BACKGROUND

Blood-brain barrier (BBB) breakdown is observed in older versus younger adults and in late-onset Alzheimer's disease versus age-matched controls, but its causes and consequences in aging are unclear. We tested the hypothesis that BBB breakdown is associated with cognitive decline and inflammation in nondemented elders.

METHODS

Cerebrospinal fluid and serum inflammatory markers were measured using sandwich immunoassays in 120 subjects. Least Absolute Shrinkage and Selection Operator-logistic regression selected cerebrospinal fluid and serum signatures that best classified BBB impairment defined by the cerebrospinal fluid albumin index ≥9. Linear regression examined changes in Clinical Dementia Rating sum of boxes as a function of BBB integrity at baseline.

RESULTS

Mean age was 70 years, mean Mini-Mental State Examination was <em>27</em>, and BBB impairment was recorded in 13.5%. BBB breakdown was associated with cognitive decline (P = .015). Cerebrospinal fluid intercellular adhesion molecule-1, vascular endothelial growth factor, <em>interleukin</em>-8, serum amyloid A, macrophage derived chemokine, and gender generated an area under the curve of 0.95 for BBB impairment, and serum IL-16, VEGF-D, IL-15, and other variables generated an AUC of 0.92 for BBB impairment. Serum <em>interleukin</em>-16, vascular endothelial growth factor-D, <em>interleukin</em>-15, and other variables generated an area under the curve of 0.92.

CONCLUSIONS

BBB breakdown is associated with more rapid cognitive decline. Inflammatory mechanisms, including cell adhesion, neutrophil migration, lipid metabolism, and angiogenesis may be implicated. Cell adhesion, neutrophil migration, high-density lipoprotein metabolism, and angiogenesis are implicated.

MicroRNAs have been implicated as a central regulator of the immune system. We have previously reported that Helicobacter pylori (H. pylori) was able to increase the expression of miR-146a, and miR-146a may negatively regulate H. pylori-induced inflammation, but the exact mechanism of how H. pylori contribute the induction of miR-146a is not clear. Here, we attempted to assess the role of H. pylori related proinflammatory cytokines including <em>interleukin</em> (IL)-8, tumor necrosis factor (TNF)-α, and <em>interleukin</em> (IL)-1β, and cytotoxin-associated gene A (CagA) virulence factor on the induction of miR-146a. We found that IL-8, TNF-α, and IL-1β could contribute to the induction of miR-146a in gastric epithelial cell HGC-<em>27</em> in NF-κB-dependent manner, while the induction of miR-146a upon H. pylori stimulation was independent of above proinflammatory cytokines. Furthermore, overexpression of miR-146a reduced H. pylori-induced IL-8, TNF-α, and IL-1β. However, CagA had no effect on the miR-146a induction. Taken together, our study suggest that proinflammatory cytokines IL-8, TNF-α, and IL-1β could contribute to the induction of miR-146a during H. pylori infection, while CagA is not necessarily required for miR-146a induction. miR-146a may function as novel negative regulators to modulate the inflammation.

Little is known about the pathogenesis of Entamoeba histolytica and how epithelial cells respond to the parasite. Herein, we characterized the interactions between E. histolytica and colonic epithelial cells and the role macrophages play in modulating epithelial cell responses. The human colonic epithelial cell lines Caco-2 and T84 were grown either as monoculture or co-cultured in transwell plates with differentiated human THP-1 macrophages for 24 h before stimulation with soluble amebic proteins (SAP). In naive epithelial cells, prolonged stimulation with SAP reduced the levels of heat shock protein (Hsp) <em>27</em> and 72. However in THP-1 conditioned intestinal epithelial cells SAP enhanced Hsp<em>27</em> and Hsp72, which was dependent on the activation of ERK MAP kinase. Hsp synthesis induced by SAP conferred protection against oxidative and apoptotic injuries. Treatment with SAP inhibited NF-kappaB activation induced by <em>interleukin</em>-1beta; specifically, the NF-kappaB-DNA binding, nuclear translocation of p65 subunit, and phosphorylation of IkappaB-alpha were reduced. Gene silencing by small interfering RNA confirmed the role of Hsp<em>27</em> in suppressing NF-kappaB activation at IkappaB kinase (IKK) level. By co-immunoprecipitation studies, we found that Hsp<em>27</em> interacts with IKK-alpha and IKK-beta, and this association was increased in SAP-treated conditioned epithelial cells. Overexpression of wild type Hsp<em>27</em> amplified the effects of SAP, whereas a phosphorylation-deficient mutant of Hsp<em>27</em> abrogated SAP-induced NF-kappaB inhibition. In conditioned epithelial cells, Hsp<em>27</em> was phosphorylated at serine 15 after prolonged exposure to SAP. This mechanism may explain the absence of colonic inflammation seen in the majority of individuals infected with E. histolytica.

