Decreased expression of the glucocorticoid receptor-GILZ pathway in Kupffer cells promotes liver inflammation in obese mice.

BACKGROUND & AIMS: Kupffer cells (KC) play a key role in the onset of inflammation in non-alcoholic steatohepatitis (NASH). The glucocorticoid receptor (GR) induces glucocorticoid-induced leucine zipper (GILZ) expression in monocytes/macrophages and is involved in several inflammatory processes. We hypothesized that the GR-GILZ axis in KC may contribute to the pathophysiology of obesity-induced liver inflammation. METHODS: By using a combination of primary cell culture, pharmacological experiments, mice deficient for the Gr specifically in macrophages and transgenic mice overexpressing Gilz in macrophages, we explored the involvement of the Gr-Gilz axis in KC in the pathophysiology of obesity-induced liver inflammation. RESULTS: Obesity was associated with a downregulation of the Gr and Gilz, and an impairment of Gilz induction by lipopolysaccharide (LPS) and dexamethasone (DEX) in KC. Inhibition of Gilz expression in isolated KC transfected with Gilz siRNA demonstrated that Gilz downregulation was sufficient to sensitize KC to LPS. Conversely, liver inflammation was decreased in obese transgenic mice specifically overexpressing Gilz in macrophages. Pharmacological inhibition of the Gr showed that impairment of Gilz induction in KC by LPS and DEX in obesity was driven by a downregulation of the Gr. In mice specifically deficient for Gr in macrophages, Gilz expression was low, leading to an exacerbation of obesity-induced liver inflammation. CONCLUSIONS: Obesity is associated with a downregulation of the Gr-Gilz axis in KC, which promotes liver inflammation. The Gr-Gilz axis in KC is an important target for the regulation of liver inflammation in obesity.

Glucocorticoid-induced leucine zipper enhanced expression in dendritic cells is sufficient to drive regulatory T cells expansion in vivo.

Tolerance induction by dendritic cells (DCs) is, in part, mediated by the activation of regulatory T cells (Tregs). We have previously shown in vitro that human DCs treated with glucocorticoids (GCs), IL-10, or TGF-ß upregulate the GC-Induced Leucine Zipper protein (GILZ). GILZ overexpression promotes DC differentiation into regulatory cells that generate IL-10-producing Ag-specific Tregs. To investigate whether these observations extend in vivo, we have generated CD11c-GILZ(hi) transgenic mice. DCs from these mice constitutively overexpress GILZ to levels observed in GC-treated wild-type DCs. In this article, we establish that GILZ(hi) DCs display an accumulation of Foxp3(+) Tregs in the spleens of young CD11c-GILZ(hi) mice. In addition, we show that GILZ(hi) DCs strongly increase the Treg pool in central and peripheral lymphoid organs of aged animals. Upon adoptive transfer to wild-type recipient mice, OVA-loaded GILZ(hi) bone marrow-derived DCs induce a reduced activation and proliferation of OVA-specific T cells as compared with control bone marrow-derived DCs, associated with an expansion of thymus-derived CD25(+)Foxp3(+) CD4 T cells. Transferred OVA-loaded GILZ(hi) DCs produce significantly higher levels of IL-10 and express reduced levels of MHC class II molecules as compared with OVA-loaded control DCs, emphasizing the regulatory phenotype of GILZ(hi) DCs in vivo. Thus, our work demonstrates in vivo that the GILZ overexpression alone is sufficient to promote a tolerogenic mode of function in DCs.

CXCR4 dysfunction in non-alcoholic steatohepatitis in mice and patients.

