UNASSIGNED

Neuroinflammation appears to be a key modulator of disease progression in amyotrophic lateral sclerosis (ALS) and thereby a promising therapeutic target. The CD4+Foxp3+ regulatory T-cells (Tregs) infiltrating into the central nervous system suppress neuroinflammation and promote the activation of neuroprotective microglia in mouse models of ALS. To our knowledge, the therapeutic association of host Treg expansion with ALS progression has not been studied in vivo.

UNASSIGNED

To assess the role of Tregs in regulating the pathophysiology of ALS in humans and the therapeutic outcome of increasing Treg activity in a mouse model of the disease.

UNASSIGNED

This prospective multicenter human and animal study was performed in hospitals, outpatient clinics, and research institutes. Clinical and function assessment, as well as immunological studies, were undertaken in 33 patients with sporadic ALS, and results were compared with 38 healthy control participants who were consecutively recruited from the multidisciplinary ALS clinic at Westmead Hospital between February 1, 2013, and December <em>31</em>, 2014. All data analysis on patients with ALS was undertaken between January 2015 and December 2016. Subsequently, we implemented a novel approach to amplify the endogenous Treg population using peripheral injections of <em>interleukin</em> 2/<em>interleukin</em> 2 monoclonal antibody complexes (IL-2c) in transgenic mice that expressed mutant superoxide dismutase 1 (SOD1), a gene associated with motor neuron degeneration.

UNASSIGNED

In patients with ALS, Treg levels were determined and then correlated with disease progression. Circulating T-cell populations, motor neuron size, glial cell activation, and T-cell and microglial gene expression in spinal cords were determined in SOD1G93A mice, as well as the association of Treg amplification with disease onset and survival time in mice.

UNASSIGNED

The cohort of patients with ALS included 24 male patients and 9 female patients (mean [SD] age at assessment, 58.9 [10.9] years). There was an inverse correlation between total Treg levels (including the effector CD45RO+ subset) and rate of disease progression (R = -0.40, P = .002). Expansion of the effector Treg population in the SOD1G93A mice was associated with a significant slowing of disease progression, which was accompanied by an increase in survival time (IL-2c-treated mice: mean [SD], 160.6 [10.8] days; control mice: mean [SD], 144.9 [10.6] days; P = .003). Importantly, Treg expansion was associated with preserved motor neuron soma size and marked suppression of astrocytic and microglial immunoreactivity in the spinal cords of SOD1G93A mice, as well as elevated neurotrophic factor gene expression in spinal cord and peripheral nerves.

UNASSIGNED

These findings establish a neuroprotective effect of Tregs, possibly mediated by suppression of toxic neuroinflammation in the central nervous system. Strategies aimed at enhancing the Treg population and neuroprotective activity from the periphery may prove therapeutically useful for patients with ALS.

OBJECTIVE

Polymorphisms in the interleukin 1 alpha (IL1A) and IL1B gene regions were previously associated with keratoconus in a Korean population. In the present study, we investigated whether the IL1A and IL1B polymorphisms are associated with keratoconus in a Japanese population.

METHODS

A total of 169 Japanese patients with keratoconus and 390 Japanese healthy controls were recruited. We genotyped one IL1A single nucleotide polymorphism (SNP; rs2071376) and two IL1B SNPs (rs1143627 and rs16944) to compare the frequencies of alleles, genotypes, and haplotypes between cases and controls.

RESULTS

Statistically significant association was observed for rs1143627 (-31 T>C) in the IL1B promoter region; the T allele of rs1143627 was associated with an increased risk of keratoconus (p=0.014, corrected p value [pc]=0.043, odds ratio=1.38). The C allele of rs16944 (-511 C>T) in the IL1B promoter region had a 1.33-fold increased risk of keratoconus, although this increase did not reach statistical significance (p=0.033, pc=0.098). The TT genotype of rs1143627 was weakly associated with an increased risk of keratoconus (p=0.033, pc=0.099, odds ratio=1.52). However, no significant differences were found in the allele and genotype frequencies between the cases and controls for rs2071376 in IL1A. Regarding haplotypic diversity, the haplotype created by the T allele of rs1143627 and C allele of rs16944 was associated with a 1.72-fold increased risk of keratoconus (p=4.0×10(-5), pc=1.6×10(-4)).

CONCLUSIONS

Our results replicate associations reported recently in a Korean population. Thus, IL1B may play an important role in the development of keratoconus through genetic polymorphisms.

BACKGROUND

Inflammation and oxidative stress (OS) are cardiovascular risk factors in patients with chronic kidney disease. N-acetylcysteine (NAC) is a thiol-containing antioxidant with anti-inflammatory properties and has been shown to reduce the number of cardiovascular events in hemodialysis patients.