The prevalence of relative adrenal insufficiency (RAI) in critically ill cirrhosis patients with severe sepsis is over 60% and associated features include poor liver function, renal failure, refractory shock, and high mortality. RAI may also develop in noncritically ill cirrhosis patients but its relationship to the clinical course has not yet been assessed. The current study was performed in 143 noncritically ill cirrhosis patients admitted for acute decompensation. Within 24 hours after hospitalization adrenal function, plasma renin activity, plasma noradrenaline and vasopressin concentration, and serum levels of nitric oxide, <em>interleukin</em>-6 and tumor necrosis factor alpha were determined. RAI was defined as a serum total cortisol increase <9 μg/dL after 250 μg of intravenous corticotropin from basal values <35 μg/dL. Patients were followed for 3 months. RAI was detected in 26% of patients (n = 37). At baseline, patients with RAI presented with lower mean arterial pressure (76 ± 12 versus 83 ± 14 mmHg, P = 0.009) and serum sodium (131 ± 7 versus 135 ± 5 mEq/L, P = 0.007) and higher blood urea nitrogen (32 ± 24 versus 24 ± 15 mg/dl, P = 0.06), plasma renin activity (7.1 ± 9.9 versus 3.4 ± 5.6 ng/mL*h, P = 0.03), and noradrenaline concentration (544 ± 334 versus 402 ± 316 pg/mL, P = 0.02). During follow-up, patients with RAI exhibited a higher probability of infection (41% versus 21%, P = 0.008), severe sepsis (<em>27</em>% versus 9%, P = 0.003), type-1 hepatorenal syndrome (HRS) (16% versus 3%, P = 0.002), and death (22% versus 7%, P = 0.01).

CONCLUSIONS

RAI is frequent in noncritically ill patients with acute decompensation of cirrhosis. As compared with those with normal adrenal function, patients with RAI have greater impairment of circulatory and renal function, higher probability of severe sepsis and type-1 HRS, and higher short-term mortality.

OBJECTIVE

Gain-of-function (GOF) mutations in the signal transducer and activator of transcription 1 (STAT1) result in unbalanced STAT signaling and cause immune dysregulation and immunodeficiency. The latter is often characterized by the susceptibility to recurrent Candida infections, resulting in the clinical picture of chronic mucocutaneous candidiasis (CMC). This study aims to assess the frequency of GOF STAT1 mutations in a large international cohort of CMC patients.

METHODS

STAT1 was sequenced in genomic DNA from 57 CMC patients and 35 healthy family members. The functional relevance of nine different STAT1 variants was shown by flow cytometric analysis of STAT1 phosphorylation in patients' peripheral blood cells (PBMC) after stimulation with interferon (IFN)-α, IFN-γ or <em>interleukin</em>-<em>27</em> respectively. Extended clinical data sets were collected and summarized for 26 patients.

RESULTS

Heterozygous mutations within STAT1 were identified in 35 of 57 CMC patients (61%). Out of 39 familial cases from 11 families, 26 patients (67%) from 9 families and out of 18 sporadic cases, 9 patients (50%) were shown to have heterozygous mutations within STAT1. Thirteen distinct STAT1 mutations are reported in this paper. Eight of these mutations are known to cause CMC (p.M202V, p.A267V, p.R<em>27</em>4W, p.R<em>27</em>4Q, p.T385M, p.K388E, p.N397D, and p.F404Y). However, five STAT1 variants (p.F172L, p.Y287D, p.P293S, p.T385K and p.S466R) have not been reported before in CMC patients.

CONCLUSIONS

STAT1 mutations are frequently observed in patients suffering from CMC. Thus, sequence analysis of STAT1 in CMC patients is advised. Measurement of IFN- or IL-induced STAT1 phosphorylation in PBMC provides a fast and reliable diagnostic tool and should be carried out in addition to genetic testing.

Type 1 regulatory T cells (Tr1 cells) are induced by <em>interleukin</em>-<em>27</em> (IL-<em>27</em>) and have critical roles in the control of autoimmunity and resolution of inflammation. We found that the transcription factors IRF1 and BATF were induced early on after treatment with IL-<em>27</em> and were required for the differentiation and function of Tr1 cells in vitro and in vivo. Epigenetic and transcriptional analyses revealed that both transcription factors influenced chromatin accessibility and expression of the genes required for Tr1 cell function. IRF1 and BATF deficiencies uniquely altered the chromatin landscape, suggesting that these factors serve a pioneering function during Tr1 cell differentiation.

Accumulating evidence indicates that <em>interleukin</em> (IL)-<em>27</em>, a member of the IL-12 family of cytokines, antagonizes pathological Th17 effector cell responses. Relatively little is known about the cytokines that regulate human Th17 cells. In this study, we investigated the effect of IL-<em>27</em> on the differentiation of human Th17 cells and on committed memory Th17 cells. We demonstrate that IL-<em>27</em> suppresses the development of human Th17 cells by downregulating retinoid orphan nuclear receptor C expression and that this inhibition is associated with the induction of the intracellular signaling factors STAT1 and induction of the suppressor of cytokine signaling protein 1. The IL-<em>27</em>-mediated inhibition of IL-17 is independent of IL-10. We show that IL-<em>27</em> inhibits differentiation of naïve T cells into IL-17(+) T cells under different Th17 polarizing conditions. IL-<em>27</em> suppresses other Th17 subset cytokines such as IL-22 and IL-21 but not tumor necrosis factor-α. Moreover, we also show that IL-<em>27</em> inhibits IL-17 production by committed Th17 memory cells, which is independent of IL-10. These studies show that IL-<em>27</em> negatively regulates both the developing and committed human Th17 responses and therefore may be a promising therapeutic approach in the treatment of Th17-mediated diseases.