Homing of inflammatory cells to the liver is key in the progression of non-alcoholic steatohepatitis (NASH). An abnormal response of CD4+ T-cells from obese mice to the chemotactic effect of CXCL12 has been reported but the mechanism involved in this process and relevance in patients are unknown. We aimed to explore the mechanism involved in the abnormal chemotaxis of CXC chemokine ligand 12 (CXCL12) in several mouse models of NASH and the relevance in the context of human non-alcoholic fatty liver disease (NAFLD). We assessed chemotactic responsiveness of CD4+ T-cells to CXCL12, the effect of AMD3100, a CXC chemokine receptor 4 (CXCR4) antagonist, in mice and lymphocytes from patients with NAFLD, and the affinity of CXCL12 for CXCR4. CXCL12-promoted migration of CD4+ T-cells from three different mouse models of NASH was increased and dependent of CXCR4. CD4+ T-cells from patients with NASH, but not from patients with pure steatosis, responded more strongly to the chemotactic effect of CXCL12, and this response was inhibited by AMD3100. Treatment with AMD3100 decreased the number of CD4+ T-cells to the liver in ob/ob mice. CXCL12 expression in the liver, CXCR4 and CXCR7 expression in CD4+ T-cells were not increased in three different mouse models of NASH. However, the affinity of CXCL12 for CXCR4 was increased in CD4+ T-cells of ob/ob mice. In conclusion, the CXCL12/CXCR4 pathway contributes in both mice and patients to the enhanced recruitment of CD4+ T-cells in NASH. An increased affinity of CXCL12 to CXCR4 rather than a higher expression of the chemokine or its receptors is involved in this process.

Proper desensitization of CXCR4 is required for lymphocyte development and peripheral compartmentalization in mice.

Desensitization controls G protein-dependent signaling of chemokine receptors. We investigate the physiologic implication of this process for CXCR4 in a mouse model harboring a heterozygous mutation of the Cxcr4 gene, which engenders a desensitization-resistant receptor. Such anomaly is linked to the warts, hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome, a human rare combined immunodeficiency. Cxcr4(+/mutant(1013)) mice display leukocytes with enhanced responses to Cxcl12 and exhibit leukopenia as reported in patients. Treatment with CXCL12/CXCR4 antagonists transiently reverses blood anomalies, further demonstrating the causal role of the mutant receptor in the leukopenia. Strikingly, neutropenia occurs in a context of normal bone marrow architecture and granulocyte lineage maturation, indicating a minor role for Cxcr4-dependent signaling in those processes. In contrast, Cxcr4(+/1013) mice show defective thymopoiesis and B-cell development, accounting for circulating lymphopenia. Concomitantly, mature T and B cells are abnormally compartmentalized in the periphery, with a reduction of primary follicles in the spleen and their absence in lymph nodes mirrored by an unfurling of the T-cell zone. These mice provide a model to decipher the role of CXCR4 desensitization in the homeostasis of B and T cells and to investigate which manifestations of patients with WHIM syndrome may be overcome by dampening the gain of CXCR4 function.

Influence of replacing tuberculin skin test with ex vivo interferon γ release assays on decision to administer prophylactic antituberculosis antibiotics before anti-TNF therapy.

BACKGROUND: The recommendations for detecting latent tuberculosis infection (LTBI) before antitumour necrosis factor (anti-TNF) therapy are based on the tuberculin skin test (TST), which lacks both specificity and sensitivity and can lead to unnecessary treatment with antibiotics. A study was undertaken to investigate the effect of replacing TST with interferon γ (IFNγ) release assays (IGRA) in screening for LTBI and deciding to begin prophylactic antituberculosis (TB) antibiotics before anti-TNF therapy in immune-mediated inflammatory diseases. METHODS: In 15 tertiary care hospitals, consecutive patients with rheumatoid arthritis, spondylarthropathies or Crohn's disease were screened for LTBI before anti-TNF therapy with TST, QuantiFERON TB Gold in tube (QTF-Gold IT) and T-SPOT.TB at the same time. The potential diagnosis of LTBI and the effect on the decision to begin antibiotic prophylaxis were assessed. RESULTS: Among 429 patients, 392 had results for the three tests. The results for TST, T-SPOT.TB and QTF Gold IT were positive for 35.2%, 15.1% and 9.9% of patients, respectively (p<0.0001). Antibiotics were required for 177 patients (45.2%) if positive TST results were included in the LTBI definition, 107 patients (27.3%) if TST results were replaced with results from one of the IGRA tests and 84 patients (21.4%) if TST results were replaced with QTF-Gold IT results (p<0.0001). The decision on the use of antibiotic prophylaxis was changed for 113 patients (28.8%, 95% CI 24.4% to 33.6%) if TST results were replaced with QTF-Gold IT results. CONCLUSIONS: Replacing TST with IGRA for determining LTBI allowed the proportion of patients with immune-mediated inflammatory diseases needing prophylactic anti-TB antibiotics before beginning anti-TNF agents to be reduced by half.

