Posttranslational modification (PTM) of islet autoantigens can cause lack of central tolerance in type 1 diabetes (T1D). Tissue transglutaminase (tTG), involved in PTM of gluten antigens in celiac disease, creates negatively charged peptides favored by T1D-predisposing HLA-DQ molecules, offering an attractive candidate modifying islet autoantigens in T1D. The highly predisposing HLA-DQ8cis/trans molecules share preferences for negatively charged peptides, as well as distinct peptide-binding characteristics that distinguish their peptide-binding repertoire. We screened islet autoantigens with the tTG substrate motif for candidate-modified epitopes binding to HLA-DQ8cis/trans and identified <em>31</em> candidate islet epitopes. Deamidation was confirmed for 28 peptides (90%). Two of these epitopes preferentially bound to HLA-DQ8cis and six to HLA-DQ8trans upon deamidation, whereas all other peptides bound equally to HLA-DQ8cis/trans. HLA-DQ8cis-restricted T cells from a new-onset T1D patient could only be generated against a deamidated proinsulin peptide, but cross-reacted with native proinsulin peptide upon restimulation. The rate of T-cell autoreactivity in recent-onset T1D patients extended from 42% to native insulin to 68% adding responses to modified proinsulin, versus 20% and 37% respectively, in healthy donors. Most patients responded by interferon-γ, whereas most healthy donors produced <em>interleukin</em>-10 only. Thus, T-cell autoreactivity exists to modified islet epitopes that differs in quality and quantity between patients and healthy donors.

This report characterizes nine new cell lines derived from patients with malignant pleural mesothelioma. The lines were initiated between July 1990 and July 1992 from solid tumors (5 lines) or effusions (4 lines) and had proliferated for a period of at least 2 months without senescence. They were characterized by cell size, doubling time, immunohistochemical analyses, electron microscopy, and chromosomal karyotyping. Growth factor/cytokine elaboration was determined using enzyme-linked immunoassays. The established lines were similar in morphology to their parent tumor (ie, epithelial or sarcomatoid). Cell sizes ranged from 59 to 81 microns, and the doubling times varied from <em>31</em> to 65 hours. The lines stained with cytokeratin and showed expected negative staining for adenomarkers including B72.3 and carcinoembryonic antigen. All cell lines exhibited aneuploidy, with modal chromosome numbers between 40 and 81 and had multiple chromosomal aberrations. Significant production of granulocyte-monocyte colony-stimulating factor, leukemia inhibitory factor, platelet-derived growth factor, and <em>interleukin</em>-6 was seen. These new cell lines derived from human mesotheliomas can now be used to aid in the design of innovative treatment strategies.

1. Recent data indicate that <em>interleukin</em> (IL)-17 may contribute to neutrophilic airway inflammation by inducing the release of neutrophil-mobilizing cytokines from airway cells. The aim of this study was to evaluate the role of mitogen activated protein kinases in IL-17 induced release of IL-8 and IL-6 in bronchial epithelial cells. 2. Transformed human bronchial epithelial cells (16HBE) were stimulated with either IL-17 or vehicle. Both groups were treated either with SB202190 (inhibitor of p38 MAP kinase), PD98059 (inhibitor of extracellular-signal-regulated kinase [ERK] pathway), Ro-<em>31</em>-7549 (protein kinase C [PKC] inhibitor), LY 294002 (a phosphatidylinositol 3-kinase [PI 3-kinase] inhibitor) or vehicle. IL-6 and IL-8 levels were measured in conditioned media by ELISA. 3. The IL-17-induced release of IL-6 and IL-8 was concentration-dependently inhibited by SB202190 and by PD98059 in bronchial epithelial cells without affecting cell proliferation or survival. 4. Ro-<em>31</em>-7549 and LY294002 had no significant effect on IL-17-induced IL-6 or IL-8 release in bronchial epithelial cells. 4. Taken together, these data indicate a role for p38 and ERK kinase pathways in IL-17-induced release of neutrophil-mobilizing cytokines in human bronchial epithelial cells. These mechanisms constitute potential pharmacotherapeutical targets for inhibition of the IL-17-mediated airway neutrophilia.

