OBJECTIVE

We conducted a prospectively randomized clinical trial to compare the efficacy of three outpatient therapy regimens in 341 patients with progressive metastatic renal cell carcinoma.

METHODS

Patients were stratified according to known clinical predictors and were subsequently randomly assigned. Treatment arms were: arm A (n = 132), subcutaneous interferon alfa-2a (sc-IFN-alpha-2a), subcutaneous interleukin-2 (sc-IL-2), and intravenous (IV) fluorouracil; arm B (n = 146): arm A treatment combined with per oral 13-cis-retinoic acid; and arm C (n = 63), sc-IFN-alpha-2a and IV vinblastine.

RESULTS

Treatment (according to the standard 8-week Hannover Atzpodien regimen) arms A, B, and C yielded objective response rates of 31%, 26%, and 20%, respectively. Arm B, but not arm A, showed a significantly improved progression-free survival (PFS) compared with arm C (P =.0248). Both arm A (median overall survival, 25 months; P =.0440) and arm B (median overall survival, 27 months; P =.0227) led to significantly improved overall survival (OS) compared with arm C (median OS, 16 months). All three sc-IFN-alpha-2a-based therapies were moderately or well tolerated.

CONCLUSIONS

Our results established the safety and improved long-term therapeutic efficacy of sc-IL-2 plus sc-INF-alpha-2a-based outpatient immunochemotherapies, compared with sc-INF-alpha-2a/IV vinblastine.

Adipose tissue is an endocrine organ capable of secreting a number of adipokines with a role in the regulation of adipose tissue and whole-body metabolism. We used two-dimensional gel electrophoresis combined with mass spectrometry to profile the secreted proteins from (pre)adipocytes. The culture medium of 3T3-L1 cells during adipocyte differentiation was screened, and 41 proteins that responded to blocking of secretion by <em>20</em> degrees C treatment and/or brefeldin A treatment were identified. Prohibitin, stress-70 protein, and adhesion-regulating molecule 1 are reported for the first time as secreted proteins. In addition, procollagen C-proteinase enhancer protein, galectin-1, cyclophilin A and C, and SF<em>20</em>/<em>IL</em>-25 are newly identified as adipocyte secreted factors. Secretion profiles indicated a dynamic environment including an actively remodeling extracellular matrix and several factors involved in growth regulation.

This study reports on the potent cytocidal and interleukin-1 releasing properties of Escherichia coli hemolysin (ECH) on human monocytes. Nanomolar concentrations of purified ECH (250-2,000 ng/ml) caused rapid and irreversible depletion of cellular ATP to levels below <em>20</em>% of controls within 60 min. Subcytocidal doses (10-<em>20</em>0 ng/ml) of ECH induced rapid release within 60-1<em>20</em> min of large amounts of interleukin 1 beta (<em>IL</em>-1 beta) from cultured monocytes. <em>IL</em>-1 beta release occurred in the presence of actinomycin D and cycloheximide, and was thus probably due to processing and export of intracellular <em>IL</em>-1 beta precursor. Incubation of toxin-producing E. coli at ratios of only 0.3-3 colony-forming units per monocyte evoked approximately 50% depletion of total cellular ATP within 90 min. Toxin producers also stimulated synthesis and release of large amounts of interleukin 1, but not of tumor necrosis factor within the same time span. In contrast, non-toxin producers caused neither cell death nor rapid interleukin 1 release. Stimulation of rapid interleukin 1 release coupled with potent cytocidal effects on cells of monocytic origin may represent pathogenetically significant events incurred by bacterial strains that produce ECH and related cytolysins.

Interleukin 24 (<em>IL</em>-24) encodes a secreted protein that exhibits significant homology to the interleukin 10 (<em>IL</em>-10) family of cytokines. Here we show that the human <em>IL</em>-24 is secreted by activated peripheral blood mononuclear cells and is the ligand for two heterodimeric receptors, <em>IL</em>-22R1/<em>IL</em>-<em>20</em>R2 and <em>IL</em>-<em>20</em>R1/<em>IL</em>-<em>20</em>R2. The latter is also the receptor for <em>IL</em>-<em>20</em>. COS cells transfected with either <em>IL</em>-24 receptor heterodimers bind the ligand with similar saturation kinetics. <em>IL</em>-24 binding to either its endogenous receptors on human keratinocytes or to ectopically expressed receptors on baby hamster kidney cells leads to activation of the signal transducers and activators of transcription. Taken together, these results provide compelling evidence for <em>IL</em>-24 being the fourth member of <em>IL</em>-10 family of cytokines to which their specific receptors have been identified.