Expression of CXCL12 receptors in B cells from Mexican Mestizos patients with systemic Lupus erythematosus.

BACKGROUND: Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease characterized by B-cell hyper-reactivity and the production of pathogenic anti-nuclear-directed auto-antibodies (Abs). B-cell ontogeny is partly dependent on the CXCL12/CXCR4 axis for which the contribution to SLE pathogenesis remains unclear. CXCR7, the novel receptor for CXCL12, is differentially expressed among memory B-cell subsets. However, its biological role in SLE remains to be explored. METHODS: Relative CXCR4 and CXCR7 expression levels were compared by quantitative PCR in leukocytes from blood samples of 41 Mexican Mestizos patients with SLE and 45 ethnicity-matched healthy subjects. Intracellular and membrane expression of both receptors was analyzed by flow cytometry in naive and Ab-secreting B cells. B-cell responsiveness to CXCL12 was investigated using Transwell-based chemotaxis assays. Data were analyzed using the Kruskal-Wallis test for comparisons of values amongst healthy controls and patients with inactive or active SLE, and non-parametrically using the Mann-Whitney U-test for multiple comparisons and unpaired samples. Correlations were determined by Spearman's ranking. RESULT: SLE leukocytes displayed reduced levels of CXCR4 and CXCR7 transcripts. In SLE patients, a significant defect in CXCR4 expression was detected at the surface of naive and Ab-secreting B cells, associated with an abnormal intracellular localization of the receptor. CXCR7 predominantly localized in cytosolic compartments of B cells from healthy and SLE individuals. Disease activity did not impact on these expression patterns. Altered receptor compartmentalization correlated with an impaired CXCL12-promoted migration of SLE B cells. CONCLUSIONS: Our data highlight a down-regulation of CXCL12 receptors on circulating B cells from SLE patients that likely influences their migratory behavior and distribution.

Housekeeping gene variability in the liver of alcoholic patients.

BACKGROUND: Quantification of gene expression using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) requires normalization to an endogenous reference gene termed housekeeping gene (HKG). Many of the commonly used HKGs are regulated and vary under experimental conditions and disease stages. Alcoholic liver disease (ALD) is associated with several different liver histological lesions that may modulate HKG expression. We investigated the variability of commonly used HGKs (18S, ß-actin, glyceraldehyde-3-phosphate [GAPDH], and arginine/serine-rich splicing factor [SFRS4]) in the liver of patients with ALD. METHODS: Fifty consecutive patients at different stages of ALD underwent liver biopsy. The stability of HKG was assessed according to liver histological lesions. RESULTS: ß-actin had the highest coefficient of dispersion (COD) (23.9). ß-actin tended to decrease with steatosis and to increase with alcoholic hepatitis; ß-actin also increased in patients with both alcoholic hepatitis and cirrhosis. GAPDH and SFRS4 COD were 2.8 and 2.1, respectively. GAPDH was decreased with steatosis and increased with alcoholic hepatitis and fibrosis. 18S had the lowest COD (1.4). Both 18S and SFRS4 levels were not significantly modified with respect to all alcohol-induced liver histological lesions. CONCLUSIONS: In patients with ALD, the most constantly expressed HKGs are 18S and SFRS4. These genes are appropriate reference genes for normalization of RT-qPCR in the liver of patients with ALD. The use of other HKGs such as ß-actin or GAPDH would lead to misinterpretation of the results.

IFNalpha kinoid vaccine-induced neutralizing antibodies prevent clinical manifestations in a lupus flare murine model.

