Serum concentrations of sIL-2R, IL-6, TGF-beta1, neopterin, and zinc in chronic hepatitis C patients treated with interferon-alpha

T lymphocytes and immunoregulatory cytokines play an important role in the host response to hepatitis C virus (HCV) infection. Zinc is required for a wide spectrum of immune functions, including T-cell activity. To determine the clinical significance of the cytokines sIL-2R, IL-6, TGF-beta1, neopterin, and of zinc in chronic heptatitis C virus (HCV) infection, we investigated their concentrations in the serum of 16 patients with chronic HCV infection before, during and at the end of therapy with interferon (IFN) alpha (Roferon A), and after 6 months follow-up. Elevated concentrations of sIL-2R, IL-6, TGF-beta1, and neopterin were found in the serum of all patients prior to therapy, as compared to healthy controls. sIL-2R patterns differed in responders and non-responders. While the mean concentration of sIL-2R (335.75 pg/ml) before therapy was about 40% higher in complete responders (n=4) than in controls (272.20 pg/ml), the mean concentration in non-responders (n=6) was 4-fold higher than in controls (1153.33 pg/ml). During therapy, sIL-2R levels in responders decreased by about 40%. Mean IL-6 concentrations in both complete and partial responders (n=6) decreased continuously during treatment, while mean concentrations in non-responders decreased for only a short time, and increased again after cessation of therapy. Mean levels of TGF-beta1 behaved similarly to those of IL-6. Only negligible differences in mean neopterin levels were found between responders and non-responders over the entire observation time. The mean serum zinc concentrations slightly decreased in all 3 patient groups, the greatest reduction occurring in 3 of the 4 responders. The present findings underscore the importance of the immune system in the pathogenesis of chronic HCV infection. Serum sIL-2R levels may be used as a serological marker of outcome following IFN-alpha treatment.     

SERUM CONCENTRATIONS OF sIL-2R, IL-6, TGF-β1, NEOPTERIN, AND ZINC IN CHRONIC HEPATITIS C PATIENTS TREATED WITH INTERFERON-ALPHA

T lymphocytes and immunoregulatory cytokines play an important role in the host response to hepatitis C virus (HCV) infection. Zinc is required for a wide spectrum of immune functions, including T-cell activity. To determine the clinical significance of the cytokines sIL-2R, IL-6, TGF-β1, neopterin, and of zinc in chronic heptatitis C virus (HCV) infection, we investigated their concentrations in the serum of 16 patients with chronic HCV infection before, during and at the end of therapy with interferon (IFN) α (Roferon A), and after 6 months follow-up. Elevated concentrations of sIL-2R, IL-6, TGF-β1, and neopterin were found in the serum of all patients prior to therapy, as compared to healthy controls. sIL-2R patterns differed in responders and non-responders. While the mean concentration of sIL-2R (335.75 pg/ml) before therapy was about 40% higher in complete responders (n=4) than in controls (272.20 pg/ml), the mean concentration in non-responders (n=6) was 4-fold higher than in controls (1153.33 pg/ml). During therapy, sIL-2R levels in responders decreased by about 40%. Mean IL-6 concentrations in both complete and partial responders (n=6) decreased continuously during treatment, while mean concentrations in non-responders decreased for only a short time, and increased again after cessation of therapy. Mean levels of TGF-β1 behaved similarly to those of IL-6. Only negligible differences in mean neopterin levels were found between responders and non-responders over the entire observation time. The mean serum zinc concentrations slightly decreased in all 3 patient groups, the greatest reduction occurring in 3 of the 4 responders. The present findings underscore the importance of the immune system in the pathogenesis of chronic HCV infection. Serum sIL-2R levels may be used as a serological marker of outcome following IFN-α treatment.     

Inhibition of classic signaling is a novel function of soluble glycoprotein 130 (sgp130), which is controlled by the ratio of interleukin 6 and soluble interleukin 6 receptor

IL-6 trans-signaling via the soluble IL-6 receptor (sIL-6R) plays a critical role in chronic inflammation and cancer. Soluble gp130 (sgp130) specifically inhibits IL-6 trans-signaling but was described to not interfere with classic signaling via the membrane-bound IL-6R. Physiological and most pathophysiological conditions are characterized by a molar excess of serum sIL-6R over IL-6 characterized by free IL-6 and IL-6 found in IL-6·sIL-6R complexes allowing both classic and trans-signaling. Surprisingly, under these conditions, sgp130 was able to trap all free IL-6 molecules in IL-6·sIL-6R·sgp130 complexes, resulting in inhibition of classic signaling. Because a significant fraction of IL-6 molecules did not form complexes with sIL-6R, our results demonstrate that compared with the anti-IL-6R antibody tocilizumab or the anti-trans-signaling monoclonal antibody 25F10, much lower concentrations of the dimeric sgp130Fc were sufficient to block trans-signaling. In vivo, sgp130Fc blocked IL-6 signaling in the colon but not in liver and lung, indicating that the colon is a prominent target of IL-6 trans-signaling. Our results point to a so far unanticipated role of sgp130 in the blockade of classic signaling and indicate that in vivo only low therapeutic concentrations of sgp130Fc guarantee blockade of IL-6 trans-signaling without affecting IL-6 classic signaling.     

