the impact of CAT1 and CAT2 haplotypes on traditional cardiovascular and metabolic markers one year after BS in a women population. The 294 women with a body mass index (BMI) >35 kg/m2 were followed-up for one year after BS, monitoring their anthropometric, metabolic and inflammatory parameters. CAT1 patients had significantly improved diastolic blood pressure (DBP) and Creactive protein (CRP) levels compared to CAT2 one year after BS. In untreated women at baseline, the change of CRP one year after BS was higher in CAT1 patients. In the population of women receiving at least one anti-lipidic, anti-hypertensive or anti-diabetic treatment at baseline, DBP and fat mass were lower one year after BS in CAT1 patients and the greater change of fat mass was associated with a higher change of adiponectin. The results highlight the beneficial impact of the CAT1 haplotype on traditional cardiovascular and metabolic parameters after BS. Our findings suggest that the CAT1 haplotype could be implicated in the level of metabolic and cardiovascular improvement after BS.

Inhibitors of BCR signalling interrupt the survival signal mediated by the micro-environment in mantle cell lymphoma.

Several studies provide evidences for mantle cell lymphoma (MCL) cell survival relying on B-cell receptor (BCR)-mediated signalling pathways, whereas the nature of this activation is unknown. Significant progress in MCL treatment is achieved through therapies targeting BCR-associated kinases, i.e., Ibrutinib and Fostamatinib, inhibitors of BTK and SYK, respectively. Our study addresses survival signals emanating from the BCR or the tumour environment and how inhibiting BCR signalling effectors might impact these survival signals. We found that BTK was constitutively activated and that SYK phosphorylation was highly increased and sustained upon BCR activation of primary MCL cells. Moreover, MCL cells from leukaemic patients secreted high amount of IL-1ß, IL-6, IL-8 and CCL5. Activation of the BCR induced (i) cell survival, (ii) STAT3 activation and (iii) increased autocrine secretion of IL-1ß, IL-6, IL-8, CCL5, IL-10, TNFα and VEGF. Specific inhibition of BTK by Ibrutinib or SYK by Fostamatinib (R406) reversed these protective effects and decreased both basal and BCR-induced autocrine cytokine secretions associated with STAT3 phosphorylation. Interestingly, targeting BTK and SYK prevented and inhibited BCR-induced MCL cell adhesion to human bone marrow stromal cells (HMSCs) in short- and long-term co-culture. We demonstrated that BCR-induced survival relies on autocrine secretion of IL-1ß, TNFα and CCL5 that might facilitate adhesion of MCL cells to HMSC. Treatment with Ibrutinib or Fostamatinib blocked the chemotactic signal thus increasing apoptosis.

RANTES/CCL5 mediated-biological effects depend on the syndecan-4/PKCα signaling pathway.

The perpetuation of angiogenesis is involved in certain chronic inflammatory diseases. The accelerated neovascularisation may result from an inflammatory status with a response of both endothelial cells and monocytes to inflammatory mediators such as chemokines. We have previously described in vitro and in vivo the pro-angiogenic effects of the chemokine Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES)/CCL5. The effects of RANTES/CCL5 may be related to its binding to G protein-coupled receptors and to proteoglycans such as syndecan-1 and -4. The aim of this study was to evaluate the functionality of syndecan-4 as a co-receptor of RANTES/CCL5 by the use of mutated syndecan-4 constructs. Our data demonstrate that site-directed mutations in syndecan-4 modify RANTES/CCL5 biological activities in endothelial cells. The SDC4S179A mutant, associated with an induced protein kinase C (PKC)α activation, leads to higher RANTES/CCL5 pro-angiogenic effects, whereas the SDC4L188QQ and the SDC4A198del mutants, leading to lower phosphatidylinositol 4,5-bisphosphate (PIP2) binding or to lower PDZ protein binding respectively, are associated with reduced RANTES/CCL5 cellular effects. Moreover, our data highlight that the intracellular domain of SDC-4 is involved in RANTES/CCL5-induced activation of the PKCα signaling pathway and biological effect. As RANTES/CCL5 is involved in various physiopathological processes, the development of a new therapeutic strategy may be reliant on the mechanism by which RANTES/CCL5 exerts its biological activities, for example by targeting the binding of the chemokine to its proteoglycan receptor.

Serum proteoglycans as prognostic biomarkers of hepatocellular carcinoma in patients with alcoholic cirrhosis.