Immunotolerance is maintained by regulatory T cells (Tregs), including CD4(+)CD25(hi), CD8(+)CD28(-), gammadelta, and CD3(+)CD56(+) [natural killer T (NKT)] cells. CD4(+)CD25(hi) cells are impaired in children with autoimmune hepatitis (AIH). Little is known about Tregs in adults with AIH. The aim of this study was to investigate the frequency and function of Treg subsets in adult patients with AIH during periods of active disease and remission. Forty-seven AIH patients (16 with active disease and <em>31</em> in remission) and 28 healthy controls were studied. Flow cytometry was used to evaluate surface markers and function-related intracellular molecules in gammadelta, CD8(+)CD28(-), NKT, and CD4(+)CD25(hi) cells. CD4(+)CD25(hi) T cell function was determined by the ability to suppress proliferation and interferon gamma (IFN-gamma) production by CD4(+)CD25(-) target cells. Liver forkhead box P3-positive (FOXP3(+)) cells were sought by immunohistochemistry. In AIH patients, particularly during active disease, CD4(+)CD25(hi) T cells were fewer, expressed lower levels of FOXP3, and were less effective at inhibiting target cell proliferation versus healthy controls. Moreover, although the numbers of CD8(+)CD28(-) T cells were similar in AIH patients and healthy controls, NKT cells were numerically reduced, especially during active disease, and produced lower quantities of the immunoregulatory cytokine <em>interleukin</em>-4 versus controls. In contrast, gammadelta T cells in AIH patients were more numerous versus healthy controls and had an inverted Vdelta1/Vdelta2 ratio and higher IFN-gamma and granzyme B production; the latter was correlated to biochemical indices of liver damage. There were few FOXP3(+) cells within the portal tract inflammatory infiltrate.

CONCLUSIONS

Our data show that the defect in immunoregulation in adult AIH is complex, and gammadelta T cells are likely to be effectors of liver damage.

BACKGROUND

Platelet-derived growth factor (PDGF) and superoxide anion (O2.-) have been implicated in vascular diseases. We investigated whether PDGF stimulates the production of O2.- in human aortic smooth muscle cells (HSMCs) and whether O2.- leads in this way to the activation of nuclear factor-kappaB (NF-kappaB) and induction of monocyte chemoattractant protein 1 (MCP-1) in PDGF-stimulated HSMCs.

RESULTS

PDGF-AB concentration- and time-dependently stimulated O2.- generation from HSMCs. The stimulatory effect of PDGF-AB was mimicked by PDGF-BB but not by PDGF-AA. The generation of O2.- by PDGF-AB was attenuated by the NAD(P)H oxidase inhibitor iodonium diphenyl, the specific protein kinase C (PKC) inhibitor Ro <em>31</em>-8220, and the phosphatidylinositol 3-kinase inhibitor wortmannin. Allopurinol and nifedipine had no effect on PDGF-AB-induced O2.- release, whereas indomethacin potentiated this response. Gel mobility shift assay revealed that PDGF-AB increased the binding activity of NF-kappaB, which contained predominantly the p50/p65 heterodimer in nuclear extracts from HSMCs. Superoxide dismutase as well as iodonium diphenyl, Ro <em>31</em>-8220, and wortmannin attenuated PDGF-AB-induced activation of NF-kappaB and expression of MCP-1 mRNA. In contrast, superoxide dismutase did not inhibit the <em>interleukin</em>-1beta-induced NF-kappaB activation.

CONCLUSIONS

The results demonstrate that PDGF stimulates O2.- generation in HSMCs via PKC-dependent and wortmannin-sensitive pathways involving flavoenzyme(s). This PDGF-induced O2.- production may be involved in vascular lesion formation by mediating, at least in part, NF-kappaB activation and MCP-1 induction.

Some studies have reported that proinflammatory polymorphisms in <em>interleukin</em>-1B (IL-1B) and IL-1 receptor antagonist (IL-1RN) genes are associated with increased gastric cancer risk. However, other studies have shown null or inverse associations. This meta-analysis reviews and summarizes published evidence for these associations. Searching the PubMed Database yielded 35 studies that reported on the association between IL-1B -511 C>T, IL-1B -<em>31</em> T>C, or IL-1RN variable number tandem repeat polymorphisms and gastric cancer risk. Q-statistics and I(2) statistics were calculated to examine heterogeneity. Summary odds ratios (OR) and 95% confidence intervals (95% CI) were calculated in the random-effects model using the DerSimonian-Laird method. For all gastric cancers, the overall ORs (95% CIs) for IL-1B -511 CT versus CC and TT versus CC genotypes were 1.07 (0.91-1.25) and 1.16 (0.95-1.42), respectively. ORs (95% CIs) for the association between IL-1B -<em>31</em> CT versus TT and CC versus TT genotypes were 0.99 (0.83-1.19) and 0.98 (0.78-1.21), respectively. For the associations between IL-1RN and gastric cancer, ORs (95% CIs) for *2/L versus LL and *2/*2 versus L/L were 1.15 (0.96-1.38) and 1.23 (0.79-1.92). For each of the examined associations, there was significant heterogeneity among studies; P(heterogeneity) < or = 0.001 and I(2) ranged from 0.54 to 0.71. Noncardia cancers showed stronger associations with IL-1B -511 CT or TT and IL1-RN *2/*2 genotypes, but limiting the analysis to intestinal-type cancers, studies conducted in Western countries, or studies in which polymorphisms were in Hardy-Weinberg equilibrium, made no material difference in the results. The overall associations between IL-1B or IL-1RN proinflammatory polymorphisms and gastric cancer were null but several studies showed an association. The sources of this variation are unclear.