The liver is the major metabolic organ and is subjected to constant attacks from chronic viral infection, uptake of therapeutic drugs, life behavior (alcoholic), and environmental contaminants, all of which result in chronic inflammation, fibrosis, and, ultimately, cancer. Therefore, there is an urgent need to discover effective therapeutic agents for the prevention and treatment of liver injury, the ideal drug being a naturally occurring biological inhibitor. Here we establish the role of IL30 as a potent antiinflammatory cytokine that can inhibit inflammation-induced liver injury. In contrast, <em>interleukin</em> (IL)<em>27</em>, which contains IL30 as a subunit, is not hepatoprotective. Interestingly, IL30 is induced by the proinflammatory signal such as IL12 through interferon-gamma (IFN-γ)/signal transducer and activator of transcription 1 signaling. In animal models, administration of IL30 by way of a gene therapy approach prevents and treats both IL12-, IFN-γ-, and concanavalin A-induced liver toxicity. Likewise, immunohistochemistry analysis of human tissue samples revealed that IL30 is highly expressed in hepatocytes, yet barely expressed in inflammation-induced tissue such as fibrous/connective tissue.

CONCLUSIONS

These novel observations reveal a novel role of IL30 as a therapeutic cytokine that suppresses proinflammatory cytokine-associated liver toxicity.

Breast cancers frequently metastasize to bone where they often cause extensive tumor-induced osteoclast-mediated osteolysis. <em>Interleukin</em>-6 (IL-6) and IL-11 are two cytokines exhibiting osteolytic properties through their potent stimulation of osteoclast formation. We investigated the expression of IL-6 and IL-11 in 99 invasive primary breast tumors by immunohistochemistry and in situ hybridization, respectively. We examined their potential as predictive factors for further development of bone metastases. 52/90 (57%) of tumor samples showed IL-6 cytoplasmic immunostaining. There was no significant association between IL-6 status and any of the classical prognostic factors. 15/89 (17%) of the tumor samples expressed IL-11 mRNA. A positive IL-11 mRNA status was associated with a low tumor grade (P=0.05). Tumors expressing IL-11 mRNA had a statistically significant (P=0.002) higher rate of bone metastases occurrence (12/15, 80%) than IL-11 negative tumors (<em>27</em>/74, 37%). Such association was not found for IL-6. Our findings demonstrate for the first time IL-11 gene expression in some primary invasive breast tumors and suggest the potential of this cytokine as possible biological predictive factor for the development of bone metastases.

Gut microbes programme their metabolism to suit intestinal conditions and convert dietary components into a panel of small molecules that ultimately affect host physiology. To unveil what is behind the effects of key dietary components on microbial functions and the way they modulate host-microbe interaction, we used for the first time a multi-omic approach that goes behind the mere gut phylogenetic composition and provides an overall picture of the functional repertoire in <em>27</em> fecal samples from omnivorous, vegan and vegetarian volunteers. Based on our data, vegan and vegetarian diets were associated to the highest abundance of microbial genes/proteins responsible for cell motility, carbohydrate- and protein-hydrolyzing enzymes, transport systems and the synthesis of essential amino acids and vitamins. A positive correlation was observed when intake of fiber and the relative fecal abundance of flagellin were compared. Microbial cells and flagellin extracted from fecal samples of 61 healthy donors modulated the viability of the human (HT29) colon carcinoma cells and the host response through the stimulation of the expression of Toll-like receptor 5, lectin RegIIIα and three <em>interleukins</em> (IL-8, IL-22 and IL-23). Our findings concretize a further and relevant milestone on how the diet may prevent/mitigate disease risk.

OBJECTIVE

This study was performed to identify morphologic features of cardiac myxomas related to embolism and to provide a better understanding of the biology of these tumors, mainly in relation to their interleukin (IL)-6 expression and DNA content.

METHODS

A total of 37 cardiac myxomas were reviewed retrospectively in a clinicopathologic study that included the correlation of echocardiographic and pathologic findings in 25 cases, together with immunohistochemical evaluation of IL-6 expression and flow cytometric DNA analysis of 35 tumors.