A major involvement of IFNalpha in the etiopathogenesis of systemic lupus erythematosus has been suggested by clinical observations, including the increase of serum levels of this cytokine in patients with active disease. Supporting this hypothesis, we have shown that expression of IFNalpha from a recombinant adenovirus (IFNalpha Adv) precipitates lupus manifestations in genetically susceptible New Zealand Black (NZB) x New Zealand White (NZW)F(1) mice (NZB/W) but not in BALB/c mice. In the present investigation, we have prepared an IFNalpha immunogen, termed IFNalpha kinoid, which, appropriately adjuvanted, induces transient neutralizing antibodies (Abs) but no cellular immune response to the cytokine and without apparent side effects. Using this preparation, we also showed that, in kinoid-vaccinated NZB/W mice, lupus manifestations, including proteinuria, histological renal lesions, and death triggered by IFNalpha Adv challenge were delayed/prevented as long as an effective threshold of anti-IFNalpha inhibitory capacity was present in the serum.

Shared reactivity of V{delta}2(neg) {gamma}{delta} T cells against cytomegalovirus-infected cells and tumor intestinal epithelial cells.

Long-lasting expansion of Vdelta2(neg) gammadelta T cells is a hallmark of cytomegalovirus (CMV) infection in kidney transplant recipients. The ligands of these cells and their role remain elusive. To better understand their immune function, we generated gammadelta T cell clones from several transplanted patients. Numerous patient Vdelta1(+), Vdelta3(+), and Vdelta5(+) gammadelta T cell clones expressing diverse Vgamma chains, but not control Vgamma9Vdelta2(+) T clones, displayed strong reactivity against CMV-infected cells, as shown by their production of tumor necrosis factor-alpha. Vdelta2(neg) gammadelta T lymphocytes could also kill CMV-infected targets and limit CMV propagation in vitro. Their anti-CMV reactivity was specific for this virus among herpesviridae and required T cell receptor engagement, but did not involve major histocompatibility complex class I molecules or NKG2D. Vdelta2(neg) gammadelta T lymphocytes expressed receptors essential for intestinal homing and were strongly activated by intestinal tumor, but not normal, epithelial cell lines. High frequencies of CMV- and tumor-specific Vdelta2(neg) gammadelta T lymphocytes were found among patients' gammadelta T cells. In conclusion, Vdelta2(neg) gammadelta T cells may play a role in protecting against CMV and tumors, probably through mucosal surveillance of cellular stress, and represent a population that is largely functionally distinct from Vgamma9Vdelta2(+) T cells.

CXCL12 expression by healthy and malignant ovarian epithelial cells.

BACKGROUND: CXCL12 has been widely reported to play a biologically relevant role in tumor growth and spread. In epithelial ovarian cancer (EOC), CXCL12 enhances tumor angiogenesis and contributes to the immunosuppressive network. However, its prognostic significance remains unclear. We thus compared CXCL12 status in healthy and malignant ovaries, to assess its prognostic value. METHODS: Immunohistochemistry was used to analyze CXCL12 expression in the reproductive tracts, including the ovaries and fallopian tubes, of healthy women, in benign and borderline epithelial tumors, and in a series of 183 tumor specimens from patients with advanced primary EOC enrolled in a multicenter prospective clinical trial of paclitaxel/carboplatin/gemcitabine-based chemotherapy (GINECO study). Univariate COX model analysis was performed to assess the prognostic value of clinical and biological variables. Kaplan-Meier methods were used to generate progression-free and overall survival curves. RESULTS: Epithelial cells from the surface of the ovary and the fallopian tubes stained positive for CXCL12, whereas the follicles within the ovary did not. Epithelial cells in benign, borderline and malignant tumors also expressed CXCL12. In EOC specimens, CXCL12 immunoreactivity was observed mostly in epithelial tumor cells. The intensity of the signal obtained ranged from strong in 86 cases (47%) to absent in 18 cases (<10%). This uneven distribution of CXCL12 did not reflect the morphological heterogeneity of EOC. CXCL12 expression levels were not correlated with any of the clinical parameters currently used to determine EOC prognosis or with HER2 status. They also had no impact on progression-free or overall survival. CONCLUSION: Our findings highlight the previously unappreciated constitutive expression of CXCL12 on healthy epithelia of the ovary surface and fallopian tubes, indicating that EOC may originate from either of these epithelia. We reveal that CXCL12 production by malignant epithelial cells precedes tumorigenesis and we confirm in a large cohort of patients with advanced EOC that CXCL12 expression level in EOC is not a valuable prognostic factor in itself. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00052468.