Pyuria is one of the main features of urinary tract infections (UTI). Nevertheless, the mechanism of polymorphonuclear leukocyte (PMN) recruitment into the urine remains to be investigated. We examined whether interleukin-8 (<em>IL</em>-8), a potent neutrophil chemoattractant and activator, was involved in pyuria seen in UTI. Of 113 patients, 112 had elevated levels of <em>IL</em>-8 in their urine (1,078.0 +/- 181.5 pg/ml), regardless of whether they had an upper or lower UTI; this was in contrast to undetectable levels (less than 16 pg/ml) in the urine of all of the <em>20</em> normal individuals and 74 control patients without UTI. A concomitant study revealed increases in urine <em>IL</em>-6, but not <em>IL</em>-1 beta, and tumor necrosis factor alpha levels in patients with UTI. In addition to gram-negative bacteria, a wide spectrum of microorganisms was capable of inducing <em>IL</em>-8 production in urine. Local production of <em>IL</em>-8 in the urinary tract was suggested by a urine <em>IL</em>-8 level that was higher than the paired serum <em>IL</em>-8 level. The urine <em>IL</em>-8 level correlated with the number of PMN in the urine, and an average of half of the chemotactic activity in urine from patients with UTI could be abrogated by anti-<em>IL</em>-8 antibody treatment in vitro. Furthermore, urine <em>IL</em>-8 purified from patients was bioactive and showed multiple forms on immunoblotting analysis. This is the first documentation of <em>IL</em>-8 in the urine of patients with UTI, and these results imply that <em>IL</em>-8 is involved in inducing PMN migration into the urinary tract.

The vertical transmission of hepatitis C virus (HCV-VT) is a major route of HCV infection in children, but the risk factors remain incompletely understood. This study analyzed the role of interleukin 28B (<em>IL</em>28B) in HCV-VT and in the spontaneous clearance of HCV among infected infants. Between 1991 and <em>20</em>09, 145 mothers were recruited for this study: 100 were HCV-RNA+ve / human immunodeficiency virus negative (HIV-ve), with 128 children, and 33 were HCV-RNA-ve/HCV antibody+ve, with 43 children. The infants were tested for HCV-RNA at birth and at regular intervals until the age of 6 years. <em>IL</em>28B (single nucleotide polymorphism rs12979860) was determined in the mothers and children. HCV-VT was assumed when children presented HCV-RNA+ve in two subsequent blood samples. HCV-VT-infected infants were categorized as: (1) transient viremia with posterior HCV-RNA-ve and without serum-conversion; (2) persistent infection with serum-conversion. Of the 31 mothers with CC polymorphism, 19 (61%) were HCV-RNA+ve, whereas among the 68 mothers with non-CC polymorphism, 56 (82%) were HCV-RNA+ve. In all, 26 of 128 (<em>20</em>%) infants born to the HCV-RNA+ve mothers acquired HCV infection, but only 9 (7%) were chronically infected. The rate of HCV-VT was higher among the mothers with higher HCV viremia. No HCV-VT was detected in the HCV-RNA-ve women. Neither the mothers' nor the childrens' <em>IL</em>-28 status was associated with an increased risk of HCV-VT. The factors influencing viral clearance among the infected children were genotype non-1 and genotype CC of <em>IL</em>28B. In logistic regression, child CC polymorphism was the only predictor of HCV-clearance in HCV genotype-1.

CONCLUSIONS

High maternal viral load is the only predictive factor of HCV-VT. <em>IL</em>28B plays no role in HCV-VT, but <em>IL</em>28B CC child polymorphism is associated independently with the spontaneous clearance of HCV genotype-1 among infected children.

Phosphorylation of p47(phox) is a key event in NADPH oxidase activation. We examined the ability of proinflammatory cytokines such as TNFalpha, <em>IL</em>-1, and G-CSF to induce this process compared with GM-CSF. Only TNF-alpha and GM-CSF induced a clear p47(phox) phosphorylation. This phosphorylation was time dependent and reached its maximum at <em>20</em> min. Two-dimensional phosphopeptide mapping of p47(phox) phosphorylated in neutrophils primed with TNF-alpha revealed partial phosphorylation of p47(phox) on the same peptide as for GM-CSF. Neutrophil incubation with TNF-alpha and subsequent addition of the chemotactic peptide fMLP resulted in more intense phosphorylation of p47(phox) sites than with each reagent alone. A neutralizing Ab against the p55 TNF receptor, contrary to a neutralizing Ab against the p75 TNF receptor, inhibited TNF-alpha-induced p47(phox) phosphorylation. Neutrophil treatment with both TNF-alpha and GM-CSF resulted in more intense phosphorylation of the same p47(phox) peptide observed with each cytokine alone, suggesting that they engaged pathways converging on common serines. This additive effect was also obtained on the priming of NADPH oxidase activity. The use of protein kinase inhibitors pointed to the involvement of a protein tyrosine kinase, but not protein kinase C. These findings show that TNF-alpha, via its p55 receptor, induces a protein tyrosine kinase-dependent selective phosphorylation of p47(phox) on specific serines. The ability of TNF-alpha and GM-CSF, two different cytokines with two different receptors to induce this specific p47(phox) phosphorylation, suggests that this event could be a common element of the priming of neutrophils by TNF-alpha and GM-CSF.