METHODS

The current study aimed to determine the effect of oral NAC (2 x 600 mg/daily) on plasma levels of inflammatory and OS markers in peritoneal dialysis (PD) patients. We performed a placebo-controlled study over 8 weeks in 30 patients (40% males, age 52 +/- 13 years) on regular PD. Before the study was started, the patients were divided into 2 groups of 15 patients matched for age and gender. 22 patients completed the study (12 on NAC, 10 on placebo). Proinflammatory cytokines [high-sensitivity C-reactive protein, interleukin-6 (IL-6), tumor necrosis factor-alpha, and pentraxin 3] and markers of OS (pentosidine, advanced oxidation protein products, homocysteine, glutathione, asymmetric dimethylarginine, and free sulfhydryls) were measured before and after treatment with NAC.

RESULTS

Treatment with NAC for 8 weeks increased mean baseline plasma NAC levels from 2.6 to 24.8 mumol/L (p = 0.007). This intervention, which caused no side effects, significantly diminished IL-6 levels, from 9.4 (4.5 - 31) to 7.6 (4.9 - 13.5) pg/mL (p = 0.006), whereas no such changes were observed in the placebo group. NAC treatment did not significantly affect the other inflammatory and OS markers.

CONCLUSIONS

Short-term oral NAC treatment resulted in reduction of circulating IL-6, suggesting that such treatment could be a useful strategy in blunting the inflammatory response in PD patients.

BACKGROUND

Gastric cancer (GC) is the most common malignancy in Oman. <em>Interleukin</em>-1beta gene (IL-1B) and <em>interleukin</em>-1 receptor antagonist gene (IL-1RN) polymorphisms have been associated with increased GC risk. No previous studies have examined their role in an Arab population. We tested the associations between polymorphisms of IL1B at positions -<em>31</em>, -511, and +3954 and the IL-1RN polymorphism [variable number of tandem repeats (VNTR) and TC polymorphism at the -2018 position] and GC in Omani Arab patients.

METHODS

Genomic DNA was extracted from peripheral blood of 245 control subjects and 118 gastric cancer patients. The DNA samples were analyzed using the TaqMan allelic discrimination test for IL-1B -<em>31</em>, -511, and +3954 polymorphisms and IL-1RN -2018 polymorphism. The VNTR of IL-1RN was genotyped using the polymerase chain reaction followed by agarose gel electrophoresis.

RESULTS

There was an association between the presence of IL-1RN*2 allele and gastric cancer [odds ratio (OR) = 2.2, 95% confidence interval (CI) = 1.0-3.3, P = 0.04). The GC risk further increased to OR = 3.5 (95% CI = 1.0-11.9) in Helicobacter pylori-positive patients. No association was found between any of the other polymorphisms studied and GC.

CONCLUSIONS

IL-1RN polymorphism increased the risk of GC in an Omani Arab population, consistent with previous reports. In contrast, the IL-1B -<em>31</em> polymorphism was not associated with an increased GC risk. These findings underscore the role of cytokine gene polymorphisms in the development of GC and further support the ethnic differences in the effect of IL-1B polymorphism on GC carcinogenesis.

This study was undertaken to determine whether infection with Helicobacter pylori strains that contain the cagE gene was associated with duodenal ulceration in children. The presence of flaA, cagA, and cagE genes was determined by polymerase chain reaction in H. pylori previously cultured from 29 children. Twelve (92%) of 13 children with duodenal ulcers were infected with cagE-positive isolates, compared with only 5 (<em>31</em>%) of 16 with gastritis alone (P<.01). Infection of gastric cells in tissue culture by cagE-positive H. pylori resulted in greater increments in <em>interleukin</em>-8 levels compared with cagE-negative strains (2.3+/-0.1 vs. 1.3+/-0.2 ng/mL in AGS cells [P<.005]; 1.5+/-0.3 vs. 0.5+/-0.2 ng/mL in KATO-III cells [P<.05]). H. pylori-containing cagE was associated with the presence of duodenal ulceration in children. Enhanced chemokine production after infection with cagE-positive H. pylori could affect disease outcome.

A phase-I/II study of recombinant <em>interleukin</em> 2 (rIL-2) was performed in <em>31</em> melanoma patients. The first dose of rIL-2 was given intrasplenically followed 4 hr later by an i.v. dose and 3 further i.v. doses on alternate days. Three courses of treatment were planned at 3-week intervals. The maximum tolerated single dose was 11 x 10(6) Cetus U/m2. Haematological and immunological data were available on 20 patients. Post-treatment response to rIL-2 therapy was evident from (i) a rapid depletion of peripheral blood lymphocytes (PBL) with a rebound at 4-7 days (2 times pre-treatment values); (ii) an increase in the number of IL-2 receptor-positive lymphocytes (4-15 times pre-treatment values); (iii) an increase in the number of "positive" patients with cytotoxic (anti-K562) peripheral blood mononuclear cells (PBMC) from 30% to 80%; (iv) amplified killing of K562 by positive patients in relation to pre-treatment values; and (v) the induction of PBMC cytotoxicity (in 45% of patients) against the NK-resistant, LAK-sensitive target, Mel I. Partial clinical responses to rIL-2 treatment were observed in 4 patients, but these were not reflected in the PBMC LAK activity or the other parameters examined.