BACKGROUND: Proteoglycans are involved in neoangiogenesis and transduction of oncogenic signals, two hallmarks of carcinogenesis. METHODS: This study sought to assess the prognostic value of serum levels of three proteoglycans (endocan, syndecan-1, and glypican-3) and VEGF in 295 patients with alcoholic cirrhosis: 170 without hepatocellular carcinoma, 58 with early hepatocellular carcinoma, and 67 with advanced hepatocellular carcinoma at inclusion. We analyzed the association between proteoglycan levels and prognosis using Kaplan-Meier and Cox methods. RESULTS: Serum levels of the three proteoglycans and VEGF were increased in patients with advanced hepatocellular carcinoma compared with those without hepatocellular carcinoma or with early hepatocellular carcinoma. In multivariate analysis, high levels of serum endocan (>5 ng/mL) were independently associated with death [HR, 2.84; 95% confidence interval (CI,) 1.18-6.84; P = 0.02], but not with hepatocellular carcinoma occurrence, in patients without hepatocellular carcinoma at baseline. High serum endocan (>5 ng/mL) and syndecan-1 (>50 ng/mL) levels were significantly associated with greater risk of tumor recurrence (P = 0.025) in patients with early hepatocellular carcinoma treated by radiofrequency ablation. In patients with advanced hepatocellular carcinoma, high serum levels of endocan (P = 0.004) and syndecan-1 (P = 0.006) were significantly associated with less favorable overall survival. However, only a high level of serum syndecan-1 (>50 ng/mL) was independently associated with greater risk of death (HR, 6.21 95% CI, 1.90-20.30; P = 0.0025). CONCLUSION: Serum endocan and syndecan-1 are easily assessable prognostic serum biomarkers of overall survival in alcoholic cirrhosis with and without hepatocellular carcinoma. IMPACT: These new biomarkers will be useful to manage patients with hepatocellular carcinoma developed on alcoholic cirrhosis.

PNPLA3 rs738409, hepatocellular carcinoma occurrence and risk model prediction in patients with cirrhosis.

BACKGROUND & AIMS: Several studies have reported an association between the genetic variant rs738409 (G) in the PNPLA3 gene and the risk of cirrhosis in various liver diseases. Our purpose was to assess the influence of this polymorphism on the risk of hepatocellular carcinoma (HCC) occurrence in two distinct longitudinal cohorts of patients with cirrhosis as well as its possible usefulness in HCC-risk model prediction. METHODS: PNPLA3 rs738409 genotypes were assessed in 279 patients with alcoholic- and 253 patients with HCV-related cirrhosis. These patients were followed-up and screened for the risk of HCC, and the influence of rs738409 on the occurrence of liver cancer was assessed using the Kaplan-Meier method, then according to the multivariate Cox model. RESULTS: In patients with HCV-related cirrhosis, rs738409 genotypes did not influence the risk of HCC development (log-rank = 0.7) or death (log-rank = 0.2). Conversely, in patients with alcoholic cirrhosis, the rs738409 (GG) genotype was an independent risk factor for HCC occurrence (HR = 1.72 [1.21-2.45], log-rank = 0.002) as well as older age, male gender, and higher BMI. Combining these features enabled HCC-risk stratification of this population into three groups with the 6-year cumulative incidence ranging from 3.4% (low risk, n = 58), 12.2% (intermediate risk, n = 163), and 51.7% (high risk, n = 58), respectively (HR = 4.3 [2.7-6.4]; log-rank <0.0001). CONCLUSIONS: This study provides key data that affirm the influence of the rs738409 (GG) genotype on the occurrence of HCC in patients with alcoholic cirrhosis. Its combination with clinical features refines the selection of patients at higher risk of liver cancer development.

Chemokine RANTES promoter dimorphisms and hepatocellular carcinoma occurrence in patients with alcoholic or hepatitis C virus-related cirrhosis.