OBJECTIVE

To assess the timing of changes in cytokines, cytokine-related markers, autoantibodies and viral antibodies in the pathogenesis of rheumatoid arthritis (RA).

METHODS

Case-control study nested in a prospective cohort of <em>31</em> 330 blood donors in Oslo, Norway. Forty-nine donors developed RA up to 23 years after their most recent blood donation. Stored sera from these donors (case sera) and a sex- and age-matched sample of 245 healthy donors (control sera), and postdiagnostic sera from 33 of the 49 RA cases, were analysed for a panel of cytokines and cytokine-related markers, autoantibodies and antibodies against Epstein-Barr virus and parvovirus B19.

RESULTS

Cytokines and cytokine-related markers were generally negative in case sera from >5 years before the diagnosis of RA. In the 5-year interval immediately before the diagnosis of RA, more case than control sera were positive (odds ratios >2) for interleukin (IL)-1 alpha, IL-1 beta, IL-1 receptor antagonist, IL-4, IL-10, tumour necrosis factor-alpha and soluble tumour necrosis factor receptor I. In postdiagnostic sera, however, 11 of 16 examined cytokines and cytokine-related markers were statistically significantly elevated compared with control sera. Seropositivity for IgG antibodies against cyclic citrullinated peptides and for IgM and IgA rheumatoid factors were seen in case sera from up to 18 years before the diagnosis of RA. IgG antibodies against Epstein-Barr virus and parvovirus B19 did not differ significantly between case and control sera.

CONCLUSIONS

Cytokines and cytokine-related markers appear to be upregulated rather late in RA pathogenesis. In contrast, IgM rheumatoid factor and IgG anti-cyclic citrullinated peptide autoantibodies may precede the diagnosis of RA by up to two decades.

OBJECTIVE

To determine the value of the erythrocyte sedimentation rate (ESR) and plasma interleukin-6 (IL-6) as biologic markers for monitoring disease activity in giant cell arteritis (GCA).

METHODS

Twenty-five patients with biopsy-proven GCA were enrolled into a prospective treatment study. Therapy was initiated with prednisone, 60 mg/day, followed by a predetermined tapering schedule. Patients were monitored monthly for clinical signs of active vasculitis and laboratory parameters indicative of inflammation, including elevated ESR (>30 mm/hour) and elevated plasma IL-6 concentrations (>6.1 pg/ml).

RESULTS

Upon initiation of corticosteroid treatment, clinical signs of GCA disappeared in all patients; however, 60% of the patients developed symptoms of recurrent disease, on 1 or more occasions, while the prednisone dosage was being reduced. These 31 disease flares diagnosed over 550 days were associated with symptoms of systemic inflammation but did not result in vascular complications. The ESR was elevated in 76% of the patients prior to initiation of treatment (median 65 mm/hour) and normalized by day 28 of therapy (median 6 mm/hour). The median ESR remained in the normal range during the followup period. Plasma IL-6 levels, which were abnormal in 92% of untreated patients (median 16 pg/ml), were partially responsive to the initial high doses of corticosteroids by day 28 (median 6 pg/ml), but levels did not completely normalize with continued therapy. Elevation of the ESR was seen during only 58% of all disease flares, but 89% of disease recurrences were associated with increased plasma IL-6 levels (P = 0.03).

CONCLUSIONS

Plasma IL-6 is more sensitive than ESR for indicating disease activity in untreated and treated GCA patients. Standard corticosteroid regimens only partially suppress vascular inflammation. Smoldering disease activity may expose GCA patients to the risk of progressive vascular disease (e.g., formation of aortic aneurysms) and chronic systemic complications such as IL-6-mediated osteopenia.

<em>Interleukin</em>-<em>31</em>, produced mainly by activated CD4(+) T cells, is a newly discovered member of the gp130/IL-6 cytokine family. Unlike all the other family members, IL-<em>31</em> does not engage gp130. Its receptor heterodimer consists of a unique gp130-like receptor chain IL-<em>31</em>RA, and the receptor subunit OSMRbeta that is shared with another family member oncostatin M (OSM). Binding of IL-<em>31</em> to its receptor activates Jak/STAT, PI3K/AKT and MAPK pathways. IL-<em>31</em> acts on a broad range of immune- and non-immune cells and therefore possesses potential pleiotropic physiological functions, including regulating hematopoiesis and immune response, causing inflammatory bowel disease, airway hypersensitivity and dermatitis. This review summarizes the recent findings on the biological characterization and physiological roles of IL-<em>31</em> and its receptors.