Variable proportions of Hodgkin's disease (HD) cases are associated with the Epstein-Barr virus (EBV), but the role of EBV in HD is not entirely clear. Hodgkin and Reed-Sternberg (HRS) cells of EBV-associated HD are characterized by expression of the EBV gene product LMP1. In other cellular environments, LMP1 has been shown to induce <em>interleukin</em> (IL)-6. In this study, 105 HD cases were tested for differences in IL-6 expression among LMP1-positive and -negative cases. Isotopic in situ hybridization and correlation with the presence of EBV gene products revealed significantly higher proportions of cases with IL-6-expressing tumour cells in LMP1-positive (<em>31</em> of 37, 84 per cent) as compared with LMP1-negative HD cases (35 of 68, 51 per cent). Thus, although not exclusive to EBV-positive HRS cells, IL-6 expression appears to be upregulated in EBV-associated HD. IL-6 receptor (CD126) expression was tested by in situ hybridization and found in a broad spectrum of cell types, regularly including HRS cells. Superinduction of IL-6 expression may be among the mechanisms by which EBV confers a growth advantage on virus-infected HRS cells and by which the virus may contribute to the morphological and clinical peculiarities of HD.

BACKGROUND

There is an increasing evidence linking inflammation to some cardiovascular conditions, such as coronary artery disease and hypertension. Similarly, there is emerging data to support the association between inflammation and atrial fibrillation (AF). We also investigated the role of systemic inflammation in different categories of AF.

METHODS

Eighty five consecutive patients with AF were enrolled in this study. AF was categorized as new onset, chronic (persistent and permanent) and lone. Age- and sex-matched 30 healthy people consisted of control group. Serum level of high sensitive C-reactive protein (hs-CRP) and interleukin-6 (IL-6) was measured.

RESULTS

Serum hs-CRP level was higher in overall AF patients than in controls (0.63+/-0.57 vs 0.23+/-0.1 mg/dL, p=0.001). Similarly, IL-6 level was also higher in all AF patients compared with controls (29+/-36 vs 11.6+/-9.7 pg/mL, p=0.008). In subgroup analysis, hs-CRP and IL-6 levels were significantly higher in both chronic (0.69+/-0.62 vs 0.23+/-0.1 mg/dL, p=0.001; 30+/-39 vs 11.6+/-9.7 pg/mL, p=0.001, respectively) and new onset AF patients (0.51+/-0.46 vs 0.23+/-0.1 mg/dL, p=0.003; 28.4+/-31 vs 11.6+/-9.7 pg/mL, p=0.009, respectively) compared with controls. These markers were not different in new onset and chronic AF subgroups. On the other hand, hs-CRP and IL-6 levels tended to be high in lone AF patients (p=0.06). The presence of AF was an independent factor for hs-CRP (OR=0.35, 95%CI=0.1-0.61, p=0.005) and IL-6 (OR=17, 95%CI=1-37, p=0.037).

CONCLUSIONS

Our results support that inflammation may have an important role in the AF pathogenesis.

Pentachlorophenol (PCP), hexachlorocyclohexane-[alpha], -beta, and -[gamma] (HCH-[alpha], -beta, and -[gamma]), polychlorinated biphenyls (PCBs), and hexachlorobenzene (HCB) are widely distributed industrial chemicals. They are suspected to induce immunologic impairments in exposed individuals. We examined dose-response relationships of blood levels of these chemicals with cellular (numbers of lymphocyte subpopulations, in vitro lymphocyte response) or humoral (plasma cytokine levels, immunoglobulin autoantibodies) immunologic dysfunctions. We studied 146 patients who had been occupationally exposed primarily to PCBs for more than 6 months. Lymphocyte subpopulations, in vitro responses to mitogens and allogeneic stimulator cells, plasma neopterin, cytokines, soluble cytokine receptors, soluble adhesion molecules, anti-Ig autoantibodies, and liver transaminases were determined. Blood levels of the different compounds were strongly correlated with one another. There were only weak dose-response relationships between blood levels of PCBs with cellular immune parameters, and of HCHs and HCB with humoral immune parameters. An exception was the statistically significant negative association of HCB with interferon-[gamma] (IFN-[gamma]), indicating that HCB has a significant impact on Th1 lymphocytes. Patients with HCB blood levels above the mean of 1,109 ng/L more often had undetectable IFN-[gamma] blood levels than patients below the mean. Patients with increased PCB 138 >> 710 ng/L) had more frequently undetectable <em>interleukin</em>-4 blood levels than patients with PCB 138 below the mean, and patients with increased PCB 101 >> <em>31</em> ng/L) more often had low DR+ cell counts in the blood (< 190/microL) than patients with PCB 101 below the mean. To assess possible cumulative effects, we compared patients who had blood levels of all compounds below background with patients who had blood levels of all compounds above background. Patients with low or absent blood levels of the compounds studied had higher IFN-[gamma] plasma levels, providing some evidence for a cumulative effect of several weakly active compounds. In conclusion, exposure to PCBs, HCB, or HCHs is associated with weak immunologic abnormalities. These results contrast with those obtained in earlier studies of blood levels of PCP, which showed a strong dose-dependent relationship with immunologic impairments. Our data suggest that long-term exposure of patients to HCB suppresses IFN-[gamma] production.