RESULTS

There were 24 female patients and 13 male patients. The mean (+/- SD) age was 52 +/- 15 years. Fifty-four percent of patients presented with dyspnea, 51% presented with increased erythrocyte sedimentation rate (ESR), and 27% presented with embolic episodes, which were significantly associated with villous surface tumors. Atrial fibrillation was registered in 19% of patients and was significantly associated with large left atrial myxomas. Echocardiography proved to be a reliable method for preoperative diagnosis and for predicting tumor size and morphology. There was no perioperative mortality or long-term recurrences. The frequency of early surgical complications was associated with a longer mean ischemic time. Seventeen percent of tumors had abnormal DNA content, and 74% of tumors showed immunohistochemical expression of IL-6. Neither of these factors showed a significant association with embolism or constitutional illness.

CONCLUSIONS

Villous surface myxomas are related to embolism, and large left atrial tumors are related to atrial fibrillation. Echocardiography is a reliable method with which to predict tumor size and morphology. Myxoma cells usually express IL-6, and some tumors have abnormal cellular DNA content. Surgical excision of the tumor is a safe and effective treatment.

<em>Interleukin</em>-10 (IL-10) is an anti-inflammatory and immune-deviating cytokine expressed in the endometrium and placenta. IL-10 null mutant (IL-10-/-) mice have been employed to examine the role of IL-10 in regulating immune events in early pregnancy and its significance in implantation and pregnancy success. The inflammatory response elicited in endometrial tissue by insemination was amplified in IL-10-/- mice, with a 66% increase in leukocytes in the endometrial stroma on Day 3 of pregnancy. Despite this, no evidence of abnormal type 1/type 2 skewing was seen in T-lymphocytes from lymph nodes draining the uterus. On Day 18 of gestation, IL-10-/- females mated with IL-10-/- males had 15% more implantation sites and <em>27</em>% more viable fetuses than pregnant wild-type (IL-10+/+) mice. Placental weight was unaffected, but fetal weight and the fetal:placental weight ratio were higher in IL-10-/- pregnancies. Similar data were obtained in allogeneic pregnancies when IL-10-/- females were mated with major-histocompatibility complex (MHC) disparate IL-10-/- males. Pups delivered by IL-10-/- mothers had increased birth weight and followed an altered growth trajectory, with growth impairment evident from early postnatal life into adulthood, which was reflected in alterations in body composition at 14 wk of age. This study shows that although IL-10 is not essential for maternal immune tolerance or successful pregnancy irrespective of MHC disparity in the fetus, maternal IL-10 is a determinant of growth trajectory in progeny in utero and after birth.

A growing body of evidence suggests an essential role of the heterodimeric cytokine, IL-<em>27</em>, for regulating immunity. IL-<em>27</em> is composed of two subunits (p28 and EBI3) and is classified as a member of the IL-12 family of cytokines. APCs have been recognized as a major cellular source of IL-<em>27</em> following activation with microbial products or IFNs (types I and II). In this review, we describe the current knowledge of the implications of IL-<em>27</em> during the pathogenesis of infectious and autoimmune diseases. Experimental studies have used genetically targeted IL-<em>27</em>RA-/- mice, EBI3-/- mice, and p28-/- mice or involved study designs with administration of bioengineered IL-<em>27</em>/IL-<em>27</em>RA homologs. Whereas many reports have described that IL-<em>27</em> suppresses inflammation, we also review the current literature, suggesting promotion of inflammation by IL-<em>27</em> in some settings. Recent advances have also been made in understanding the cross-talk of cleavage products of the complement system with IL-<em>27</em>-mediated immune responses. Additional data on IL-<em>27</em> have been obtained recently by observational studies in human patients with acute and chronic inflammatory diseases. Collectively, the findings from the past decade identify IL-<em>27</em> as a critical immunoregulatory cytokine, especially for T cells, whereas some controversy is fueled by results challenging the view of IL-<em>27</em> as a classical silencer of inflammation.

OBJECTIVE

To determine the progression-free survival at 12 weeks, to evaluate the toxic effects, and to analyze the biological activity of thalidomide in patients with relapsed multiple myeloma (MM) after high-dose chemotherapy and stem cell transplantation.

METHODS

From 1999 to 2001, we performed a multicenter prospective phase 2 study in patients with MM that relapsed after high-dose chemotherapy and stem cell transplantation to evaluate the efficacy of oral thalidomide, with dose escalation from 200 to 600 mg/d over 12 weeks and a subsequent maintenance phase of 200 mg/d for up to 1 year. Outcome was correlated with serum and plasma levels of vascular endothelial growth factor and serum levels of tumor necrosis factor alpha, soluble intercellular adhesion molecule 1, interferon gamma, interleukin (IL) 2, and IL-6 during treatment.

RESULTS

Thirty patients were treated (19 men and 11 women; median age, 58 years). The median number of prior therapies was 5, and the median duration from diagnosis of MM to study enrollment was 4.3 years. The 12-week progression-free survival rate was 67% (95% confidence interval [CI], 48%-86%). The observed response rate (partial response plus minor response) was 43% (95% CI, 28%-60%) with a median duration of 6 months. Attributable toxicities included constipation, fatigue, rash, and neuropathy, which was dose limiting in 8 patients (27%). Dose escalation from 200 to 600 mg/d was achieved in 50% of patients. Although responses were observed with lower doses, possibly eliminating the need to escalate the dose, responses were also seen in patients who completed the dose escalation. Some patients had disease progression while receiving the maintenance dose of 200 mg/d. Analysis of biomarker assays did not identify any biomarker associated with greater response, but a significant increase in levels of soluble intercellular adhesion molecule 1, IL-2, and interferon gamma was seen with thalidomide therapy.