Serum levels of interleukin-6 type cytokines and soluble interleukin-6 receptor in patients with rheumatoid arthritis.

We investigated the serum concentrations of interleukin-6 (IL-6) and two IL-6 family of cytokines (leukaemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) as well as IL-6 soluble receptor (sIL-6R) using an enzyme-linked immunosorbent assay (ELISA) in 66 patients with rheumatoid arthritis (RA) and 24 healthy controls. We examined a possible association between the serum levels of these peptides and RA activity according to the Mallya and Mace scoring system and Ritchie''s index. We also evaluated the correlation between the serum levels of IL-6, LIF, CNTF and sIL-6R and duration of the disease and calculated sIL-6R/IL-6 ratio in RA patients and in the control group. IL-6 and sIL-6R were detectable in all 66 patients with RA and 24 normal individuals. LIF was also found in the serum of all patients with RA and in 16 (66.7%) normal individuals. In contrast CNTF was measurable only in 15 (22.7%) patients with RA and 24 (33.3%) normal individuals. The highest IL-6 and sIL-6R levels were found in the patients with Stages 3 and 4 of RA activity and the lowest in the control group. In contrast there were no statistically significant differences between the LIF and CNTF levels in RA patients and normal individuals. We found positive correlation between IL-6 and sIL-6R concentrations and Ritchie''s index and a lack of such correlation with LIF and CNTF. IL-6 serum level correlated positively with the disease duration, but sIL-6R, LIF and CNTF did not. Serum sIL-6R/IL-6 ratio was significantly lower in RA patients than in healthy controls. In conclusion, an increase in the serum levels of IL-6 and sIL-6R, but not LIF and CNTF concentrations, may be useful markers for RA activity.     

Variability of serum levels of tumor necrosis factor-alpha, interleukin 6, and soluble interleukin 6 receptor over 2 years in young women

Serum levels of tumor necrosis factor-alpha (TNF-alpha), interleukin 6 (IL-6), and soluble interleukin 6 receptor (sIL-6R) have been studied as risk factors of cardiovascular disease in longitudinal studies. However, it is unknown about their long-term intra-individual variations and whether single measurements of these cytokines and receptor are reliable biomarkers in epidemiological studies. In this study, serum levels of TNF-alpha, IL-6, and sIL-6R were assayed by ELISAs in 36 young, healthy women from whom three blood samples were collected at 12-month intervals over 2 years, and the intraclass correlation coefficients (ICC) were estimated. The ICC of 0.73 (95% CI=0.49-0.79) for TNF-alpha was comparable to those of other commonly used biomarkers, justifying its use in epidemiological studies. The ICC of 0.48 (95% CI=0.25-0.58) for IL-6 was not optimal. However, IL-6 has been demonstrated as a consistent risk factor for cardiovascular disease, suggesting it could still be a useful biomarker if its disease association is substantial. The ICC of 0.36 (95% CI=0.10-0.47) for sIL-6R was relatively low, and multiple samples would need to be collected in prospective studies for this receptor.     

Relationship between circulating interleukin-10 (IL-10) with interleukin-6 (IL-6) type cytokines (IL-6, interleukin-11 (IL-11), oncostatin M (OSM)) and soluble interleukin-6 (IL-6) receptor (sIL-6R) in patients with multiple myeloma