BACKGROUND: This study explores the influence of two functional genetic polymorphisms in the regulated on activation in normal T-cell expressed and secreted(RANTES) promoter on the risk of hepatocellular carcinoma (HCC) occurrence in patients with alcoholic or Hepatitis C Virus (HCV)-related cirrhosis. METHODS: RANTES C-28G and G-403A promoter dimorphisms and RANTES serum levels were assessed in 243 HCV-infected patients and 253 alcoholic patients, included at the time of diagnosis of cirrhosis and prospectively followed-up. RESULTS: During a mean follow-up time of 76 months, 137 (27.6%) patients developed HCC and 170 (34.2%) died or were transplanted. During follow-up, patients with alcoholic cirrhosis and bearing two copies of the RANTES G-403 variant (2G-403 genotype, n = 156/253) had a higher rate of HCC occurrence compared with patients carrying at least one RANTES A-403 allele (26.3% vs. 8.2%, P = 0.0004). The RANTES 2G-403 genotype was a risk factor for HCC occurrence [HR = 3.0 (1.3-5.8); first quartile time to HCC occurrence: 60 vs. 120 months; LogRank = 0.007] and death [HR = 1.4 (1.0-2.0); median time to death: 55 vs. 79 months; LogRank = 0.01] in this subgroup. Carriage of the RANTES 2G-403 genotype was not associated with HCC development or death in patients with HCV-related cirrhosis. The RANTES C-28G dimorphism did not influence the occurrence of death or HCC in either cohort of patients. CONCLUSION: This study suggests an influence of the chemokine RANTES G-403A dimorphism on the occurrence of HCC in patients with alcoholic cirrhosis. IMPACT: Our findings provide clues for future studies on RANTES gene in relation to HCC susceptibility.

Constitutive and B-cell receptor-induced activation of STAT3 are important signaling pathways targeted by bortezomib in leukemic mantle cell lymphoma.

BACKGROUND: The deregulation of several transcription factors contribute to the aggressive course of mantle cell lymphoma. This study focuses on survival signals emanating from the tumor environment and involving the signal transducer and activator of transcription (STAT) 3 through cytokines or antigen recognition. DESIGN AND METHODS: Primary mantle cell lymphoma cells were isolated from 20 leukemic patients. The phosphorylation status of STAT3 was evaluated by immunoblottting and immunofluorescence, the levels of cytokine secretion by enzyme-linked immunosorbent assay and the cell survival signals by apoptosis and cell viability assays. RESULTS: STAT3 was constitutively phosphorylated in the Jeko-1 mantle cell lymphoma cell line and in 14 out of 20 (70%) cases of leukemic mantle cell lymphoma as the result of an autocrine secretion of interleukin-6 and/or interleukin-10. In addition, B-cell receptor engagement resulted in an increase of both in vitro cell survival and STAT3 phosphorylation in primary mantle cell lymphoma cells. Inhibition of the Janus-activated kinase/STAT3 pathway increased spontaneous apoptosis and suppressed B-cell receptor-induced cell survival in all cases analyzed. The impact of in vitro exposure to the proteasome inhibitor bortezomib was next evaluated in primary mantle cell lymphoma cells. Bortezomib induced apoptosis and a decrease of both interleukin-6/interleukin-10 secretion and STAT3 phosphorylation. In addition, bortezomib inhibited B-cell receptor-triggered STAT3 phosphorylation and cell survival. CONCLUSIONS: We demonstrated that STAT3 was activated in primary mantle cell lymphoma cells either constitutively through a cytokine autocrine loop or in response to B-cell receptor engagement, both processes leading to a survival signal inhibited by bortezomib. STAT3 appears, therefore, to play a pivotal role in mantle cell lymphoma and represents a promising therapeutic target.

Monocyte chemoattractant protein-1 (MCP-1)/CCL2 secreted by hepatic myofibroblasts promotes migration and invasion of human hepatoma cells.

The aim of our study was to investigate whether myofibroblasts and the chemokine monocyte chemoattractant protein-1 (MCP-1)/CCL2 may play a role in hepatocellular carcinoma progression. We observed that hepatic myofibroblast LI90 cells express MCP-1/CCL2 mRNA and secrete this chemokine. Moreover, myofibroblast LI90 cell-conditioned medium (LI90-CM) induces human hepatoma Huh7 cell migration and invasion. These effects are strongly reduced when a MCP-1/CCL2-depleted LI90-CM was used. We showed that MCP-1/CCL2 induces Huh7 cell migration and invasion through its G-protein-coupled receptor CCR2 and, to a lesser extent, through CCR1 only at high MCP-1/CCL2 concentrations. MCP-1/CCL2's chemotactic activities rely on tyrosine phosphorylation of focal adhesion components and depend on matrix metalloproteinase (MMP)-2 and MMP-9. Furthermore, we observed that Huh7 cell migration and invasion induced by the chemokine are strongly inhibited by heparin, by beta-D-xyloside treatment of cells and by anti-syndecan-1 and -4 antibodies. Finally, we developed a 3-dimensional coculture model of myofibroblast LI90 and Huh7 cells and demonstrated that MCP-1/CCL2 and its membrane partners, CCR1 and CCR2, may be involved in the formation of mixed hepatoma-myofibroblast spheroids. In conclusion, our data show that human liver myofibroblasts act on hepatoma cells in a paracrine manner to increase their invasiveness and suggest that myofibroblast-derived MCP-1/CCL2 could be involved in the pathogenesis of hepatocellular carcinoma.