CONCLUSIONS

The optimal thalidomide dose varies, and adverse effects can be dose limiting. The dose of thalidomide therapy should be based on the individual patient to ensure that it is well tolerated and that a response is achieved.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive epithelial malignancy. It is the most common neoplasm arising in the upper aerodigestive tract. <em>Interleukin</em> (IL) 12 and IL-18 are cytokines which have a major anti-tumour activity via stimulation of a T-helper type 1 (Th1) immune response. <em>Interleukin</em> 10, a potent antagonist of IL-12, is a cytokine which possesses immunosuppressive activity mainly produced via T-helper type 2 (Th2) cells. Studies of other types of cancer have shown that the level of IL-12 in serum or tissues is suppressed and/or the IL-10 level is increased, suggesting that there is an impaired cell-mediated anti-tumour response. The aim of this study was to measure pre-operative serum cytokine concentrations in HNSCC patients in order to detect any changes in IL-10, IL-12 and IL-18, compared with non-tumour controls. The relationship between cytokine levels and standard clinicopathological features, including tumour site, tumour stage and presence of nodal metastasis, was also examined. Fifty-seven patients with primary HNSCC were prospectively recruited, together with 40 non-tumour control patients with a similar age and sex distribution. Serum cytokine levels were measured using commercial quantitative enzyme-linked immunosorbent assay. The HNSCC patients had significantly lower IL-12 levels (median; interquartile range) than controls (42.8 pg/ml, 26.2-61.6 vs 52.3 pg/ml, 37.5-113.7; p=0.018). Also, patients were more likely to have detectable IL-10 levels than were controls, as IL-10 was positive in <em>27</em>/55 patients but in only 9/39 controls (p=0.011). Furthermore, IL-10 detectability varied according to primary site, being more commonly observed in hypopharyngeal and laryngeal tumours, and IL-10 was more likely to be detected with advanced tumour stage (T3 and T4). No differences in IL-18 levels were observed between patients and controls (p=0.169). These results suggest (in agreement with studies on other solid malignancies) that HNSCC causes a significant change in the serum levels of specific Th1 and Th2 cytokines, producing an in vivo environment that is unlikely to promote an effective cell-mediated anti-tumour response.

<em>Interleukin</em>-6 (IL-6) is produced in response to inflammatory and noninflammatory stress and acts as the principal regulator of the acute-phase protein response. IL-6 stimulates the hypothalamic-pituitary-adrenal axis and may be involved in the thyroid function abnormalities observed in nonthyroidal illness (NTI). This study examined the effects of single-dose IL-6 (3 microg/kg subcutaneously [s.c.]) in healthy human subjects: 19 received IL-6 and 13 received control saline injection. The dose of IL-6 was chosen on the basis of previous studies indicating that the peak IL-6 level after injection reaches concentrations observed with major stress such as abdominal surgery. Plasma levels of thyrotropin (TSH), free thyroxine (FT4), total T4, 3,5-3'-L-triiodothyronine (T3), 3,3'-5'-L-triiodothyronine or reverse T3 (rT3), and thyroxine-binding globulin (TBG) were measured over a 4-hour period and 24 hours after IL-6 injection. Plasma TSH levels were <em>27</em>% lower 240 minutes after IL-6 relative to control levels (0.93 +/- 0.10 v 1.28 +/- 0.18 mIU/mL, P = .001), but recovered by 24 hours. Plasma FT4 was elevated at 240 minutes compared with the controls (1.16 +/- 0.04 v 1.03 +/- 0.03 ng/dL, P = .0002). T4 levels were also elevated at 240 minutes (7.8 +/- 0.36 v 7.05 +/- 0.37 microg/dL, P = .0003). TBG levels were not significantly changed at this time point. At 24 hours, T3 levels were 19% lower than the control values (87.6 +/- 5.1 v 108.5 +/- 5.4 ng/dL, P = .0002); plasma rT3 levels were elevated by 21% compared with control levels (30.6 +/- 1.7 v 24.3 +/- 1.3 ng/dL, P = .002), while FT4 levels returned to normal. The changes in T3/rT3 levels were reminiscent of the pattern observed in NTI that may be due to inhibition of type-1 5'-deiodinase. Cortisol levels were greatly elevated after IL-6 compared with control values; peak levels were observed 120 minutes after IL-6 injection (28.7 +/- 1.6 v 9.5 +/- 1.0 ng/dL, P < .0001). This elevation in cortisol may have contributed to the suppression of TSH levels and inhibition of type-1 5'-deiodinase activity. Alternatively, IL-6 may have suppressed TSH secretion via a direct suprapituitary action. The elevation of T4 and FT4 levels may have been due to inhibition of T4 degradation at the liver and/or by direct action of IL-6 on the thyroid gland. These findings demonstrate the potent effects of IL-6 on thyroid hormone metabolism in healthy individuals, and suggest that IL-6 may act directly or indirectly at two or more sites on thyroid hormone secretion and metabolism.