This study addresses the hypothesis that clinical manifestations of chronic fatigue syndrome (CFS) are due in part to abnormal production of or sensitivity to cytokines such as <em>interleukin</em>-1beta (IL-1beta) and IL-6 under basal conditions or in response to a particular physical stress: 15 min of exercise consisting of stepping up and down on a platform adjusted to the height of the patella. The study involved 10 CFS patients and 11 age-, sex-, and activity-matched controls: of these, 6 patients and 4 controls were tested in both the follicular and the luteal phases of the menstrual cycle, and the remainder were tested in only one phase, for a total of <em>31</em> experimental sessions. Prior to exercise, plasma concentrations of the acute phase reactant alpha2-macroglobulin were 29% higher in CFS patients (P < 0.008) compared to controls. Secretion of IL-6 was generally higher for CFS patients (approximately 38%), however, this difference was statistically significant only if all values over a 3-day period were analyzed by repeated-measures ANOVA (P = 0.035). IL-6 secretion correlated with plasma alpha2-macroglobulin in control subjects at rest (R = 0.767, P = 0.001). Immediately after exercise, the CFS patients reported greater ratings of perceived exertion (P=0.027) compared to the healthy control subjects. Ratings of perceived exertion correlated with IL-1beta secretion by cells from healthy control subjects (R = 0.603, P = 0.022), but not from CFS patients, and IL-1beta secretion was not different between groups. Exercise induced a slight (< 12%) but significant (P = 0.006) increase in IL-6 secretion, but the responses of the CFS patients were not different than controls. Furthermore, no significant exercise-induced changes in body temperature or plasma alpha2-macroglobulin were observed. These data indicate that under basal conditions, CFS is associated with increased IL-6 secretion which is manifested by chronically elevated plasma alpha2-macroglobulin concentrations. These modest differences suggest that cytokine dysregulation is not a singular or dominant factor in the pathogenesis of CFS.

High-fat diet (HFD) induced obesity is associated with low-grade inflammation, insulin resistance (IR), and glucose intolerance. The objective of this study is to assess the effect of <em>interleukin</em> 10 (IL10), an anti-inflammatory cytokine, on blocking HFD-induced obesity and obesity-associated metabolic disorders by hydrodynamic delivery of IL10-containing plasmid. Animals fed a regular chow or HFD received two injections (one on day 1 and the other on day <em>31</em>) of plasmids containing green fluorescence protein (GFP) or mouse IL10 (mIL10) gene. Blood concentration of mIL10 reached ~200 ng/ml on day 7 in animals receiving mIL10 plasmid DNA. The transfection efficiency of liver cells was the same in animals fed a regular chow or HFD. No difference was seen in animals on regular chow when injected with plasmids containing either gfp or mIL10 gene. Overexpression of mIL10 prevented weight gain of animals on HFD. Intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance tests (ITT) showed that mIL10 maintained insulin sensitivity and prevented glucose intolerance. The mechanistic study reveals that mIL10 suppressed macrophage infiltration and reduced the development of crown-like structures in adipose tissue (AT). Collectively, these results suggest that maintaining a higher level of IL10 through gene transfer could be an effective strategy in preventing diet-induced obesity.

Naturally occurring apoptotic cells have been demonstrated in the postnatal cerebellum of rodents (Wood et al. [1993] Neuron 11:621-632; Krueger et al. [1995] J. Neurosci. 15:3366-3374). The nature of these cells differs among species: they are considered to be granule cells in mouse and astrocytes in rat. We labeled proliferating and apoptotic cells in the postnatal human cerebellar cortex by using antibodies against the Ki-67/proliferating cell nuclear antigen and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method for fragmented DNA. We also immunocytochemically detected some proteins encoded by genes modulating apoptosis and specific markers of neuronal/glial differentiation. Proliferating cells were observed from birth to 4 months, representing <em>31</em>-35% of cells within the external granular layer (EGL). Apoptotic cells were detected during the first 3 months and corresponded to 5-7% of EGL cells. Much lower percentages were calculated in other cortical layers and white matter. The balance between proliferation and apoptosis was quantitatively favorable to the latter during the first postnatal week. Expression of BCL-2, CPP32, and <em>interleukin</em>-1beta-converting enzyme (ICE) proteins was spatially and developmentally regulated in parallel with apoptosis. Apoptotic cells were often CPP32/ICE immunoreactive but negative for BCL-2. Some apoptotic cells were positive for vimentin and, less frequently, for alpha-internexin or type-III beta tubulin, but never expressed the glial fibrillary acidic protein. This study demonstrates that apoptosis is a significant phenomenon in early postnatal development of human cerebellar cortex and shares some of the regulatory mechanisms described in other vertebrates.