Blockade of B7-H1 improves myeloid dendritic cell-mediated antitumor immunity.

Suppression of dendritic cell function in cancer patients is thought to contribute to the inhibition of immune responses and disease progression. Molecular mechanisms of this suppression remain elusive, however. Here, we show that a fraction of blood monocyte-derived myeloid dendritic cells (MDCs) express B7-H1, a member of the B7 family, on the cell surface. B7-H1 could be further upregulated by tumor environmental factors. Consistent with this finding, virtually all MDCs isolated from the tissues or draining lymph nodes of ovarian carcinomas express B7-H1. Blockade of B7-H1 enhanced MDC-mediated T-cell activation and was accompanied by downregulation of T-cell interleukin (IL)-10 and upregulation of IL-2 and interferon (IFN)-gamma. T cells conditioned with the B7-H1-blocked MDCs had a more potent ability to inhibit autologous human ovarian carcinoma growth in non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice. Therefore, upregulation of B7-H1 on MDCs in the tumor microenvironment downregulates T-cell immunity. Blockade of B7-H1 represents one approach for cancer immunotherapy.

[CXCR4, a therapeutic target in rare immunodeficiencies?]. / CXCR4, une cible thérapeutique dans certains déficits immunitaires rares?

Currently, more than 200 primary immunodeficiency diseases have been discovered. In most cases, genetic defects affect the expression or the function of proteins involved in immune development and homeostasis. Some orphan immuno-hematological disorders are characterized by an abnormal leukocyte trafficking, a notion predictive of an anomaly of the chemokine/chemokine receptor system. In this review, we focus on recent advances in the characterization of dysfunctions of the CXCL12 (SDF-1)/CXCR4 signaling axis in two rare human immunodeficiencies, one associated with a loss of CXCR4 function, the Idiopathic CD4(+) T-cell Lymphocytopenia, and the other with a gain of CXCR4 function, the WHIM syndrome.

Harmful effect of adipose tissue on liver lesions in patients with alcoholic liver disease.

BACKGROUND & AIMS: Adipose tissue is an important source of cytokines. Excess weight is an independent risk factor for steatosis, acute alcoholic hepatitis (AAH), and cirrhosis in patients with alcoholic liver disease (ALD). In this study, we investigated the role of adipose tissue in human ALD. PATIENTS AND METHODS: Fifty patients with ALD underwent liver and abdominal subcutaneous adipose tissue biopsies and supplied blood samples for the investigation of cytokine gene expression and secretion, as well as liver histology. RESULTS: The levels of TNF-alpha and IL-10 in adipose tissue were higher in patients with AAH. IL-10 level in adipose tissue was also correlated with fibrosis score. TNF-alpha gene expression in adipose tissue was correlated with Maddrey score, blood C-reactive protein (CRP) concentration and liver IL-6 concentration. IL-6 production levels in the liver were higher in patients with AAH and correlated with AAH score, liver histological lesions, liver TNF-alpha concentration, Maddrey score, and blood CRP concentration. Plasma concentrations of soluble forms of TNF-receptor were correlated with inflammatory lesions in the liver, Maddrey score and fibrosis score. CONCLUSION: In patients with ALD, inflammation occurs not only in the liver, but also in the adipose tissue. Adipose tissue inflammation is correlated with the severity of pathological features in the liver. Our findings may account for the harmful interactions between body mass index, AAH, fibrosis, and cirrhosis in alcoholic patients.

Identification of glucocorticoid-induced leucine zipper as a key regulator of tumor cell proliferation in epithelial ovarian cancer.