We investigated the serum concentration of the interleukin-10 (IL-10), along with cytokines of interleukin-6 (IL-6) family (IL-6, IL-11 and oncostatin M - OSM), as well as soluble receptor for IL-6 (sIL-6R), in 121 patients with multiple myeloma (MM) and 28 healthy subjects. We studied the interactions between IL-10 and other cytokines, and the receptor. The correlation between IL-10 and some clinical and laboratory parameters associated with the disease activity were also analysed. The IL-10 was detectable in all patients with multiple myeloma and in all controls. The IL-10 concentration was significantly increased in myeloma patients compared with healthy persons (mean - 7.09 and 2.1 pg/ml, respectively) (p = 0.008). The level of IL-10 correlated positively with the advanced stage of disease estimated according to the Salmon and Durie classification (I versus III stage - p = 0.03). Higher values of IL-10 were found in patients with the light chain disease, hypercalcaemia, and correlated with the elevated concentrations of C-reactive protein (CRP). IL-6 was detected in 117 of the 121 patients and in all controls. The concentration of IL-6 was statistically increased in MM patients compared with control group (mean - 16.06 and 4.49 pg/ml, respectively) (p = 0.01). We found a positive correlation between IL-10 and IL-6 serum levels in MM patients. The relationship, expressed as Spearman's rank sum coefficient (rho = 0.249, p = 0.006) was significant. IL-11 was detected in 26 of the 121 MM patients and in 3 of the 28 healthy subjects at the mean concentration of 1.2 and 0.6 pg/ml respectively (p > 0.05). OSM was at detectable levels in 51 of the 121 patients and in only 4 of the 28 controls (mean - 3.84 and 0.1 pg/ml, p = 0. 002). The correlation between IL-10 and IL-11 levels in MM patients was not significant, but there was a strong statistical correlation between IL-10 and OSM concentrations (rho= 0.327, p = 0.0002). The serum concentration of sIL-6R was measurable in all patients and all controls (mean - 66.00 and 39.57 ng/ml respectively), but the difference between these groups was not significant. We found significant, positive correlation between the levels of IL-10 and sIL-6R (rho= 0.233, p = 0.01). In conclusion, we state that the serum concentrations of IL-10, IL-6, OSM and sIL-6R in MM patients may be a useful markers for the evaluation of the disease activity.     

Tumour necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6) and their soluble receptors (sTNF-alpha-Rp55 and slL-6R) serum levels in systemic lupus erythematodes

We investigated a possible association between serum concentrations of tumour necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6) and their soluble receptors (sTNF-alpha-Rp55 and sIL-6R) using an enzyme-linked immunosorbent assay (ELISA) in 55 patients with systemic lupus erythematodes (SLE) and 16 healthy controls. We also examined a possible association between the serum levels of these peptides and SLE activity, as well as TNF-alpha and IL-6 concentrations and the levels of their soluble receptors. The median concentrations of TNF-alpha, sTNF-alpha-Rp55 and IL-6 were significantly higher in SLE patients than in normal individuals. In contrast, there was no difference between the serum level of sIL-6R in both groups. We found positive correlations between the serum concentrations of TNF-alpha and IL-6 as well as their soluble receptors and disease activity. There were also correlations between TNF-alpha and sTNF-alpha-Rp55 as well as IL-6 and sIL-6R serum levels in SLE patients but there were no such correlations in the normal control group. In conclusion, an increase in the serum levels of TNF-alpha, sTNF-alpha-Rp55 and IL-6 may become useful markers for SLE activity. Patients with SLE have sIL-6R serum concentration similar to that as in normal individuals. However, it correlates with disease activity and the level of IL-6.     

Serum IL-1beta,sIL-2R,IL-6, IL-8 and TNF-alpha in schizophrenic patients,relation with symptomatology and responsiveness to risperidone treatment.

Activation of the inflammatory response system and varied levels of cytokines in acute schizophrenia have been suggested by recent studies. Psychopharmacologic agents can differentially effect cytokine production, which suggests that therapeutic function of neuroleptics may involve immunomodulation. The present study was carried out to examine: (i) serum concentrations of interleukin (IL)-1beta, soluble interleukin-2 receptor (sIL-2R), IL-6, IL-8 and tumour necrosis factor (TNF)-alpha in schizophrenic patients; (ii) their relation with psychopathological assessment; and (iii) the relation of the initial cytokine levels with responsiveness to risperidone therapy. Thirty-four drug-free schizophrenic patients with acute exacerbation and 23 age- and gender-matched healthy controls were recruited for this study. Psychopathological assessments at admission and throughout risperidone treatment for 60 days were recorded. Serum cytokine concentrations were determined with chemilumunescence assays. According to our results, serum IL-1beta, sIL-2R, IL-6, IL-8 and TNF-alpha concentrations adjusted for age, gender, body mass index and smoking were no different in patients with schizophrenia and controls and among subtypes of schizophrenia. However, the initial TNF-alpha concentrations had a significant effect on Brief Psychiatric Rating Scale and Scale Assessment of Positive Symptoms scores. The initial cytokine concentrations of the patients responsive to risperidone were not significantly different from those of non-responsive patients. The present study demonstrates that plasma levels of IL-1beta, sIL-2R, IL-6, IL-8 and TNF-alpha adjusted for confounding factors are not altered in drug-free schizophrenic patients at acute exacerbation. We suggest that, if cytokine production is altered in schizophrenia, these alterations may not be detectable in systemic circulation. According to our results, the therapeutic effect of risperidone is not related to basal levels of the aforementioned cytokines. However, serum TNF-alpha may contribute to symptomatology in schizophrenia     