BACKGROUND

Bronchopulmonary dysplasia (BPD) is closely associated with ventilator-induced lung injury (VILI) in very preterm infants. The greatest risk of VILI may be in the immediate period after birth, when the lungs are surfactant deficient, still partially filled with liquid and not uniformly aerated. However, there have been very few studies that have examined this immediate post-birth period and identified the initial injury-related pathways that are activated. We aimed to determine if the early response genes; connective tissue growth factor (CTGF), cysteine rich-61 (CYR61) and early growth response 1 (EGR1), were rapidly induced by VILI in preterm lambs and whether ventilation with different tidal volumes caused different inflammatory cytokine and early response gene expression.

METHODS

To identify early markers of VILI, preterm lambs (132 d gestational age; GA, term approximately 147 d) were resuscitated with an injurious ventilation strategy (V(T) <em>20</em> mL/kg for 15 min) then gently ventilated (5 mL/kg) for 15, 30, 60 or 1<em>20</em> min (n = 4 in each). To determine if early response genes and inflammatory cytokines were differentially regulated by different ventilation strategies, separate groups of preterm lambs (125 d GA; n = 5 in each) were ventilated from birth with a V(T) of 5 (VG5) or 10 mL/kg (VG10) for 135 minutes. Lung gene expression levels were compared to levels prior to ventilation in age-matched control fetuses.

RESULTS

CTGF, CYR61 and EGR1 lung mRNA levels were increased approximately 25, 50 and 1<em>20</em>-fold respectively (p < 0.05), within 30 minutes of injurious ventilation. VG5 and VG10 caused significant increases in CTGF, CYR61, EGR1, IL1- , IL-6 and IL-8 mRNA levels compared to control levels. CTGF, CYR61, IL-6 and IL-8 expression levels were higher in VG10 than VG5 lambs; although only the IL-6 and CYR61 mRNA levels reached significance.

CONCLUSIONS

CTGF, CYR61 and EGR1 may be novel early markers of lung injury and mechanical ventilation from birth using relatively low tidal volumes may be less injurious than using higher tidal volumes.

OBJECTIVE

In an ongoing effort to identify diagnostic ovarian cancer biomarkers, SEREX (serological analysis of recombinant cDNA expression libraries) technique was employed resulting in detection of <em>20</em> known genes, nine ESTs and one novel sequence. Interleukin-8 (<em>IL</em>-8) was one of ovarian cancer-associated antigens identified by SEREX screening. The objective of this study was, therefore, to evaluate the potential importance of circulating anti-<em>IL</em>-8 antibody as ovarian cancer biomarker.

METHODS

We developed and optimized a new immunofluorescent bead-based assay for detection of anti-IL-8 antibody in blood serum. Circulating IL-8 and anti-IL-8 IgG concentrations were measured in blood sera from 44 patients with early stage (I-II) ovarian cancer, 50 patients with late stage (III-IV) ovarian cancer, 37 patients with benign pelvic masses, and 80 healthy women using the bead-based assay.

RESULTS

Our data indicate that serum contains IL-8 cytokine, anti-IL-8 antibody, and IL-8:anti-IL-8 complexes. We found that concentrations of IL-8 and anti-IL-8 antibody were elevated in sera of patients with ovarian cancer as compared with healthy controls. Logistic regression analysis of circulating concentrations of anti-IL-8 IgG in patients with stages I-II ovarian cancer versus healthy controls allowed for prediction of early ovarian cancer with 98% specificity, 65.5% sensitivity, 80.3% of patients correctly classified. Combining IL-8 and anti-IL-8 IgG with CA 125 resulted in increased classification power as compared to individual markers analyzed separately.

CONCLUSIONS

Thus, IL-8 and anti-IL-8 autoantibody might potentially serve as additional biomarkers for ovarian cancer.

OBJECTIVE

To evaluate the efficacy of epigallocatechin-3-gallate (EGCG), a potent antiinflammatory molecule, in regulating interleukin-1beta (IL-1beta)-induced production of the chemokines RANTES (CCL5), monocyte chemoattractant protein 1 (MCP-1/CCL2), epithelial neutrophil-activating peptide 78 (ENA-78/CXCL5), growth-regulated oncogene alpha (GROalpha/CXCL1), and matrix metalloproteinase 2 (MMP-2) activity in rheumatoid arthritis (RA) synovial fibroblasts.

METHODS

Fibroblasts obtained from RA synovium were grown, and conditioned medium was obtained. Cell viability was determined by MTT assay. RANTES, MCP-1, ENA-78, and GROalpha produced in culture supernatants were measured by enzyme-linked immunosorbent assay. MMP-2 activity was analyzed by gelatin zymography. Western blotting was used to study the phosphorylation of protein kinase C (PKC) isoforms and nuclear translocation of NF-kappaB.