Hypoxia has been implicated in the pathogenesis of preeclampsia, a hypertensive disorder of pregnancy. However, in vivo evidence and mechanistic understanding remain elusive. Preeclampsia is associated with impaired placental angiogenesis. We have recently shown that <em>interleukin</em> (IL)-10 can support trophoblast-driven endovascular crosstalk. Accordingly, we hypothesize that pathological levels of oxygen coupled with IL-10 deficiency induce severe preeclampsia-like features coupled with elevated production of antiangiogenic factors, apoptotic pathways, and placental injury. Exposure of pregnant wild-type and IL-10(-/-) mice to 9.5% oxygen resulted in graded placental injury and systemic symptoms of renal pathology, proteinuria (wild-type 645.15 ± 115.73 versus 198.09 ± 93.45; IL-10(-/-) 819.<em>31</em> ± 127.85 versus 221.45 ± 82.73 μg/mg/24 hours) and hypertension (wild-type 118.37 ± 14.45 versus 78.67 ± 14.07; IL-10(-/-) 136.03 ± 22.59 versus 83.97 ± 18.25 mm Hg). Recombinant IL-10 reversed hypoxia-induced features in pregnant IL-10(-/-) mice confirming the protective role of IL-10 in preeclampsia. Hypoxic exposure caused marked elevation of soluble fms-like tyrosine kinase 1 (110.8 ± 20.1 versus 44.7 ± 11.9 ng/mL) in IL-10(-/-) mice compared with their wild-type counterparts (81.6 ± 13.1 versus 41.2 ± 8.9 ng/mL), whereas soluble endoglin was induced to similar levels in both strains (approximately 380 ± 50 versus 180 ± <em>31</em> ng/mL). Hypoxia-induced elevation of p53 was associated with marked induction of proapoptotic protein Bax, downregulation of Bcl-2, and trophoblast-specific apoptosis in utero-placental tissue. Collectively, we conclude that severe preeclampsia pathology could be triggered under certain threshold oxygen levels coupled with intrinsic IL-10 deficiency, which lead to excessive activation of antiangiogenic and apoptotic pathways.

OBJECTIVE

Patients with metastatic or recurrent Ewing's sarcoma family of tumors and alveolar rhabdomyosarcoma have <25% 5-year survival in most studies. This study administered a novel immunotherapy regimen aimed at consolidating remission in these patients.

METHODS

Fifty-two patients with translocation positive, recurrent, or metastatic Ewing's sarcoma family of tumors or alveolar rhabdomyosarcoma underwent prechemotherapy cell harvest via apheresis for potential receipt of immunotherapy. Following completion of standard multimodal therapy, 30 patients ultimately initiated immunotherapy and were sequentially assigned to three cohorts. All cohorts received autologous T cells, influenza vaccinations, and dendritic cells pulsed with peptides derived from tumor-specific translocation breakpoints and E7, a peptide known to bind HLA-A2. Cohort 1 received moderate-dose recombinant human interleukin-2 (rhIL-2), cohort 2 received low-dose rhIL-2, and cohort 3 did not receive rhIL-2.

RESULTS

All immunotherapy recipients generated influenza-specific immune responses, whereas immune responses to the translocation breakpoint peptides occurred in 39%, and only 25% of HLA-A2(+) patients developed E7-specific responses. Toxicity was minimal. Intention-to-treat analysis revealed a 31% 5-year overall survival for all patients apheresed (median potential follow-up 7.3 years) with a 43% 5-year overall survival for patients initiating immunotherapy.

CONCLUSIONS

Consolidative immunotherapy is a scientifically based and clinically practical approach for integrating immunotherapy into a multimodal regimen for chemoresponsive cancer. Patients receiving immunotherapy experienced minimal toxicity and favorable survival. The robust influenza immune responses observed suggest that postchemotherapy immune incompetence will not fundamentally limit this approach. Future studies will seek to increase efficacy by using more immunogenic antigens and more potent dendritic cells.

OBJECTIVE

Single nucleotide polymorphisms (SNPs) in the gene that encodes interleukin (IL)-28B predict response of patients with chronic hepatitis C to antiviral therapy. We investigated the roles of polymorphisms rs12979860 and rs8099917 on the early virologic response of treatment-naïve patients.

METHODS

SNPs were identified by real-time polymerase chain reaction analysis of samples from 682 patients (genotype [GT]1=372, GT2/3=208, GT4=102) who were treated with 180 μg pegylated interferon-α2a and 400 or 800 mg (GT2/3, depending on the protocol) or 1000-1200 mg (GT1/4) ribavirin/day. The duration of treatment was 24 (GT2/3) or 24-72 weeks (GT1/4).