OBJECTIVE

Allograft rejection involves multiple effector mechanisms. <em>Interleukin</em>(IL)-12 family members play a critical role in influencing helper T-cell differentiation and inflammatory processes, and their respective role in orchestrating inflammation of autoimmune or infectious origin starts to be unravelled. We highlight recent findings on the function of the different IL-12 family members: IL-12p70, IL-23, IL-<em>27</em> and IL-35 and discuss their possible involvement in influencing the balance between graft rejection and tolerance.

RESULTS

The capacity of dendritic cells to produce IL-12 and IL-23 strongly influences the outcome of CD4 T-cell responses. While the IL-12/interferon-gamma axis has classically been involved in autoimmune pathologies and acute graft rejection, it is now clear that it also displays immunoregulatory properties. In contrast, IL-23 promotes the function of proinflammatory IL-17-producing cells in both mice and humans. Both IL-<em>27</em> and IL-35 have recently emerged as important regulators of adaptive immune responses.

CONCLUSIONS

The contribution of the IL-12/interferon-gamma pathway to acute graft rejection may be more complicated than initially thought. As our understanding of the IL-12 family is rapidly growing and changing, the respective role of its members in orchestrating innate and adaptive immune responses toward alloantigens should be addressed.

BACKGROUND: Lymphocyte activation culminates in blastogenesis, cell cycle progression, DNA replication and mitosis. These complex cellular changes are programmed almost simultaneously by multiple ligands and receptors that trigger specific signal transduction pathways and transcription factors. Until now, the discovery of the genes regulated by each ligand/receptor pair has been hampered by the technologies available. RESULTS: To identify <em>interleukin</em>-2 (IL-2)-responsive genes, human peripheral blood mononuclear cells (PBMC) were pre-activated with anti-CD3, rested, and restimulated with IL-2 for 4 hr. Gene expression was analyzed using Affymetrix U95Av2 oligonucleotide arrays. To determine the most stringent parameters to score a gene as a bona fide IL-2 target, the expression of 19 known IL-2-regulated genes was examined first. All were induced at least 2-fold, with a difference in fluorescent intensity of>>/= 100 at p < 0.05. An additional 53 unique genes met these criteria. To determine which of these were immediate/early IL-2 targets in T cells, purified T cells were stimulated with IL-2 for 4 hr in the presence of cycloheximide to prevent secondary gene expression. Of the 72 genes identified in PBMCs, 20 were detected as immediate/early IL-2-regulated genes in purified T cells. In addition, <em>27</em> unique genes were IL-2-regulated in T cells but not in PBMCs. CONCLUSIONS: For a successful reductionist approach to the analysis of gene expression in lymphocyte activation, it is necessary to examine purified cell populations and immediate/early gene expression regulated by each ligand/receptor pair involved. This approach should allow the discovery of genes regulated by all of the ligand/receptor pairs involved in lymphocyte activation.

Proinflammatory cytokines such as <em>interleukin</em> 6 (IL-6), tumour necrosis factor alpha (TNF-alpha) and IL-1beta are considered to be involved in the pathogenesis of multiple myeloma (MM). In the present study, we examined a G/C polymorphism at position -174 in the promoter region of IL-6, a biallelic polymorphism at position -308 in the promoter region of TNF-alpha, the TaqI restriction fragment length polymorphism in exon 5 of IL-1beta and a variable number of identical tandem repeat polymorphisms in intron 2 of IL-1 receptor antagonist (IL-1Ra) genes. The alleles of these loci are known to influence the level of production of the cytokines and the IL-1Ra. Seventy-three patients with MM, <em>27</em> with monoclonal gammopathy of undetermined significance (MGUS) and 129 healthy individuals were included. No difference was found between patients and healthy controls or between MM and MGUS patients in the distributions of genotypes and frequencies of alleles of the IL-6 (-174), TNF-alpha (-308), IL-1beta TaqI and IL-1Ra gene polymorphisms. No associations between the polymorphisms at the loci under study and clinical factors such as age, sex, clinical stage at onset and M-protein type were observed. Our results indicate that the cytokine (IL-6, TNF-alpha and IL-1beta) and IL-Ra gene polymorphisms do not confer susceptibility to the development of MM.

BACKGROUND

Behcet's disease (BD) is a chronic systemic disorder characterised by oral and genital ulcerative lesions, ocular and cutaneous manifestations. Cytokines are the major mediators of immunologic and inflammatory reactions. Nitric oxide is reactive nitrogen intermediate which plays a key role in pathogenesis of many inflammatory and autoimmune skin diseases. The study was conducted to determine serum interleukin-2 (IL-2), interleukin-6 (IL-6), tumour necrosis factor (TNF)-alpha and nitric oxide levels in relation to the pathogenesis of Behcet's disease.