BACKGROUND: Little is known about the molecules that contribute to tumor progression of epithelial ovarian cancer (EOC), currently a leading cause of mortality from gynecological malignancies. Glucocorticoid-Induced Leucine Zipper (GILZ), an intracellular protein widely expressed in immune tissues, has been reported in epithelial tissues and controls some of key signaling pathways involved in tumorigenesis. However, there has been no report on GILZ in EOC up to now. The objectives of the current study were to examine the expression of GILZ in EOC and its effect on tumor cell proliferation. RESULTS: GILZ expression was measured by immunohistochemical staining in tissue sections from 3 normal ovaries, 7 benign EOC and 50 invasive EOC. GILZ was not detected on the surface epithelium of normal ovaries and benign tumors. In contrast, it was expressed in the cytoplasm of tumor cells in 80% EOC specimens. GILZ immunostaining scores correlated positively to the proliferation marker Ki-67 (Spearman test in univariate analysis, P < 0.00001, r = 0.56). They were also higher in tumor cells containing large amounts of phosphorylated protein kinase B (p-AKT) (unpaired t test, P < 0.0001). To assess the effect of GILZ on proliferation and AKT activation, we used the BG-1 cell line derived from ovarian tumor cells as a cellular model. GILZ expression was either enhanced by stable transfection or decreased by the use of small interfering (si) RNA targeting GILZ. We found that GILZ increased cell proliferation, phospho-AKT cellular content and AKT kinase activity. Further, GILZ upregulated cyclin D1 and phosphorylated retinoblastoma (p-Rb), downregulated cyclin-dependent kinase inhibitor p21, and promoted the entry into S phase of cell cycle. CONCLUSION: The present study is the first to identify GILZ as a molecule produced by ovarian cancer cells that promotes cell cycle progression and proliferation. Our findings clearly indicate that GILZ activates AKT, a crucial signaling molecule in tumorigenesis. GILZ thus appears as a potential key molecule in EOC.

Obesity-induced lymphocyte hyperresponsiveness to chemokines: a new mechanism of Fatty liver inflammation in obese mice.

BACKGROUND & AIMS: Hepatic lipid retention (steatosis) predisposes hepatitis. We investigated the mechanisms of lymphocyte homing to fatty liver and the role of lipopolysaccharide (LPS) in the onset of inflammation in ob/ob mice. METHODS: We decreased intestinal bacterial compounds by oral antibiotic treatment to test the role of endogenous LPS in liver inflammation. Adoptive transfer of lymphocytes was used to study the respective contributions of steatosis and lymphocytes to liver inflammation. We tested lymphocyte response to chemokines by in vitro chemotaxis assays in ob/ob, their lean controls, and "non-obese ob/ob" mice, generated by controlling caloric intake to distinguish between the effects of obesity and leptin deficiency. RESULTS: Antibiotic treatment decreased liver infiltration with CD4(+) T, CD8(+) T, natural killer (NK)T, B, and NK cells. Adoptive transfer of lymphocytes from ob/ob or control mice showed that (1) steatosis increased lymphocyte recruitment to the liver; (2) CD4(+) T, CD8(+) T, and B cells from ob/ob mice had a greater propensity to migrate specifically to the liver. This migration was enhanced by LPS. These results were also observed in a model of high-fat diet-induced obesity. CD4(+) T and B cells were hyperresponsive to CXCL12 and CXCL13, respectively. Weight normalization in "non-obese ob/ob" mice decreased liver inflammation, lymphocyte response to chemokines, and homing to the liver. CONCLUSIONS: Our study provides the first evidence that liver inflammation in mice with genetic or diet-induced obesity results from both steatosis and lymphocyte hyperresponsiveness to chemokines expressed in the liver. These abnormalities are reversible with weight normalization.

Stimulation of the primary anti-HIV antibody response by IFN-alpha in patients with acute HIV-1 infection.