RESULTS

Patients with C/C also had higher rates of rapid virologic response (RVR) (GT1, 38.3% vs 11.6%; GT4, 76.5% vs 23.5%; both P<.001) and sustained virologic responses (SVRs) (GT1, 79.1% vs 43.2%; GT4, 85.3% vs 44.1%; both P<.001). In patients with GT2/3, the RVR was more frequent in carriers of C/C (75.3% vs 52.6%, P<.01), but SVR rates were similar between those with C/C and T (80.5% vs 74.4%, P=.31). Results for rs8099917 were comparable. The positive predictive value of rs12979860 C/C for SVR was higher than that of rs8099917 T/T (80.5% vs 71.6%). Overall, RVR was the best predictor of SVR. In patients who did not have GT1, IL28B polymorphisms did not affect the SVR if RVR data were included in the multivariate analysis.

CONCLUSIONS

An early virologic response to pegylated interferon and ribavirin is more likely among carriers of rs12979860 C/C and rs8099917 T/T, which might underlie their high rates of SVR. Determination of the IL28B genotype and whether patients have an RVR might be used in future studies of patients with hepatitis C virus genotype 1 or 4.

Early clinical trials using T lymphocyte-depleted human marrow for transplantation have reported that such grafts reduce, to varying degrees, both the incidence and the severity of graft-v-host disease (GVHD). However, to date, no clear estimates have been made as to what degree of T cell depletion is necessary to prevent GVHD in every case. To address this problem, we used a limiting dilution assay (LDA) to quantitate residual clonable T lymphocytes in human T cell-depleted bone marrow in <em>31</em> HLA-identical transplants for leukemia. The number of phytohemagglutinin -<em>interleukin</em> 2-responsive T lymphocytes determined by LDA and expressed as T cell per kilogram recipient weight was found to correlate with the subsequent development of GVHD: no patients who received less than 1 X 10(5) T cell per kilogram developed GVHD (N = 24). Of the seven patients who received 1 X 10(5) to 4.4 X 10(5) T cell per kilogram, four patients developed grade I or II skin GVHD. This study thus provides a quantitative estimate of the number of T lymphocytes necessary to initiate clinically detectable GVHD in an HLA-identical host.

BACKGROUND

Leptin regulates food intake and modulates immunity and inflammation. A positive feedback mechanism has been described between tumour necrosis factor (TNF) and leptin, and it has been suggested that leptin potentiates inflammation in patients with rheumatoid arthritis (RA).

OBJECTIVE

To assess whether inflammation correlates with leptin concentrations in patients with RA, and whether anti-TNF treatment modulates leptin concentrations in these patients.

METHODS

Leptin, IL6 and CRP were measured (at baseline and after 2 weeks of treatment) in the blood of <em>31</em> patients with RA starting either anti-TNF treatment or placebo, and in 18 healthy controls.

RESULTS

In patients with RA, plasma leptin concentrations at baseline correlated inversely with the degree of inflammation as assessed by C reactive protein (CRP; r(s)(2) = 0.21, p<0.01) or interleukin (IL) 6 concentrations (r(s)(2) = 0.22, p<0.008). Mean (SD) leptin concentrations did not differ between patients with RA and controls (6.0 (4.6) v 4.2 (2.8) ng/ml in men; 15.1 (7.9) v 13.4 (5.2) ng/ml in women). Short course anti-TNF treatment for 2 weeks did not modify leptin concentrations, despite significant reduction of CRP and IL6.

CONCLUSIONS

A significant inverse correlation between inflammation and leptin concentrations was found in patients with active RA, although plasma leptin concentrations did not significantly differ from those in healthy controls. This suggests that active chronic inflammation may lower plasma leptin concentrations. Two weeks' treatment with anti-TNF did not change plasma leptin concentrations and longer treatment may be needed to see an effect on leptin.

Interferon (IFN)-gamma plays an important role in the pathogenesis of sepsis. Production of IFN-gamma is stimulated by synergistic effects of <em>interleukin</em> (IL)-18, IL-12, and IL-15. To investigate the regulation of IFN-gamma production during severe gram-negative infection, the plasma concentrations of IFN-gamma, IL-18, IL-12, and IL-15 were measured in 83 patients with suspected melioidosis. The diagnosis was confirmed in 62 patients, <em>31</em> of whom had blood cultures positive for Burkholderia pseudomallei, of whom 12 died. Compared with healthy controls, patients had elevated levels of IFN-gamma, IL-18, IL-12p40, and IL-15 on admission, with significantly higher levels in blood culture-positive patients, and these levels remained elevated during the 72-h study period. In whole blood stimulated with heat-killed B. pseudomallei, anti-IL-12 had a stronger inhibitory effect than anti-IL-18 and anti-IL-15 on IFN-gamma production. This effect of anti-IL-12 was further enhanced by anti-IL-18. These data suggest that during gram-negative sepsis, IFN-gamma production is controlled at least in part by endogenous IL-18, IL-12, and IL-15.