Myeloperoxidase and superoxide dismutase 2 polymorphisms comodulate the risk of hepatocellular carcinoma and death in alcoholic cirrhosis.

UNLABELLED: Alcohol increases reactive oxygen species (ROS) formation in hepatocyte mitochondria and by reduced nicotinamide adenine dinucleotide phosphate oxidases and myeloperoxidase (MPO) in Kupffer cells and liver-infiltrating neutrophils. Manganese superoxide dismutase (MnSOD) converts superoxide anion into hydrogen peroxide, which, unless detoxified by glutathione peroxidase or catalase (CAT), can form the hydroxyl radical with iron. Our aim was to determine whether Ala16Val-superoxide dismutase 2 (SOD2), G-463A-MPO, or T-262C-CAT dimorphisms modulate the risks of hepatocellular carcinoma (HCC) and death in alcoholic cirrhosis. Genotypes and the hepatic iron score were assessed in 190 prospectively followed patients with alcoholic cirrhosis. During follow-up (61.1 +/- 2.7 months), 51 patients developed HCC, and 71 died. The T-262C-CAT dimorphism did not modify hepatic iron, HCC, or death. The GG-MPO genotype did not modify iron but increased the risks of HCC and death. The hazard ratio (HR) was 4.7 (2.1-10.1) for HCC and 3.6 (1.9-6.7) for death. Carriage of one or two Ala-SOD2 allele(s) was associated with higher liver iron scores and higher risks of HCC and death. The 5-year incidence of HCC was 34.4% in patients with both the GG-MPO genotype and one or two Ala-SOD2 alleles, 5.1% in patients with only one of these two traits, and 0% in patients with none of these traits. Corresponding 5-year death rates were 37.6%, 11.6%, and 5%. CONCLUSION: The combination of the GG-MPO genotype (leading to high MPO expression) and at least one Ala-SOD2 allele (associated with high liver iron score) markedly increased the risks of HCC occurrence and death in patients with alcoholic cirrhosis.

Efficient killing of SW480 colon carcinoma cells by a signal transducer and activator of transcription (STAT) 3 hairpin decoy oligodeoxynucleotide--interference with interferon-gamma-STAT1-mediated killing.

The signal transducers and activators of transcription (STATs) convey signals from the membrane to the nucleus in response to cytokines or growth factors. STAT3 is activated in response to cytokines involved mostly in cell proliferation; STAT1 is activated by cytokines, including interferon-gamma, involved in defence against pathogens and the inhibition of cell proliferation. STAT3, which is frequently activated in tumour cells, is a valuable target with respect to achieving inhibition of tumour cell proliferation. Indeed, its inhibition results in cell death. We previously observed that inhibition of the transcription factor nuclear factor-kappaB, a key regulator of cell proliferation, with decoy oligodeoxynucleotides results in cell death. We used a similar approach for STAT3. A hairpin STAT3 oligodeoxynucleotide was added to a colon carcinoma cell line in which it induced cell death as efficiently as the STAT3 inhibitor stattic. The hairpin STAT3 oligodeoxynucleotide co-localized with STAT3 within the cytoplasm, prevented STAT3 localization to the nucleus, blocked a cyclin D1 reporter promoter and associated with STAT3 in pull-down assays. However, the same cells were efficiently killed by interferon-gamma. This effect was counteracted by the STAT3 oligodeoxynucleotide, which was found to efficiently inhibit STAT1. Thus, although it can inhibit STAT3, the hairpin STAT3 oligodeoxynucleotide appears also to inhibit STAT1-mediated interferon-gamma cell killing, highlighting the need to optimize STAT3-targeting oligodeoxynucleotides.

[Biology of arterial ageing and arteriosclerosis]. / Biologie du vieillissement artériel et artériosclérose.

Arterial ageing - arteriosclerosis - is characterised by both thickening and stiffening of the walls of large and medium arteries. The molecular and cellular mechanisms (i.e. endothelial dysfunction, matrix remodelling, ...) involved in this process are complex, and at least in part common to atherosclerotic injury. Arterial stiffness is strongly associated with cardiovascular disease and an increased risk of morbidity and mortality. The aim of this review is to provide an update on the pathophysiology and the biological process of arterial ageing and to underline the main difference with atherosclerosis damage process in particularly during the calcification step.