METHODS

Serum IL-2, IL-6, and TNF-alpha levels were measured with test kits by enzyme-linked immunosorbent assay (ELISA) method, while serum nitric oxide levels were determined with a test kit by colorimetric method. Serum IL-2, IL-6, TNF-alpha and nitric oxide concentrations in 27 patients with Behcet's disease and in 16 healthy controls were determined by extrapolation from their standard curves. The significance of the mean differences between the 2 groups was assessed by the Mann-Whitney U test.

RESULTS

The serum levels of IL-2, IL-6, TNF-alpha, and nitric oxide concentrations in patients with BD were significantly higher than those of the controls (P <0.001).

CONCLUSIONS

Our results suggest that elevated levels of IL-2, IL-6, TNF-alpha, and nitric oxide in Behcet's disease appear to be related to the disease.

Amiodarone, an iodine-rich cardiac drug, may induce thyrotoxicosis (AIT), which can occur in patients with preexisting thyroid abnormalities and in subjects with apparently normal thyroid glands. The pathogenesis of AIT is often due to iodine-induced excessive thyroid hormone synthesis, especially in patients with underlying thyroid disease. In some instances, however, AIT may be related to a destructive process due to amiodarone-induced thyroiditis, resulting in thyroid cell damage and thyroid hormone release into the circulation. Another thyroid inflammatory process, subacute thyroiditis, has been recently reported to be associated with markedly increased serum <em>interleukin</em>-6 (IL-6) levels. To investigate the significance of serum IL-6 levels in AIT, we evaluated in a cross-sectional study the following subjects: <em>27</em> AIT patients, 15 with no apparent thyroid abnormalities (AIT-) and 12 with nodular goiter and/or thyroid autoimmune disease (AIT+); 14 euthyroid patients receiving chronic amiodarone therapy; 10 patients with amiodarone-induced hypothyroidism; 56 patients with spontaneous hyperthyroidism due to Graves' disease (n = 35) or toxic adenoma/nodular goiter (n = 21); 20 subjects with nontoxic goiter; and 50 healthy controls. Serum free thyroid hormone concentrations did not differ in patients with amiodarone-induced or spontaneous hyperthyroidism. Mean (+/- SE) serum IL-6 values were as follows: AIT-, 573.5 +/- 78.7 fmol/L (range, 149.4-1145.1); AIT+, 152.7 +/- 46.3 fmol/L (range, < 25-505.6); euthyroid patients receiving chronic amiodarone therapy, 51.4 +/- 10.0 fmol/L (range, < 25-122.5); amiodarone-induced hypothyroidism, 43.8 +/- 8.4 fmol/L (range, < 25-84.3); Graves' disease, 108.2 +/- 18.2 fmol/L (range, < 25-250); toxic adenoma/nodular goiter, 97.6 +/- 10.3 fmol/L (range, < 25-168.9); nontoxic goiter, 47.3 +/- 7.1 fmol/L (range, < 25-106.6); and controls, 37.8 +/- 6.2 fmol/L (range, < 25-99.4). Serum IL-6 values in AIT- patients were markedly higher (P < 0.0001) than those in all other groups. Values in AIT+, although slightly higher, did not significantly differ from those in patients with spontaneous hyperthyroidism. AIT- patients had low 24-h thyroidal radioiodine uptake (RAIU), whereas AIT+ had inappropriately low normal to high (9-58%) RAIU values in the presence of excess iodine. The presence of markedly elevated serum IL-6 concentrations and low thyroidal RAIU values in patients with AIT without underlying thyroid disease suggests the presence of amiodarone-induced thyroiditis as the etiology of thyrotoxicosis. Treatment of 2 such patients with prednisone was associated with a dramatic reduction and prompt normalization of IL-6 and thyroid hormone values.(ABSTRACT TRUNCATED AT 400 WORDS)