Type I IFNs are needed for the production of antiviral antibodies in mice; whether they also stimulate primary antibody responses in vivo during human viral infections is unknown. This was assessed in patients acutely infected with HIV-1 and treated with IFN-alpha2b. Patients with acute HIV-1 infection were randomized to receive antiretroviral therapy alone (Group A, n=60) or combined for 14 weeks with pegylated-IFN-alpha2b (Group B, n=30). Emergence of anti-HIV antibodies was monitored during 32 weeks by Western blot (WB) analyses of serum samples. IFN-alpha2b treatment stimulated the production of anti-HIV antibodies. On Week 32, 19 weeks after the last IFN-alpha2b administration, there were 8.5 (6.5-10.0) HIV WB bands (median, interquartile range) in Group B and 7.0 (5.0-10.0) bands in Group A (P=0.054), and band intensities were stronger in Group B (P<0.05 for p18, p24, p34, p40, and p55 HIV antigens). IFN-alpha2b treatment also increased circulating concentrations of the B cell-activating factor of the TNF family (P<0.001) and ex vivo production of IL-12 (P<0.05), reflecting its effect on innate immune cells. Withdrawal of antiretroviral treatment on Week 36 resulted in a lower rebound of HIV replication in Group B than in Group A (P<0.05). Therefore, type I IFNs stimulate the emerging anti-HIV immune response in patients with acute HIV-1 infection, resulting in an improved control of HIV replication. Type I IFNs are thus critical in the development of efficient antiviral immune responses in humans, including the production of antiviral antibodies.

Platelet-derived growth factor expression and function in idiopathic pulmonary arterial hypertension.

RATIONALE: Platelet-derived growth factor (PDGF) promotes the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs), and may play a role in the progression of pulmonary arterial hypertension (PAH), a condition characterized by proliferation of PASMCs resulting in the obstruction of small pulmonary arteries. OBJECTIVES: To analyze the expression and pathogenic role of PDGF in idiopathic PAH. METHODS: PDGF and PDGF receptor mRNA expression was studied by real-time reverse transcription-polymerase chain reaction performed on laser capture microdissected pulmonary arteries from patients undergoing lung transplantation for idiopathic PAH. Immunohistochemistry was used to localize PDGF, PDGF receptors, and phosphorylated PDGFR-beta. The effects of imatinib on PDGF-B-induced proliferation and chemotaxis were tested on human PASMCs. MEASUREMENTS AND MAIN RESULTS: PDGF-A, PDGF-B, PDGFR-alpha, and PDGFR-beta mRNA expression was increased in small pulmonary arteries from patients displaying idiopathic PAH, as compared with control subjects. Western blot analysis revealed a significant increase in protein expression of PDGFR-beta in PAH lungs, as compared with control lungs. In small remodeled pulmonary arteries, PDGF-A and PDGF-B mainly localized to PASMCs and endothelial cells (perivascular inflammatory infiltrates, when present, showed intensive staining), PDGFR-alpha and PDGFR-beta mainly stained PASMCs and to a lesser extent endothelial cells. Proliferating pulmonary vascular lesions stained phosphorylated PDGFR-beta. PDGF-BB-induced proliferation and migration of PASMCs were inhibited by imatinib. This effect was not due to PASMC apoptosis. CONCLUSIONS: PDGF may play an important role in human PAH. Novel therapeutic strategies targeting the PDGF pathway should be tested in clinical trials.

Glucocorticoid-induced leucine zipper: A key protein in the sensitization of monocytes to lipopolysaccharide in alcoholic hepatitis.

UNLABELLED: Glucocorticoid-induced leucine zipper (GILZ), a recently identified protein induced by glucocorticoids (GCs), inhibits the nuclear factor kappaB pathway and the activation of monocytes/macrophages by lipopolysaccharides (LPS). This study aimed to elucidate the contribution of GILZ to the pathogenesis of alcoholic hepatitis (AH): we (1) assessed GILZ expression in the livers of patients with AH and (2) treated patients with severe AH with GCs (prednisolone 40 mg/day) and studied the effect of GILZ modulation on circulating monocyte function. We quantified GILZ expression in the livers of 42 consecutive alcoholic patients (21 with and 21 without AH). GILZ messenger RNA (mRNA) levels were lower in the livers of patients with AH versus those without AH (P < 0.05). We collected circulating monocytes from patients with severe AH before and 48 hours after GC treatment to quantify GILZ expression and cytokine secretion. GC treatment induced significantly higher levels of GILZ mRNA than that observed before treatment and impaired LPS-induced tumor necrosis factor-alpha (TNF-alpha) and regulated upon activation, normal T cell-expressed secretion (RANTES) by these monocytes. We transfected circulating monocytes with GILZ small interfering RNA (siRNA), specifically blocking GILZ expression, to demonstrate the role of GILZ in mediating GC effect. GILZ siRNA abrogated the effect of GC treatment on LPS-induced TNF-alpha and RANTES secretion. CONCLUSION: Low expression of GILZ may contribute to liver inflammation in AH. GCs enhance GILZ expression, abrogating macrophage sensitivity to LPS and proinflammatory cytokine secretion. These findings may explain the beneficial effect of GC treatment in patients with severe AH.