RESULTS

EGCG was nontoxic to RA synovial fibroblasts. Treatment with EGCG at 10 microM or 20 microM significantly inhibited IL-1beta-induced ENA-78, RANTES, and GROalpha, but not MCP-1 production in a concentration-dependent manner. EGCG at 50 microM caused a complete block of IL-1beta-induced production of RANTES, ENA-78, and GROalpha, and reduced production of MCP-1 by 48% (P < 0.05). Zymography showed that EGCG blocked constitutive, IL-1beta-induced, and chemokine-mediated MMP-2 activity. Evaluation of signaling events revealed that EGCG preferentially blocked the phosphorylation of PKCdelta and inhibited the activation and nuclear translocation of NF-kappaB in IL-1beta-treated RA synovial fibroblasts.

CONCLUSIONS

These results suggest that EGCG may be of potential therapeutic value in inhibiting joint destruction in RA.

The undecapeptide substance P is thought to mediate both vasodilatation and augmented vascular permeability when released from sensory nerve endings in the skin. Substance P also induces mast cell degranulation in vitro or in vivo. However, the extent to which substance P-induced changes in vascular permeability are mast cell-dependent is unclear. We investigated this issue by injecting substance P and certain related peptides (substance P1-4, substance P4-11) into the skin of genetically mast cell-deficient WBB6F1-W/W or WCB6F1- SI/SId mice the congenic normal (+/+) mice, and W/W mice which had undergone selective local repair of their mast cell deficiency by intradermal injection of <em>IL</em>-3-dependent mast cells generated in vitro from the bone marrow cells of the congenic +/+ mice. Substance P induced significant augmentation of vascular permeability and significant cutaneous swelling when injected into normal mice at doses as low as 2 pmol i.d. Substance P also induced granulocyte infiltration, although the infiltrate were modest and were seen at doses of peptide from 5 to more than <em>20</em>-fold higher than those required for induction of tissue swelling. The effects of substance P on tissue swelling, vascular permeability, and granulocyte infiltration were virtually entirely mast cell dependent. By contrast, substance P1-4 was inactive in our assays at 25 nmol/site, and substance P4-11 induced modest augmentation of vascular permeability, which was at least in part mast cell independent.

The late asthmatic reaction (LAR), consecutive to bronchial allergen challenge, is characterized both by the influx of various cells in proximal and distal airways and by the enhancement of bronchial hyperresponsiveness. However, the exact conditions for the development of the inflammatory reaction during the LAR remain to be specified. Since monokines play a key role in inflammatory processes, particularly in the lung, the production of tumor necrosis factor-alpha (TNF-alpha), interleukin; 1-beta (<em>IL</em>-1-beta) and interleukin-6 (<em>IL</em>-6) by alveolar macrophages (AM), collected 18 to <em>20</em> hours after exposure to allergen, was evaluated in 15 allergic subjects with asthma submitted to a challenge test with Dermatophagoides pteronyssinus (N = 6) or with wheat flour (N = 9) and in three healthy subjects. After bronchial provocation test, four patients presented no bronchial response (group 1), and six patients, a single early reaction (group 2). In contrast, five patients developed successively an immediate plus a late response (group 3). The monokine production was compared to that from nine allergic subjects with asthma studied at baseline (group 0) and from 11 unchallenged healthy subjects (control subjects). Measurements of cytokines were evaluated for TNF-alpha and <em>IL</em>-1-beta by a specific immunoradiometric assay, whereas <em>IL</em>-6 levels were appreciated by the proliferation of 7TD1 cells. No detectable amounts of TNF-alpha, <em>IL</em>-1-beta, and <em>IL</em>-6 were in bronchial alveolar lavage fluid, even after a tenfold concentration. In contrast, a significant increase of TNF-alpha (10,642 +/- 3127 U/ml) and <em>IL</em>-6 (1250 +/- 427 U/ml) concentrations was noted in AM supernatants from patients exhibiting an LAR (group 3) compared to cells recovered from groups 2, 1, and 0 and to challenged or unchallenged control subjects (805 +/- 244, 995 +/- 521, 1269 +/- 524, 688 +/- 85, and 445 +/- 74 pg of TNF-alpha per milliliter, respectively; 190 +/- 64, 114 +/- 91, 242 +/- 95, 80 +/- 9, and 54 +/- 19 U/ml of <em>IL</em>-6 per milliliter, respectively). No modification of <em>IL</em>-1-beta contents could be detected between the different groups. A significant correlation was detected between concentrations of TNF and <em>IL</em>-6 (r = 0.92; p less than 0.001). These results demonstrate TNF-alpha and <em>IL</em>-6 secretion by AM consecutively to the development of LAR in allergic subjects with asthma, confirming that AMs are activated after allergen challenge.(ABSTRACT TRUNCATED AT 400 WORDS)