<em>Interleukin</em>-<em>31</em> (IL-<em>31</em>) is a newly discovered cytokine associated with chronic skin inflammation and pruritus. Patients with atopic dermatitis, chronic spontaneous urticaria, allergic contact dermatitis, prurigo nodularis, primary cutaneous lymphoma and mastocytosis exhibit increased serum levels of IL-<em>31</em> protein and elevated IL-<em>31</em> mRNA in the skin. Interestingly, in some of these diseases, IL-<em>31</em> serum levels correlate with disease activity. In the present review, we particularly focus on studies investigating IL-<em>31</em> as a novel diagnostic biomarker indicating the severity of allergic diseases. We highlight a recent study on IL-<em>31</em> in mastocytosis, which reports on elevated serum levels of IL-<em>31</em> in adults correlating with the severity of disease categories, tryptase levels and percentage of bone marrow infiltration. We conclude that growing knowledge about IL-<em>31</em>, its receptors and signaling pathways serves to better understand the pathogenesis of allergic diseases and may lead to the development of novel treatment approaches.

Genetic factors play an important role in determining bone mass and several genes are involved in this process. <em>Interleukin</em>-6 (IL-6) is a candidate gene for regulation of bone mineral density (BMD) and it has been suggested recently that novel IL-6 -174 G/C allelic variants may be associated with peak BMD in young men and with bone resorption in elderly women. In this study, we assessed the relationships between IL-6 gene polymorphism, peak BMD, rate of postmenopausal BMD loss, and bone turnover in women. BMD was measured by dual-energy X-ray absorptiometry in 255 healthy premenopausal women, aged <em>31</em>-57 years. BMD loss at the forearm was measured over 4 years in 298 healthy untreated postmenopausal women, 50-88 years (mean 64 years). We also measured levels of serum osteocalcin, bone alkaline phosphatase, and N-propeptide of type I collagen for bone formation and three markers of bone resorption, including urinary and serum C-terminal cross-linking telopeptide of type I collagen and urinary N-terminal telopeptide of type I collagen, in both pre- and postmenopausal women at baseline. In premenopausal women we found a significant association between IL-6 genotypes and BMD at the whole body (analysis of variance [ANOVA], p = 0.03), femoral neck (p = 0.03), trochanter (p = 0.014), Ward's triangle (p = 0.03), and total hip (p = 0.006), with subjects having the CC genotype showing 3%-7% higher BMD levels than their GG counterparts. However, after matching women with CC and GG genotypes for body height the differences decreased (2%-4%), and were no longer significant (p = 0.10-0.23). In postmenopausal women the mean rate of loss at the ultradistal radius was significantly associated with IL-6 genotypes (ANOVA, p = 0.049), with women having the CC genotype showing a significantly greater rate of bone loss (p < 0.05) compared with their GC and GG counterparts. After adjustment for weight changes, the difference in the rate of ultradistal radius bone loss between genotypes decreased and was not significant (p = 0.06 for CC vs. GG). A similar trend was observed for distal radius bone loss (p = 0.10, ANOVA), but not for the middle radius. We found no significant association between genotypes, bone turnover markers in premenopausal women, and either bone turnover or BMD in postmenopausal women. We conclude that this new functional IL-6 polymorphism was weakly associated with level of peak BMD and the rate of forearm trabecular postmenopausal bone loss in this cohort of healthy French women. IL-6 genotypes accounted only for a small proportion of the interindividual variation of both peak BMD and rate of bone loss and were not significant after adjustment for height and changes in body weight, respectively, suggesting that part of the effect may have been due to the differences in body size. Larger long-term studies are necessary to assess adequately the relationships between IL-6 genotype, rate of bone loss, and risk of fracture.

OBJECTIVE

To test the following hypotheses: 1) osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL) serum levels in patients with Paget's disease are related to disease activity and are different from those in healthy individuals; 2) interleukin-6 (IL-6), a cytokine that has been shown to have higher levels in Paget's disease, modulates these factors; and 3) the antiresorptive effect of bisphosphonates in Paget's disease of bone may be mediated through these local factors.