Atopy is characterized by an immune system that is biased to T helper cell, type 2 (Th2) activation. This condition predisposes to asthma, a disease in which a Th2 activation was found in blood and lungs. However, most blood studies have considered purified cells, which might give an incomplete view of immune reactions. In this study, we assessed in whole blood cultures the Th1/Th2 paradigm in atopy and asthma. Sixty-nine subjects (<em>31</em> atopic asthmatics, six nonatopic asthmatics, 13 atopic nonasthmatics, and 19 control subjects) were included in this study. <em>Interleukin</em>-4 (IL-4), interferon gamma (IFN-gamma), and IL-12 were assayed in stimulated whole blood culture supernatants by using a flow cytometer microsphere-based assay. Intracellular IL-4 and IFN-gamma were detected in T cells and CD8(+) T cells by flow cytometry. Atopy was characterized by a higher production of IL-4, which was correlated to total IgE levels, and by an impairment of the T-cell capacity to produce IFN-gamma. This impairment was correlated to the number of positive skin tests. In asthma, the overproduction of IL-4 was still found if atopy was present. Unexpectedly, an overproduction of IFN-gamma was found, which was related to an increased capacity of CD8(+) T cells to produce IFN-gamma. The number of IFN-gamma-producing CD8(+) T cells was related to asthma severity, to bronchial hyperresponsiveness, and to blood eosinophilia. In addition, this number was correlated to IL-12 production. These results show that in addition to the well-known Th2 inflammation in asthma, there are IFN-gamma-producing CD8(+) T cells in the blood, possibly controlled by IL-12.

Both virus-mediated damage to airway tissues and induction of pro-inflammatory cytokines such as <em>interleukin</em>-8 (IL-8) could contribute to symptom severity during viral respiratory infections in children. To test the hypothesis that IL-8 contributes to the pathogenesis of respiratory symptoms during naturally acquired respiratory viral infections in children, nasal wash samples collected from infants with acute viral infections (n = 198) or from healthy uninfected infants (n = <em>31</em>) were analysed for IL-8. Nasal wash IL-8 was positively related to age in uninfected children (rs = 0.36, p < 0.05). Respiratory syncytial virus (RSV) infection caused more severe respiratory symptoms compared to infections with influenza A, parainfluenza viruses, or rhinoviruses. In addition, RSV, parainfluenza and rhinovirus infections increased levels of IL-8 in nasal lavage fluid, and there were some differences in the ability of the viruses to induce IL-8 production (RSV>influenza, p < 0.05). Finally, there were significant correlations between nasal wash IL-8 levels and symptom scores during infections with rhinovirus (rs = 0.56, p < 0.001) or influenza A (rs = 0.45, p < 0.05), but not with parainfluenza virus or RSV. These findings provide evidence of a close relationship between the generation of IL-8 and symptoms during acute community-acquired infections with rhinovirus or influenza A. In contrast, for RSV and parainfluenza infections, factors in addition to IL-8 production appear to contribute to the generation of clinical symptoms.

BACKGROUND

MicroRNAs (miRNAs) play important regulatory roles in development and stress response in plants. Wild soybean (Glycine soja) has undergone long-term natural selection and may have evolved special mechanisms to survive stress conditions as a result. However, little information about miRNAs especially miRNAs responsive to aluminum (Al) stress is available in wild soybean.

RESULTS

Two small RNA libraries and two degradome libraries were constructed from the roots of Al-treated and Al-free G. soja seedlings. For miRNA identification, a total of 7,287,655 and 7,035,914 clean reads in Al-treated and Al-free small RNAs libraries, respectively, were generated, and 97 known miRNAs and <em>31</em> novel miRNAs were identified. In addition, 49 p3 or p5 strands of known miRNAs were found. Among all the identified miRNAs, the expressions of 30 miRNAs were responsive to Al stress. Through degradome sequencing, 86 genes were identified as targets of the known miRNAs and five genes were found to be the targets of the novel miRNAs obtained in this study. Gene ontology (GO) annotations of target transcripts indicated that 52 target genes cleaved by conserved miRNA families might play roles in the regulation of transcription. Additionally, some genes, such as those for the auxin response factor (ARF), domain-containing disease resistance protein (NB-ARC), leucine-rich repeat and toll/<em>interleukin</em>-1 receptor-like protein (LRR-TIR) domain protein, cation transporting ATPase, Myb transcription factors, and the no apical meristem (NAM) protein, that are known to be responsive to stress, were found to be cleaved under Al stress conditions.

CONCLUSIONS

A number of miRNAs and their targets were detected in wild soybean. Some of them that were responsive to biotic and abiotic stresses were regulated by Al stress. These findings provide valuable information to understand the function of miRNAs in Al tolerance.