A noninvasive method to characterize inflammation and infection in the airways of nonexpectorating children with cystic fibrosis (CF) is needed for clinical and research purposes. Accordingly, we performed sputum inductions by administering 3% saline to 11 healthy control children and <em>20</em> children with CF, composed of 7 sputum producers (capable of spontaneously expectorating sputum) and 13 nonproducers. Induced sputum weights were comparable in each group, whereas the amount of induced sputum collected from the CF producers was over 10-fold higher than the spontaneously expectorated samples. We found a significant increase in indices of airway inflammation, including total cell counts, absolute neutrophil counts, interleukin-8 (<em>IL</em>-8) levels, and neutrophil elastase activity in the CF subjects compared with the healthy control subjects. These same indices in the induced sputum specimens from CF producers were significantly correlated with levels in the matched expectorated sputum specimens. Sputum total protein concentration was elevated in the CF groups, whereas urea and albumin levels were not significantly different. Salivary analysis, performed separately, revealed higher levels of <em>IL</em>-8 and total protein in the CF groups. Airway infection, as assessed by quantitative counts of CF-related bacterial pathogens, was also higher in the CF subjects. The same bacterial pathogens, in similar colony counts, were isolated from both the induced and expectorated sputum samples from the CF producers. We conclude that airway inflammation and infection, assessed through sputum induction, are significantly increased in children with CF as compared with healthy children. Furthermore, induced sputum samples are similar to spontaneously expectorated samples in describing both inflammation and infection in the CF airway.

Cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage-CSF (M-CSF), neutrophil-activating peptide-1/interleukin-8 (NAP-1/<em>IL</em>-8), and interleukin-6 (<em>IL</em>-6) are pivotal in the regulation of hematopoiesis and immune responses. In mesenchymal cells, their expression is induced by tumor necrosis factor alpha (TNF) and other agents. We now show that, while induction of cytokine expression by TNF in human lung fibroblasts was parallel, glucocorticoid hormones differentially affected their production. Dexamethasone (1 mumol/L) concordantly repressed expression of GM-CSF, NAP-1/<em>IL</em>-8 and <em>IL</em>-6. RNA and protein levels were reduced to approximately 5%, <em>20</em>%, and 30% of control cells, respectively, as determined by Northern blot analyses and immunoassays. A 50% reduction of RNA levels for all three cytokines occurred in the range of 1 hour. In contrast, dexamethasone (1 mumol/L) did not decrease M-CSF RNA levels and protein release. M-CSF RNA and protein levels were maintained even when dexamethasone (1 mumol/L) was present for the whole duration of a 48-hour TNF stimulation. Further experiments showed that dexamethasone downregulates expression of GM-CSF, NAP-1/<em>IL</em>-8, and <em>IL</em>-6 mainly by decreasing the mRNA stability of these cytokines, and that the dexamethasone-mediated repression of cytokine expression depends on ongoing protein and RNA syntheses. Our study suggests that glucocorticoid hormones repress expression of a set of cytokine genes important in conditions of stress. However, they seem not to affect M-CSF expression, which is likely to be more crucial in maintaining long-term functions of myeloid cells.

The outcome of SARS-CoV2 infection in patients who have received a kidney allograft and are being treated with immunosuppression is unclear. We describe <em>20</em> kidney transplant recipients (median age 59 years [inter quartile range 51-64 years], median age of transplant 13 years [9-<em>20</em> years], baseline eGFR 36.5 [23-47.5]) with SARS-CoV2 induced pneumonia. At admission, all had immunosuppression withdrawn and were started on methylprednisolone 16 mg/day, all but one was commenced on antiviral therapy and hydroxychloroquine with doses adjusted for kidney function. At baseline, all patients presented fever but only one complained of difficulty in breathing. Half of patients showed chest radiographic evidence of bilateral infiltrates while the other half showed unilateral changes or no infiltrates. During a median follow-up of seven days, 87% experienced a radiological progression and among those 73% required escalation of oxygen therapy. Six patients developed acute kidney injury with one requiring hemodialysis. Six of 12 patients were treated with tocilizumab, a humanized monoclonal antibody to the <em>IL</em>-6 receptor. Overall, five kidney transplant recipients died after a median period of 15 days [15-19] from symptom onset. These preliminary findings describe a rapid clinical deterioration associated with chest radiographic deterioration and escalating oxygen requirement in renal transplant recipients with SARS-Cov2 pneumonia. Thus, in this limited cohort of long-term kidney transplant patients, SARS-CoV-2 induced pneumonia is characterized by high risk of progression and significant mortality.

BACKGROUND

The pathogenesis of hidradenitis suppurativa (HS) is largely unknown and the disease is difficult to treat. Patients are in high need of an effective treatment. Although it is not known whether the levels of tumour necrosis factor (TNF)-α are aberrant in HS skin, anti-TNF-α biologics are used, with variable clinical efficacy.