Comparison of in vitro-specific blood tests with tuberculin skin test for diagnosis of latent tuberculosis before anti-TNF therapy.

INTRODUCTION: Latent tuberculosis infection (LTBI) is detected with the tuberculin skin test (TST) before anti-TNF therapy. We aimed to investigate in vitro blood assays with TB-specific antigens (CFP-10, ESAT-6), in immune-mediated inflammatory diseases (IMID) for LTBI screening. PATIENTS AND METHODS: Sixty-eight IMID patients with (n = 35) or without (n = 33) LTBI according to clinico-radiographic findings or TST results (10 mm cutoff value) underwent cell proliferation assessed by thymidine incorporation and PKH-26 dilution assays, and IFNgamma-release enzyme-linked immunosorbent spot (ELISPOT) assays with TB-specific antigens. RESULTS: In vitro blood assays gave higher positive results in patients with LTBI than without (p<0.05), with some variations between tests. Among the 13 patients with LTBI diagnosed independently of TST results, 5 had a negative TST (38.5%) and only 2 a negative blood assays result (15.4%). The 5 LTBI patients with negative TST results all had positive blood assays results. Ten patients without LTBI but with intermediate TST results (6-10 mm) had no different result than patients with TST result 0.3) and lower results than those with LTBI (p<0.05) on CFP-10+ESAT-6 ELISPOT and CFP-10 proliferation assays. CONCLUSION: Anti-TB blood assays are beneficial for LTBI diagnosis in IMID. Compared with TST, they show a better sensitivity, as seen by positive results in 5 patients with certain LTBI and negative TST, and better specificity, as seen by negative results in most patients with intermediate TST as the only criteria of LTBI. In the absence of clinico-radiographic findings for LTBI, blood assays could replace TST for antibiotherapy decision before anti-TNF.

CXCL12 and vascular endothelial growth factor synergistically induce neoangiogenesis in human ovarian cancers.

Ovarian carcinomas have a poor prognosis, often associated with multifocal i.p. dissemination accompanied by intense neovascularization. To examine tumor angiogenesis in the tumor microenvironment, we studied malignant ascites and tumors of patients with untreated ovarian carcinoma. We observed that malignant ascites fluid induced potent in vivo neovascularization in Matrigel assay. We detected a sizable amount of vascular endothelial cell growth factor (VEGF) in malignant ascites. However, pathologic concentration of VEGF is insufficient to induce in vivo angiogenesis. We show that ovarian tumors strongly express CXC chemokine stromal-derived factor (SDF-1/CXCL12). High concentration of CXCL12, but not the pathologic concentration of CXCL12 induces in vivo angiogenesis. Strikingly, pathologic concentrations of VEGF and CXCL12 efficiently and synergistically induce in vivo angiogenesis. Migration, expansion, and survival of vascular endothelial cells (VEC) form the essential functional network of angiogenesis. We further provide a mechanistic basis for explaining the interaction between CXCL12 and VEGF. We show that VEGF up-regulates the receptor for CXCL12, CXCR4 expression on VECs, and synergizes CXCL12-mediated VEC migration. CXCL12 synergizes VEGF-mediated VEC expansion and synergistically protects VECs from sera starvation-induced apoptosis with VEGF. Finally, we show that hypoxia synchronously induces tumor CXCL12 and VEGF production. Therefore, hypoxia triggered tumor CXCL12 and VEGF form a synergistic angiogenic axis in vivo. Hypoxia-induced signals would be the important factor for initiating and maintaining an active synergistic angiogeneic pathway mediated by CXCL12 and VEGF. Thus, interrupting this synergistic axis, rather than VEGF alone, will be a novel efficient antiangiogenesis strategy to treat cancer.