BACKGROUND

Chronic obstructive pulmonary disease (COPD) is associated with eosinophilic airway inflammation in 10-20% of patients. Benralizumab, an anti-interleukin-5 receptor α monoclonal antibody, depletes blood and sputum eosinophils. We aimed to establish whether benralizumab reduces acute exacerbations of COPD in patients with eosinophilia and COPD.

METHODS

We did this randomised, double-blind, placebo-controlled, phase 2a study between Nov 18, 2010, and July 13, 2013, at 26 sites in the UK, Poland, Germany, Canada, the USA, Denmark, and Spain. Adults aged 40-85 years, with moderate-to-severe COPD, at least one acute exacerbation of COPD, and a sputum eosinophil count of 3·0% or more within the previous year, were randomly assigned (1:1) via computer-generated permuted block randomisation (block size of four), with an interactive voice or web-response system, to receive placebo or 100 mg benralizumab subcutaneously, every 4 weeks (three doses), then every 8 weeks (five doses) over 48 weeks. Study site personnel included in study assessments, participants, and data analysts, were masked to treatment allocation. The primary endpoint was the annualised rate of acute exacerbations of COPD at week 56, defined as the number of acute exacerbations divided by total duration of person-year follow-up. Secondary and exploratory endpoints included COPD-specific Saint George's Respiratory Questionnaire (SGRQ-C), Chronic Respiratory Questionnaire self-administered standardised format (CRQ-SAS), pre-bronchodilator forced expiratory volume in 1 second (FEV1), and safety. We did a prespecified subgroup analysis by baseline blood eosinophil count. Analyses were by intention to treat and per-protocol. This trial is registered with ClinicalTrials.gov, number NCT01227278.

RESULTS

We randomly assigned 101 patients to receive placebo (n=50) or benralizumab (n=51), of whom 88 (87%) patients completed the study. Six patients who completed the study were excluded from the per-protocol population because of major protocol violations; the per-protocol population thus included 82 patients. Benralizumab did not reduce the annualised rate of acute exacerbations of COPD compared with placebo in the per-protocol population, with rates of 0·95 (0·68-1·29; n=40) versus 0·92 (0·67-1·25; n=42). Mean pre-bronchodilator FEV1 change from baseline to week 56 was -0·06 L (SD 0·24) with placebo, and 0·13 L (0·41) with benralizumab (p=0·014). Numerical, albeit non-significant, improvement in acute exacerbations of COPD, SGRQ-C, CRQ-SAS, and FEV1 were greater in benralizumab-treated patients with baseline blood eosinophil concentrations of 200 cells per μL or more or 300 cells per μL or more. Incidence of treatment-emergent adverse events was similar between the two groups, with the most common events being respiratory disorders (31 [62%] of 50 patients given placebo vs 32 [63%] of 51 given benralizumab) and infections (28 [56%] vs 27 [53%]). A higher incidence of serious treatment-emergent adverse events were recorded in patients in the benralizumab group than in those in the placebo group (14 vs nine patients), although none of these events were considered by the investigator to be benralizumab related.

CONCLUSIONS

Compared with placebo, benralizumab did not reduce the rate of acute exacerbations of COPD. However, the results of prespecified subgroup analysis support further investigation of benralizumab in patients with COPD and eosinophilia.

BACKGROUND

MedImmune.

OBJECTIVE

To investigate whether immunologic abnormalities in patients with Behçet's disease (BD) are related to abnormalities of the Th1/Th2 ratio.

METHODS

Th1/Th2 cytokine production by peripheral blood lymphocytes (PBL) from <em>31</em> patients with BD, 11 patients with inflammatory arthritis, and 10 healthy blood donors was evaluated by intracellular immunofluorescence staining. Serum <em>interleukin</em>-12 (IL-12) levels were measured using an enzyme amplified-sensitivity immunoassay. The effect of recombinant IL-12 (rIL-12) on spontaneous and Fas-mediated apoptosis of phytohemagglutinin (PHA)-stimulated PBL was evaluated by flow cytometry using propidium iodide (PI) staining and a bromodeoxyuridine (BrdU)/PI procedure.

RESULTS

Intracellular immunofluorescence staining of IL-2, IL-4, and interferon-gamma (IFNgamma) in CD3+ lymphocytes from BD patients demonstrated a strong polarization of the immune response toward the Th1 pathway that correlated with the progression of BD. Peripheral Th1 cells were significantly increased in patients with active disease (n = 14) as compared with those in patients in complete remission (n = 17), patients with inflammatory arthritis, and normal donors. In addition, serum IL-12 levels were correlated with peripheral Th1 lymphocytes and disease progression. Apoptotic analysis revealed that PHA-activated PBL from patients with active disease were highly sensitive to spontaneous and Fas-mediated activation-induced cell death. However, addition of rIL-12 to complete medium prevented this spontaneous and Fas-induced apoptosis and enhanced the proliferation of Th1 lymphocytes.