OBJECTIVE

To determine the cytokine profile in lesional and perilesional HS skin.

METHODS

We cultured <em>20</em> lesional and 10 normal-appearing perilesional HS skin samples, seven psoriasis and six healthy control skin samples in a transwell culture system. Two distinct cytokine bead arrays were used to measure the spectrum of inflammatory cytokines in the culture supernatant. Results from HS skin samples were compared with those of healthy and psoriasis skin.

RESULTS

The proinflammatory cytokines interleukin (IL)-1β and TNF-α as well as the anti-inflammatory cytokine IL-10 were significantly elevated in HS skin. Elevated levels of these cytokines were also found in perilesional HS skin. Fold increases relative to control skin of IL-1β, TNF-α and IL-10 in HS were 31, 5 and 34, compared with psoriasis: 4, 1 and 2, respectively. Levels of all three cytokines showed a trend towards a positive correlation with disease severity. IL-2, IL-4, IL-5 and interferon-γ were hardly detectable in HS or healthy control skin.

CONCLUSIONS

This study shows for the first time that IL-1β, TNF-α and IL-10 levels are elevated in HS skin. These data provide a rationale for therapies with biologics targeting cytokines such as TNF-α and IL-1.

Epidemiologic and experimental findings implicate maternal infection in the etiology of injury to brain white matter, which may lead to cerebral palsy in preterm newborns. In the present study, inflammation and brain damage in 1- and 7-d-old rats were investigated after maternal inflammation. Intraperitoneal injection of 300 microg/kg of Escherichia coli lipopolysaccharide was administered to pregnant Wistar rats at d 19 and <em>20</em> of gestation (LPS group). Control females received a saline injection. Proinflammatory cytokines <em>IL</em>-1beta, tumor necrosis factor-alpha, and <em>IL</em>-6 expression in the fetal brain were determined by reverse transcription quantitative polymerase chain reaction. Brain injury was examined in 16-mum coronal brain sections by GFAP, MBP, caspase-3 immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Expression of <em>IL</em>-1beta was significantly increased 3 d after maternal administration (P1). A significant increase in cell death occurred at P1 and P7 in specific brain areas, i.e. in the subventricular striatal zone at P1, and in 1) the periventricular striatum, 2) the periventricular white matter, and 3) the germinative ventricular zone at P7. We also observed typical astrogliosis and strong hypomyelination in the external and internal capsule in the LPS group at P7. These results demonstrate that maternal LPS treatment induces persistent fetal inflammatory reactions associated with significant white matter injury in progeny at P1 and P7. This model should be relevant for the study of the pathophysiological mechanisms involved in cerebral white matter damage in preterm human newborns and in the development of therapeutic strategies.

OBJECTIVE

BUILDER-1 and BUILDER-2 aimed to assess the efficacy and safety of tocilizumab (TCZ) in patients with ankylosing spondylitis (AS).

METHODS

BUILDER-1 was a two part, phase II-III parallel-group trial in patients with AS naive to antitumour necrosis factor (aTNF) treatment. Patients in part 1 received TCZ 8 mg/kg or placebo for 12 weeks. In part 2 (beginning after part 1 enrolment ended), newly enrolled patients received TCZ 4 or 8 mg/kg or placebo for 24 weeks. The same treatment arms were used in BUILDER-2, a phase III study in aTNF-inadequate responders. The primary endpoint for both studies was the proportion of patients achieving <em>20</em>% improvement in the Assessments in Axial SpondyloArthritis international Society (ASAS). Secondary and exploratory endpoints included ASAS40 response rates, Bath Ankylosing Spondylitis Disease Activity Index improvement, changes in joint counts, enthesitis score and C reactive protein (CRP).

RESULTS

102 patients were randomised in BUILDER-1 part 1; 99 (48 TCZ, 51 placebo) completed 12 weeks. Week 12 ASAS<em>20</em> response rates were 37.3% and 27.5% in the TCZ and placebo arms, respectively (p=0.2823). Secondary and exploratory endpoints did not differ between treatment arms. CRP levels declined with TCZ treatment, suggesting adequate IL-6 receptor blockade. As a result, BUILDER-1 part 2 and BUILDER-2 were terminated. TCZ safety results were consistent with previous observations in rheumatoid arthritis, except for a cluster of anaphylactic and hypersensitivity events at Bulgarian study sites. No apparent explanation for this clustering could be found.

CONCLUSIONS

BUILDER-1 failed to demonstrate TCZ efficacy in treating aTNF-naive patients with AS.

BACKGROUND

The aim of this study was to examine the impact of elemental diet on mucosal inflammation in Crohn's disease (CD), mainly by cytokine measurements.

METHODS

Twenty-eight consecutive patients with active CD were treated with an elemental diet (Elental) for 4 weeks. The mucosal biopsies were obtained from the terminal ileum and large bowel before and after treatment. As a control group, mucosal biopsies were obtained from <em>20</em> patients without inflammation. Mucosal cytokine concentrations were measured by enzyme-linked immunosorbent assay.