METHODS

The study group comprised 31 patients with Paget's disease who received 400 mg/day of oral tiludronate for 3 months. Serum levels of OPG, RANKL, IL-6, bone alkaline phosphatase (AP), N-terminal type I procollagen propeptide, urinary N-terminal crosslinking telopeptide of type I collagen, and urinary alpha-C-terminal crosslinking telopeptide of type I collagen were measured at baseline and 1 month after the end of therapy. In addition, the RANKL:OPG ratio was calculated, and disease activity was evaluated at baseline by quantitative bone scintigraphy.

RESULTS

Mean baseline OPG values were higher in patients with Paget's disease than in healthy control subjects (P < 0.005), but RANKL and IL-6 values and RANKL:OPG ratios in the 2 groups were similar. OPG concentrations decreased significantly after treatment with tiludronate (P < 0.005), whereas no significant changes were observed in serum RANKL values. No correlation was found between either bone markers or quantitative scintigraphic indices and serum levels of OPG, RANKL, IL-6, and RANKL:OPG ratios. Serum OPG decreased significantly only in those patients with baseline OPG values >4.1 pM/liter.

CONCLUSIONS

Serum OPG increases in Paget's disease and decreases after treatment with tiludronate, especially in patients with the highest OPG values. In contrast, RANKL serum levels and RANKL:OPG ratios are unmodified in patients with Paget's disease. Although serum OPG, RANKL, and IL-6 values were unrelated to disease activity, the increase in OPG may reflect a protective mechanism of the skeleton to compensate for increased bone resorption.

We have reported that proteasomes are expressed at abnormally high levels in various hematopoietic tumor cells (Kumatori, A., Tanaka, K., Inamura, N., Sone, S., Ogura, T., Matsumoto, T., Tachikawa, T., Shin, S., and Ichihara, A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7071-7075). In the present study, we examined changes in the expressions of proteasomes during growth of peripheral T-lymphocytes from healthy adults and differentiation of human leukemic cell lines. Up-regulation of mRNAs encoding multiple proteasome subunits was observed during proliferation of resting T-cells induced by mitogens such as phytohemagglutinin and <em>interleukin</em>-2. In contrast, in vitro terminal differentiation into monocytic, granulocytic, and erythroid cells of various immature leukemic cell lines, such as HL-60 promyelocytic leukemia cells and K562 erythroleukemia cells, by various inducing agents caused rapid and marked down-regulation of proteasomes expression, independently of the cell type, direction of differentiation, or type of signal. The syntheses of proteasome subunits of 21-<em>31</em> kDa and their associated components of 35-110 kDa, measured by [35S]methionine incorporation, were much higher in mitogen-activated T-cells and unstimulated HL-60 cells, which grow rapidly, than in resting and differentiated cells, indicating apparent correlations of the mRNA levels of proteasomes with their translational activities. However, immunochemically, no detectable difference in the cellular contents of proteasomes was found in these cells in induced and uninduced states for proliferation and differentiation, suggesting accelerated turnover of proteasomes in rapidly proliferating cells. Inhibition of proteasome expression by an antisense oligodeoxynucleotide for the largest proteasome subunit, C2, caused partial arrest of cell cycle progression of T-lymphocytes, suggesting that up-regulation of proteasomes is indispensable for proliferation of the cells. We also observed that the nuclear fraction of proteasomes increased in proliferating T-cells and that proteasomes moved rapidly between the nucleus and cytoplasm during differentiation of HL-60 cells.

The clade B human immunodeficiency virus, type 1 (HIV-1) Tat (trans-acting regulatory protein) induces <em>interleukin</em>-10 (IL-10) production in monocytes. IL-10, an anti-inflammatory cytokine, down-regulates proinflammatory cytokines and suppresses the immune response, leading to a rapid progression from HIV-1 infection to AIDS. Nine clades of HIV-1 are responsible for the majority of infections worldwide. Recent studies demonstrate that different HIV-1 clades have biological differences in relation to transmission, replication, and disease progression. In this study, we show that the cysteine to serine mutation at position <em>31</em>, found in >90% of HIV-1 clade C Tat proteins, results in a marked decrease in IL-10 production in monocytes compared with clade B Tat. Additionally, the C<em>31</em>S mutation found in C Tat is responsible for the inability of these Tat proteins to produce high IL-10 levels in monocytes due to its inability to induce intracellular calcium flux through L-type calcium channels. Moreover, we show that p38alpha/p38beta and phosphoinositide 3-kinase are crucial to Tat-induced IL-10 production. These findings provide further evidence that HIV-1 clades differ in their biological properties that may impact HIV-1 pathogenesis and disease progression.