CONCLUSIONS

Taken together, these results indicate that a strong Th1 immune response occurs in active BD and suggest that IL-12 plays a substantial part in the pathogenesis of BD. By preventing spontaneous and Fas-induced cell death, in fact, it results in an abnormal growth of autoreactive Th1 lymphocytes that could contribute to the prolonged inflammatory autoimmune condition of BD.

OBJECTIVE

Tofacitinib, an orally administered Janus kinase inhibitor, blocks signaling through γ-chain-containing cytokines (interleukins 2, 4, 7, 9, 15, and 21). We performed a phase 2 trial to measure its efficacy in patients with moderate-to-severe active Crohn's disease.

METHODS

Patients (N = 139; age, ≥18 y) with moderate-to-severe active Crohn's disease were assigned randomly to groups given 1 mg (n = 36), 5 mg (n = 34), or 15 mg (n = 35) tofacitinib or placebo (n = 34), twice daily for 4 weeks, at 48 centers in 12 countries. The primary end point was the proportion of clinical responders at week 4 (decrease from baseline in the Crohn's Disease Activity Index score of ≥70 points [Response-70]). Secondary end points included clinical remission (Crohn's Disease Activity Index score of <150 points) at week 4.

RESULTS

A clinical response was observed in 36% (P = .467), 58% (P = .466), and 46% (P ≥ .999) of patients given the 1-, 5-, and 15-mg doses of tofacitinib, compared with 47% of patients given placebo. Clinical remission was observed in 31% (P = .417), 24% (P = .776), and 14% (P = .540) of patients given the 1-, 5-, and 15-mg doses of tofacitinib, compared with 21% of patients given placebo. The 15-mg dose of tofacitinib reduced levels of C-reactive protein and fecal calprotectin from baseline. Adverse and serious adverse events were similar among groups. Dose-dependent increases in low- and high-density lipoprotein cholesterol were observed in patients given the 5- or 15-mg doses of tofacitinib.

CONCLUSIONS

There were no significant differences in the percentage of patients with moderate-to-severe active Crohn's disease who achieved clinical responses (Response-70) or clinical remission after 4 weeks' administration of tofacitinib (1, 5, or 15 mg) or placebo twice daily. However, a large percentage of patients given placebo achieved Response-70 or remission. Reductions in C-reactive protein and fecal calprotectin levels among patients given the 15-mg dose of tofacitinib indicate its biologic activity. ClinicalTrials.gov number: NCT00615199.

OBJECTIVE

To evaluate immunoglobulin heavy chain (IgH) gene rearrangements, cytokines and chemokines, and infectious agents in primary intraocular B-cell lymphoma (PIOL) cells, in order to better diagnose and understand PIOL.

METHODS

We studied ocular specimens from 57 patients with PIOL at the National Eye Institute from 1991 to 2001. Specimens were analyzed for IgH gene rearrangements using microdissection and polymerase chain reaction (PCR). We measured vitreal interleukin (IL)-10 and IL-6 levels by enzyme-linked immunosorbent assay. IL-10 mRNA was studied in PIOL cells using microdissection and reverse transcribed (RT)-PCR. Chemokine and chemokine receptor expression was examined by using immunohistochemistry. Infectious DNA of human herpetic virus-8 (HHV-8), Epstein-Bar virus (EBV), and Toxoplasma gondii was detected by using microdissection and PCR and was confirmed with Southern blot hybridization.

RESULTS

IgH rearrangement(s) were demonstrated in all 50 tested cases. Cytokine levels were measured in the vitreous of 39 patients. Thirty-one had measurable cytokine levels: 24 of 31 had elevation of IL-10 relative to that of IL-6, and, in contrast, only 7 of 31 had elevation of IL-6 relative to IL-10. IL-10 mRNA was abundant in lymphoma cells of 6 examined cases. Lymphoma cells expressed chemokine receptors of CXCR4 and CXCR5 in three tested cases. HHV-8 DNA was found in 6 of 32 cases (18.8%), EBV DNA in 2 of 21 (9.5%), and T gondii DNA in 2 of 16 (12.5%).

CONCLUSIONS

Molecular analyses detecting IgH rearrangements and vitreal levels of IL-10 and IL-6 are useful adjuncts for PIOL diagnosis. A role for specific infectious agents is hypothesized in the pathogenesis of some cases of PIOL. B-cell chemokine is likely involved in attracting PIOL cells into the eye.

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.

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 <em>interleukin</em> 28B (IL28B) in HCV-VT and in the spontaneous clearance of HCV among infected infants. Between 1991 and 2009, 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. IL28B (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 <em>31</em> 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 (20%) 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' IL-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 IL28B. 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. IL28B plays no role in HCV-VT, but IL28B CC child polymorphism is associated independently with the spontaneous clearance of HCV genotype-1 among infected children.