RESULTS

After treatment, clinical remission was achieved in <em>20</em> patients (71%). Endoscopic healing and improvement rates were 44% and 76% in the terminal ileum and 39% and 78% in the large bowel, respectively. Histologic healing and improvement rates were 19% and 54% in the terminal ileum and <em>20</em>% and 55% in the large bowel, respectively. Before treatment, the mucosal concentrations of interleukin (IL)-1beta, IL-1 receptor antagonist (IL-1ra), IL-6, IL-8, and tumor necrosis factor-alpha in the ileum and large bowel were significantly higher than in controls. These cytokine concentrations decreased to the levels of control after treatment. IL-1ra/IL-1beta ratio in the ileum and large bowel was significantly lower than in controls before treatment. The ratio increased to the level of controls after treatment. The endoscopic and histologic healing of the mucosal inflammation was associated with a decline of the mucosal cytokines and an increase of the IL-1ra/IL-1beta ratio.

CONCLUSIONS

The elemental diet (Elental) reduced mucosal cytokine production and corrected an imbalance between proinflammatory and anti-inflammatory cytokines in CD.

The widely accepted interleukin-28B (<em>IL</em>-28B) rs12979860 C/T polymorphism and the more recently proposed vitamin D serum concentration are two novel predictors of the response to antiviral treatment in chronic hepatitis C virus (HCV) infection. This study aimed to verify whether the <em>IL</em>-28B rs12979860 C/T polymorphism and pretreatment serum vitamin D levels have independent or complementary roles in predicting the rates of sustained viral response (SVR). The present study included 211 consecutive, treatment-naïve chronic HCV patients who had their pretreatment serum 25-OH vitamin D level and <em>IL</em>-28B rs12979860 C/T genotype determined. Overall, SVR was achieved by 134/211 (63.5%) patients and by 47/110 (42.7%) patients infected with difficult-to-treat HCV genotypes. On multivariate analysis, SVR was predicted by the HCV genotype, the <em>IL</em>-28B rs12979860 C/T polymorphism, and gamma-glutamyl transpeptidase, HCV RNA, cholesterol, and 25-OH vitamin D serum levels, with an area under the receiver operating characteristic (ROC) curve of 0.827. When difficult-to-treat HCV genotypes were analyzed separately, the SVR was predicted by the <em>IL</em>-28B rs12979860 C/T polymorphism, viral load, and serum vitamin D level, with an area under the ROC curve of 0.836. Moreover, by categorizing these latter patients into four groups-C/C homozygotes with vitamin D levels>><em>20</em> ng/mL (group A) or ≤<em>20</em> ng/mL (group B) and C/T heterozygotes or T/T homozygotes with vitamin D levels>><em>20</em> ng/mL (group C) or ≤<em>20</em> ng/mL (group D)-a significant linear trend was observed, with SVR rates in the following descending order: group A, 18/21 (85.7%); group B, 6/11 (54.5%); group C, 14/38 (36.8%); and group D, 9/40 (22.5%) (P < 0.0001).

CONCLUSIONS

Vitamin D serum levels are complementary to the IL-28B rs12979860 C/T polymorphism in enhancing the correct prediction of the SVR in treatment-naïve chronic hepatitis C.

BACKGROUND

A single-nucleotide polymorphism (SNP) in the promoter region of the interleukin-6 (IL-6) gene at position -174 (G>C) has been reported to be associated with a variety of major diseases, such as Alzheimer disease, atherosclerosis, and cardiovascular disease, cancer, non-insulin-dependent diabetes mellitus, osteoporosis, sepsis, and systemic-onset juvenile chronic arthritis. However, authors of previous in vitro and in vivo studies have reported conflicting results regarding the functionality of this polymorphism. We therefore aimed to clarify the role of the -174 SNP for the induction of IL-6 in vivo.

METHODS

We vaccinated 20 and 18 healthy individuals homozygous for the -174 C and G alleles, respectively, with 1 mL of Salmonella typhii vaccine. IL-1beta, IL-6, and tumor necrosis factor-alpha (TNF-alpha) were measured in the blood at baseline and up to 24 h after vaccination.

RESULTS

Individuals with the G genotype had significantly higher plasma IL-6 values at 6, 8, and 10 h after vaccination than did individuals with the C genotype (P <0.005). There were no differences between the two genotypes regarding serum concentrations of IL-1beta and TNF-alpha before or after vaccination.

CONCLUSIONS

The -174 G>C SNP in the promoter region of the IL-6 gene is functional in vivo with an increased inflammatory response associated with the G allele. Considering the central role of IL-6 in a variety of major diseases, the present finding might be of major relevance.