In this study, we investigated the effects of proteasome inhibibors (MG132 and lactacystin) on <em>interleukin</em> (IL)-8 induction. In human epithelial A549 cells, MG132 and lactacystin induced IL-8 release within the range of 0.1-30 microM. The effect of MG132 resulted from IL-8 gene transcription and was blocked by PD 98059, but was unaffected by GF109203X, Ro <em>31</em>-8220, or SB 203580. Mutational analysis of the 5' flanking region of the IL-8 gene revealed that activator protein (AP)-1-binding element, but not that element responsive to nuclear factor (NF)-IL-6 or NF-kappaB, was necessary for MG132 stimulation. Consistent with this, MG132 and lactacystin increased the DNA-binding and reporter activities of AP-1, but reduced cytokine-elicited kappaB activation. Moreover, AP-1 stimulation was associated with increased extracellular signal-related kinase (ERK), mitogen-activated protein/ERK kinase (MEK), and c-Jun N-terminal kinase (JNK) phosphorylation, whereas IL-8 activity was sensitive to the dominant-negative mutants of JNK1, JNK2, SEK, ASK, ERK2, and Ras, but not those of MEKK1, TAK, and p38 mitogen-activated protein kinase. In addition, activations of the IL-8 gene and AP-1 by MG132 and lactacystin were inhibited by GSH and NAC. Herein we present a novel action of proteasome inhibitors, possibly through ROS production, of targeting the upstream signaling molecules, ERK and JNK, which leads to AP-1 activation and IL-8 gene expression.

OBJECTIVE

To investigate the effect of short term energy restriction combined with physical activity on serum concentrations of Interleukin-6 (IL-6) in obese children and adolescents.

METHODS

Longitudinal intervention study of 3.8-5 MJ daily with exercise.

METHODS

Forty-nine white obese children and adolescents (31 girls, age 11.9+/-1.8 y; 18 boys, age 11.6+/-1.7 y).

METHODS

Indexes of obesity, IL-6, leptin, estradiol, systolic and diastolic blood pressure, heart rate at baseline and after 3 weeks.

RESULTS

All determined parameters decreased significantly during the 3 week program (IL-6: 3.9+/-4.7 vs 2.0+/-2.2 pg/ml; P<0.05). Body mass index (BMI) fat mass, percentage fat mass (indexes of obesity), and leptin were not related to IL-6 before the program. In contrast, IL-6 concentrations correlated significantly with indexes of obesity and leptin after weight loss. IL-6 concentrations did not correlate with estradiol, systolic and diastolic blood pressure, and heart rate. Changes in IL-6 concentrations correlated significantly with changes in BMI (r=0.25, P<0.05).

CONCLUSIONS

An improved body composition induced by restriction of energy intake and increase in physical activity is associated with more favorable serum concentrations of IL-6 in obese children and adolescents.

The function of two alpha-helical regions of mouse <em>interleukin</em>-2 were analyzed by saturation substitution analysis. The functional parts of the first alpha-helix (A) was defined as residues <em>31</em>-39 by the observation that proline substitutions within this region inactivate the protein. Four residues within alpha-helix A, Leu<em>31</em>, Asp34, Leu35 and Leu38, were found to be crucial for biological activity. Structural modeling suggested that these four residues are clustered on one face of alpha-helix A. Residues <em>31</em> and 35 had to remain hydrophobic for the molecule to be functional. At residue 38 there was a preference for hydrophobic side chain residues, while at residue 34 some small side chain residues as well as acidic or amide side chain residues were functionally acceptable. Inactivating changes at residue 34 had no effect upon the ability of the protein to interact with the p55 receptor. Disruption of the fifth alpha-helix (E), which had little effect upon biological activity, resulted in an inability of the protein to interact with the p55 receptor. Mutagenesis of the alpha-helix E region demonstrated that alpha-helicity and the nature of the side chain residues in this region were unimportant for biological activity. The region immediately proximal to alpha-helix E was important only for the single intramolecular disulfide linkage.

Gastric carcinogenesis is a complex, multistep process, which may be influenced by many factors and is the second most common type of malignancy and the second most-common cause of mortality in the word. <em>Interleukin</em>-1 is up-regulated in the presence of Helicobacter pylori and is important for initiating and amplifying the inflamatory response to this infection. Recently <em>interleukin</em>-1 polymorphisms have been associated with the development of gastric adenocarcinoma. In this study we investigated the presence of H. pylori and host genotypes that are highly associated with gastric alterations. DNA samples were extracted and PCR-RFLP was utilized for genotyping IL-1B (-511) polymorphisms, PCR-VNTR was utilized for genotyping IL-1RN, and PCR-CTPP was utilized for genotyping IL-1B (-<em>31</em>), the presence of H. pylori was detected by the urease test. Our results indicate a correlation between H. pylori infection and the development of gastric cancer. We did not find an association between the presence of genotype T (thymine) in bases -511 and -<em>31</em> and gastric adenocarcinoma. We also did not find any association between this polymorphism and specific type of tumor (diffuse type and intestinal type).