Cell death in NF-kappaB-dependent tumour cell lines as a result of NF-kappaB trapping by linker-modified hairpin decoy oligonucleotide.

The transcription factor NF-kappaB is frequently activated in cancer, and is therefore a valuable target for cancer therapy. Decoy oligodeoxynucleotides (ODNs) inhibit NF-kappaB by preventing its binding to the promoter region of target genes. Few studies have used NF-kappaB-targeting with ODNs in cancer. Using a hairpin NF-kappaB-decoy ODN we found that it induced growth inhibition and cell death in NF-kappaB-dependent tumour cell lines. The ODN colocalized with the p50 subunit of NF-kappaB in cells and directly interacted with it in nuclear extracts. In TNFalpha-treated cells the ODN and the p50 subunit were found in the cytoplasm suggesting that the complex did not translocate to the nucleus. Transcriptional activity of NF-kappaB was efficiently inhibited by the ODN, whereas a scrambled ODN was without effect on transcription. Thus, ODN-mediated inhibition of NF-kappaB can efficiently promote cell death in cancer cells providing a potentially powerful approach to tumour growth inhibition.

Effect of tumor necrosis factor alpha and infliximab on apoptosis of B lymphocytes infected or not with Epstein-Barr virus.

Chronic inflammation and immunosuppressive therapies increase the risk of non-Hodgkin's lymphoma associated or not with Epstein-Barr virus (EBV) infection. A possible link between infliximab treatment and increased risk of lymphoma has been suggested. Indeed, infliximab induces apoptosis of monocytes and activated T lymphocytes, but its effect on B lymphocytes infected or not with EBV is unknown. Secreted tumor necrosis factor (TNF) alpha and the expression level of TNF receptor 1 (TNFR1) and TNFR2 were compared in EBV-positive and negative B-cell lines. The impact of TNFalpha and infliximab on apoptosis of EBV-positive cells was analyzed regarding the activity of NF-kappaB. Increased expression of TNFalpha in EBV-positive cells suggested that infliximab could affect their survival. However, TNFalpha or infliximab incubation had no effect on apoptosis of EBV-positive cells. Loss of NF-kappaB activity sensitized lymphoblastoid cell lines to TNFalpha-induced apoptosis, but no direct effect of infliximab on apoptosis was detected. On the basis of our in vitro data, neither TNFalpha nor infliximab has a direct effect on apoptosis of B lymphocytes and EBV-positive cell lines. Thus, if an increased incidence of lymphoma were induced by TNFalpha blockers, it would not involve a direct effect on B cells but rather an impaired immune surveillance by T cells.

Latent membrane protein 1 regulates STAT1 through NF-kappaB-dependent interferon secretion in Epstein-Barr virus-immortalized B cells.

Constitutive activation of signal transducer and activator of transcription 1 (STAT1) is a distinctive feature of Epstein-Barr virus (EBV)-immortalized B cells (lymphoblastoid cell lines [LCLs]). The expression of STAT1 in these cells is modulated by the latent membrane protein 1 (LMP1), but the mechanism of STAT1 activation has remained unclear. We demonstrate that the tyrosine phosphorylation of STAT1 in LCLs results from an indirect pathway encompassing an NF-kappaB-dependent secretion of interferons (IFNs). The cell culture supernatant of LCLs induced tyrosine phosphorylation of STAT1 in cells with no constitutively activated STAT1. Moreover, removal of supernatant from LCLs was sufficient to decrease the phosphorylation of STAT1. Inhibition of NF-kappaB activity by different pharmacological inhibitors (i.e., parthenolide, MG132 and BAY 11-7082) and by overexpressed mutated IkappaBalpha prevented the activation of STAT1. To identify the factors involved, we performed macroarray cDNA profiling with or without inhibition of NF-kappaB. The expression of several cytokines was NF-kappaB dependent among those alpha and gamma IFNs (IFN-alpha and IFN-gamma), known activators of STAT1. By real-time PCR and enzyme-linked immunosorbent assay we show that IFN-alpha and IFN-gamma are expressed and released by LCLs in an NF-kappaB-dependent manner. Finally, the blocking of the IFN-alpha and IFN-gamma by neutralizing antibodies led to the complete inhibition of tyrosine phosphorylation of STAT1. Taken together, our results clearly show that LMP1-induced tyrosine phosphorylation of STAT1 is almost exclusively due to the NF-kappaB-dependent secretion of IFNs. Whether this response, which is usually considered to be antiviral, is in fact required for the persistence of the virus remains to be elucidated.