We have recently identified 8-difluoromethoxy-1-ethyl-6-fluoro-1, 4-dihydro-7-[4-(2-methoxyphenyl)-1-piperazinyl]-4-oxoquinoline-3-carb oxylic acid (K-12) as a potent and selective inhibitor of human immunodeficiency virus type 1 (HIV-1) transcription. In the search for more effective derivatives and their mode of action, we have found 7-(3,4-dehydro-4-phenyl-1-piperidinyl)-1, 4-dihydro-6-fluoro-1-methyl-8-trifluoromethyl-4-oxoquinoline-3- carboxylic acid (K-37) and 8-difluoromethoxy-1,4-dihydro-6-fluoro-7-(3, 4-dehydro-4-phenyl-1-piperidinyl)1-[4,(1,2, 4-triazol-1-yl)methylphenyl]-4-oxoquinoline-3-carboxylic acid (K-38) to be more potent inhibitors of HIV-1 replication than K-12. The EC50 values of K-37 and K-38 for HIV-1IIIB were <em>27</em> and 3.8 nM in peripheral blood mononuclear cells, respectively. These values were approximately 3- and 24-fold lower than the EC50 of K-12. K-38 was also a more potent inhibitor of HIV-1 replication in chronically infected cells, such as tumor necrosis factor alpha-stimulated OM-10. 1 cells. K-37 and K-38 proved to be more cytotoxic than K-12 for a variety of cell lines as well as peripheral blood mononuclear cells. These compounds were more inhibitory of Tat-induced HIV-1 long terminal repeat-driven gene expression than K-12, which suggests that their mechanism of action is attributable in part to the inhibition of Tat function. Interestingly, K-37 and K-38 could suppress the production of tumor necrosis factor alpha and <em>interleukin</em> 6 in phytohemagglutinin-stimulated peripheral blood mononuclear cells and the expression of intercellular adhesion molecule 1 in tumor necrosis factor alpha-stimulated human umbilical vein endothelial cells at their nontoxic concentrations. In contrast, another K-12 derivative, 1, 4-dihydro-8-dimethylaminomethyl-6-fluoro-7-[4-(2-methoxyphenyl)-1-pip eradinyl]-1-methyl-4-oxoquinoline-3-carboxylic acid (K-42), had anti-HIV-1 activity and cytotoxicity profiles similar to those of K-12, but K-42 scarcely inhibited the cytokine production and intercellular adhesion molecule 1 expression.

Isoforms of heat shock protein (Hsp) <em>27</em> were used as intracellular markers to study tumor necrosis factor/<em>interleukin</em>-1 (TNF/IL-1) regulation of protein phosphatases in primary human fibroblasts. These isoforms were rapidly phosphorylated to varying degrees when fibroblasts were treated with either TNF, IL-1, okadaic acid, calyculin A, ARS, epidermal growth factor, fibroblast growth factor, H2O2, buthionine sulfoximine, N-ethylmaleimide, diethylmaleimide, or iodoacetate. However, inhibitors of protein kinases A and C, tyrosyl protein kinases, and general protein kinases had no effect on the enhanced phosphorylation of these isoforms in TNF, IL-1, okadaic acid, or calyculin A-stimulated cells, suggesting that the activation of protein kinases by itself is insufficient to produce these changes. Isoforms of 32P-labeled Hsp<em>27</em> were dephosphorylated during cold-chases with excess phosphate in the absence but not in the presence of TNF/IL-1 or inhibitors of protein phosphatases suggesting that inactivation of protein phosphatase(s) plays a role in TNF/IL-1 signal transduction. Assays of phosphatase activity of cytosolic fractions from TNF or okadaic acid treated human fibroblasts showed an inactivation of protein phosphatase activity against the 32P-labeled Hsp<em>27</em> protein substrates. In vitro assays of partially purified phosphatase activity from primary human fibroblasts with Hsp<em>27</em> substrate also showed the protein phosphatase activity to be inhibited by ARS. Like okadaic acid, ARS mimics TNF in inducing specific patterns of cellular protein phosphorylation. Taken together these findings are consistent with the hypothesis that a SH-dependent protein phosphatase is inactivated during the early events of TNF/IL-1 signal transduction, hence inhibitors of protein phosphatases and SH modifying compounds can mimic the early effects of TNF/IL-1 on cells.

Endotoxin (lipopolysaccharide [LPS]) stimulates the production of cytokines, which mediate many of the metabolic effects associated with infection. In LPS-sensitive C57B1/6 mice, LPS doses as low as 0.01 micrograms per mouse decreased adipose tissue lipoprotein lipase (LPL) activity by greater than 50%. In LPS-resistant C3H/HeJ mice, which do not produce cytokines in response to LPS, doses of LPS as high as 10 micrograms per mouse did not affect LPL activity in adipose tissue. In muscle of C57Bl/6 mice, LPL activity was decreased by <em>27</em>% after 10 micrograms of LPS, whereas in C3H/HeJ mice there was no effect. These results indicate that the LPS-induced decrease in both adipose and muscle LPL activity is mediated by cytokines. Tumor necrosis factor (TNF), <em>interleukin</em> (IL)-1, leukemia-inhibiting factor (LIF), interferon alfa, and interferon gamma all decreased adipose tissue LPL activity in intact mice. In skeletal and cardiac muscle, only IL-1 and interferon gamma decreased LPL activity, whereas TNF, LIF, and interferon alfa had no effect. Inhibition of TNF activity blocked the increase in serum triglycerides that is characteristically observed after LPS but did not affect the ability of LPS to decrease adipose tissue LPL activity. Inhibition of IL-1 activity with IL-1 receptor antagonist partially inhibited the increase in serum triglycerides; however, the ability of LPS to decrease LPL activity in either adipose or muscle tissue was not affected. These data indicate that although TNF and IL-1 play a role in mediating the increase in serum triglyceride levels, these cytokines do not play a crucial role in the inhibition of either adipose or muscle LPL activity.(ABSTRACT TRUNCATED AT 250 WORDS)