OBJECTIVE

To evaluate whether T cell activation, as reflected by levels of soluble interleukin 2 receptor (sIL2R), soluble CD30 (sCD30), IL-10 and B cell activator of the tumour necrosis factor family (BAFF) at diagnosis and during initial follow-up, is predictive for persistent or renewed antineutrophil cytoplasmic antibody (ANCA) positivity and clinical relapse in patients with vasculitis associated with proteinase 3-antineutrophil cytoplasmic antibodies (PR3-ANCA).

METHODS

87 Patients with PR3-ANCA-associated vasculitis and at least 2 years of follow-up were included in the study. At diagnosis, and at 3, 6, 12, 18 and 24 months after diagnosis, cytoplasmic ANCA titres were detected by indirect immunofluorescence (IIF), and PR3-ANCA, sIL2R, sCD30, IL-10 and BAFF levels were assessed by ELISA. 31 healthy volunteers provided plasma samples for comparison. Levels of immune markers were related to ANCA positivity and relapse during follow-up.

RESULTS

Plasma levels of sIL2R, sCD30 and BAFF were higher in patients than in controls at all time points. Plasma levels of sIL2R, sCD30 and IL-10 were higher at diagnosis and relapse than during remission. At 18 months, sCD30 (p<0.001) and sIL2R levels (p = 0.01) were significantly higher in PR3-ANCA-positive patients (detected by ELISA) than in PR3-ANCA-negative patients. ANCA-positive patients detected by ELISA or IIF at 24 months had significantly higher plasma sCD30 levels (p = 0.02 and p = 0.03, respectively) than ANCA-negative patients.

CONCLUSIONS

Increased T cell activation in patients with ANCA-associated vasculitis in remission during and after immunosuppressive treatment is associated with persistent or renewed ANCA positivity.

Incubation of eosinophils (EOS) with alveolar macrophage (AM) supernatants isolated from asthmatic subjects followed by stimulation with the calcium ionophore A2<em>31</em>87 resulted in enhancement of the capacity of EOS to elaborate leukotriene C4 (LTC4) (mean enhancement 169 +/- 37%, n = <em>31</em>). Pretreatment of EOS with AM supernatants derived from normal individuals did not enhance LTC4 generation as compared with control medium. Enhancement was maximal when EOS were preincubated with a 1:6 dilution of AM supernatants for 5 min at 37 degrees C and were then stimulated with 5 microM A2<em>31</em>87 for 15 min. Separation of AM supernatants by size-exclusion HPLC using a TSK G3000 SW column resulted in a peak of enhancing activity with an estimated molecular mass of approximately 30,000 D. Further purification by anion exchange HPLC using a TSK DEAE 5PW column (pH 7.4) resolved the activity into a minor peak at 0.17 M NaCl and a major peak at 0.2 M NaCl. The activities were distinct from <em>interleukin</em>-1 and tumor necrosis factor. Resolution of the major peak of activity by reverse-phase HPLC using a C18 spherisorb ODS column and a slope gradient of 0 to 100% acetonitrile/0.1% trifluoroacetic acid demonstrated a single peak of activity that eluted at 41% acetonitrile. The enhancing activity was sensitive to trypsin and heat and was neutralized by a specific antibody to granulocyte-macrophage colony-stimulating factor (GM-CSF). Pretreatment of EOS with recombinant GM-CSF primed the cells for enhanced LTC4 generation following subsequent stimulation with A2<em>31</em>87. GM-CSF may play a role in the amplification of the eosinophilic inflammation in asthmatic airways.

OBJECTIVE

Cytokine dysregulation has been implicated in AIDS pathogenesis and the gastrointestinal tract, containing approximately 40% of the body's lymphoid tissue, is likely to act both as a reservoir of viral infection and a site for immune dysregulation. In this study evidence of cytokine dysregulation in intestinal mucosa has been sought using the reverse transcriptase polymerase chain reaction (RT-PCR) to amplify cytokine mRNA.

METHODS

RT-PCR was performed on intestinal biopsies obtained from 50 HIV-infected patients and <em>31</em> controls. Tissue was obtained at diagnostic endoscopy and total RNA extracted using an RNAzol technique. Following RT, cDNA was amplified using primers specific for beta-actin, <em>interleukin</em> (IL)-1 beta, tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma, IL-2, IL-4, IL-10 and IL-13.

RESULTS

There was a significant increase in the expression of the proinflammatory cytokines IL-1 beta and IFN-gamma in the HIV-infected compared with the control small intestinal samples (P < 0.01). IL-10 was significantly reduced in the respective groups' large intestine (P < 0.02). The expression of IL-2 was also reduced in both the small and large intestinal HIV samples although this was not significant. IL-13 mRNA was only detected in one control patient.

CONCLUSIONS

Dysregulation of cytokine gene expression occurs in the intestinal mucosa of patients with HIV infection and is characterized by increased expression of proinflammatory cytokine mRNA. Further studies are needed to localize the cellular origin of such dysregulation and to quantify the degree of abnormality.