Endotoxin-mediated cholestasis stems from impaired hepatobiliary transport of bile acids and organic anions due to altered expression and activity of transporters, including Oatp, Mrp, Ntcp, and Bsep. However, the mechanisms by which the Oatp and Mrp genes are down-regulated are largely unknown. Using in vivo and in vitro murine models of inflammation, we examined the role of cytokines and bile acids in regulating Oatp and Mrp. Endotoxin (lipopolysaccharide, LPS), interleukin (<em>IL</em>)-6, <em>IL</em>-1beta, tumor necrosis factor (TNF)-alpha, cholic acid, taurocholate, or taurodeoxycholate was administered in vivo to mice or in vitro to Hepa 1-6 mouse hepatoma cells. Mrp, Oatp, and Bsep mRNA levels were measured by reverse transcription-polymerase chain reaction. Mrp efflux activity was measured using 5-carboxyfluorescein. In vivo, LPS treatment profoundly suppressed hepatic mRNA levels of Mrp2, Mrp3, Oatp1, Oatp2, and Bsep to 15, 60, 44, 30, and 32% of controls, respectively (p < 0.05), but did not significantly alter Mrp1 expression. <em>IL</em>-6 or <em>IL</em>-1beta administration suppressed Mrp2, Oatp1, Oatp2, and Bsep mRNA levels to <em>20</em> to 60% controls (p < 0.05). TNF-alpha administration affected mRNA levels of Mrp2, Mrp3, and Oatp2 but not Oatp1 or Bsep. Bile acid treatment increased the in vivo expression of Bsep but not Mrp or Oatp. Likewise, significantly lower mRNA levels of Mrp2 with a corresponding decrease in cellular efflux of 5-carboxyfluorescein was seen in vitro in <em>IL</em>-6- and <em>IL</em>-1beta-treated Hepa 1-6 cells, whereas bile acids did not have significant effects. In conclusion, cytokines are key mediators in regulating hepatic expression of anion transporters in inflammatory cholestasis, whereas bile acids likely play a minor role.

The potential use of the modified vaccinia virus Ankara (MVA) strain as a live recombinant vector to deliver antigens and elicit protective immune responses against infectious diseases demands a comprehensive understanding of the effect of MVA infection on human host gene expression. We used microarrays containing more than 15,000 human cDNAs to identify gene expression changes in human HeLa cell cultures at 2, 6, and 16 h postinfection. Clustering of the 410 differentially regulated genes identified 11 discrete gene clusters with altered expression patterns after MVA infection. Clusters 1 and 2 (accounting for 16.59% [68 of 410] of the genes) contained 68 transcripts showing a robust induction pattern that was maintained during the course of infection. Changes in cellular gene transcription detected by microarrays after MVA infection were confirmed for selected genes by Northern blot analysis and by real-time reverse transcription-PCR. Upregulated transcripts in clusters 1 and 2 included <em>20</em> genes implicated in immune responses, including interleukin 1A (<em>IL</em>-1A), <em>IL</em>-6, <em>IL</em>-7, <em>IL</em>-8, and <em>IL</em>-15 genes. MVA infection also stimulated the expression of NF-kappaB and components of the NF-kappaB signal transduction pathway, including p50 and TRAF-interacting protein. A marked increase in the expression of histone family members was also induced during MVA infection. Expression of the Wiskott-Aldrich syndrome family members WAS, WASF1, and the small GTP-binding protein RAC-1, which are involved in actin cytoskeleton reorganization, was enhanced after MVA infection. This study demonstrates that MVA infection triggered the induction of groups of genes, some of which may be involved in host resistance and immune modulation during virus infection.

BACKGROUND

Ovarian cancer represents an important problem in gynecologic oncology. A growing tumor induces the host endothelial cells to proliferate and supply the requisite vascular support allowing tumor development. Vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) have been demonstrated to induce angiogenesis in epithelial tumors in vivo.

METHODS

This study included 24 tumors from patients with epithelial ovarian cancer in different stages, in addition to 20 tissue samples of benign ovarian lesions as a control group. VEGF has been measured in the cytosolic fractions using enzyme immunoassay and confirmed by Western blot analysis. Tissue IL-8 mRNA was assessed using reverse transcriptase polymerase chain reaction and immunohistochemistry for its protein.

RESULTS

VEGF mean rank was significantly higher in ovarian cancer tumors compared to benign lesions (P < 0.001). Moreover, it was increased with advanced stages (P < 0.05) and in patients with poor survival (P < 0.05). Eight samples were positive for IL-8 mRNA, seven of them were in malignant group, with highest frequency in stages III and IV of the disease (6/12, 50%). IL-8 correlated with poor survival of the patients (P < 0.05). Log rank of Kaplan-Meier survival analysis was significant for FIGO stage, VEGF, and IL-8 (P < 0.05).

CONCLUSIONS

These results indicate that VEGF and IL-8 are related to the malignant transformation process and can be considered as indicators of poor prognosis in epithelial ovarian cancer patients.