Subcutaneous administration of recombinant glycosylated interleukin 6 in patients with cancer: pharmacokinetics, pharmacodynamics and immunomodulatory effects

This is the first report of the serum profile of a glycosylated recombinant form of human IL-6 (rhIL-6) administered subcutaneously (1-10 microg/kg/day) in a phase I/II trial as a thrombopoietic agent in patients with advanced cancer. The pharmacodynamic effects of IL-6 were also examined. Detailed pharmacokinetic measurements were made in four patients. Peak concentrations at 5-8 h and a median t0.5 of ca. 5 h were similar to those previously reported for non-glycosylated IL-6. However, higher peak concentrations and apparent differences in effective dose levels to those previously reported with the non-glycosylated form were seen. Indications of an apparent attenuation in circulating IL-6 concentrations with continuing injections were seen in eight of 10 patients examined but anti-IL-6 antibody generation was seen in only two patients. Soluble interleukin 6 receptor concentrations generally decreased. No major changes in T cell subsets were seen but expression of CD25 and CD54 by T lymphocytes significantly increased, accompanied by marked increases in soluble CD25 (sIL-2R) and CD54 (sICAM-1). No consistent change in B cells, monocytes or NK cells were seen. No evidence for induction of TNF-alpha was found. This study demonstrates similar biological effects of glycosylated rhIL-6 to those reported for the non-glycosylated form but illustrates several apparent differences which are discussed further.     

Effect of soluble interleukin-6 receptor alpha and interleukin-6 secreted by polymorphonuclear leukocytes on tumor necrosis factor-alpha expression and its production by peripheral blood mononuclear cells

BACKGROUND: It has recently been shown that soluble interleukin-6 receptor (sIL-6R) alone or complexed with interleukin (IL)-6, besides their regulatory role in a wide variety of both normal and abnormal biologic reactions mediated by IL-6, could be an effective stimulator of the cell function. AIMS: The key question of the present study is whether the sIL-6Ralpha or sIL-6R with IL-6 released by polymorphonuclear leukocytes (PMN) can influence cytokine secretion such as tumor necrosis factor-alpha (TNF-alpha) by peripheral blood mononuclear cells (PBMC), which together with PMN develop the inflammatory and immune response of a host. METHODS: Cells were isolated from heparinized whole blood of healthy persons. The PMN were cultured for 1 h at 37 degrees C in 5% CO(2). After incubation, the culture supernatant of PMN was removed and was added to PBMC. The PBMC were cultured for 1 h at 37 degrees C in the same conditions. In the culture supernatants and lysates of PMN, we examined the concentrations of sIL-6R by enzyme-linked immunosorbent assay (ELISA). TNF-alpha was measured at both protein and mRNA levels. Protein levels were determined by ELISA. To examine TNF-alpha mRNA expression, we isolated mRNA from PBMC after culture, using TRIZOL Reagent. The quantity of mRNA TNF-alpha was determined by the Quantikine mRNA assay. RESULTS AND CONCLUSION: The results obtained revealed that sIL-6R with IL-6 secreted by PMN may play a regulatory role in the immune response by modulating the TNF-alpha expression and its production by PBMC. This may have a significant influence on an early phase of the inflammation and other reactions mediated by TNF-alpha.     

Shedding of Endogenous Interleukin-6 Receptor (IL-6R) Is Governed by A Disintegrin and Metalloproteinase (ADAM) Proteases while a Full-length IL-6R Isoform Localizes to Circulating Microvesicles

Generation of the soluble interleukin-6 receptor (sIL-6R) is a prerequisite for pathogenic IL-6 trans-signaling, which constitutes a distinct signaling pathway of the pleiotropic cytokine interleukin-6 (IL-6). Although in vitro experiments using ectopically overexpressed IL-6R and candidate proteases revealed major roles for the metalloproteinases ADAM10 and ADAM17 in IL-6R shedding, the identity of the protease(s) cleaving IL-6R in more physiological settings, or even in vivo, remains unknown. By taking advantage of specific pharmacological inhibitors and primary cells from ADAM-deficient mice we established that endogenous IL-6R of both human and murine origin is shed by ADAM17 in an induced manner, whereas constitutive release of endogenous IL-6R is largely mediated by ADAM10. Although circulating IL-6R levels are altered in various diseases, the origin of blood-borne IL-6R is still poorly understood. It has been shown previously that ADAM17 hypomorphic mice exhibit unaltered levels of serum sIL-6R. Here, by quantification of serum sIL-6R in protease-deficient mice as well as human patients we also excluded ADAM10, ADAM8, neutrophil elastase, cathepsin G, and proteinase 3 from contributing to circulating sIL-6R. Furthermore, we ruled out alternative splicing of the IL-6R mRNA as a potential source of circulating sIL-6R in the mouse. Instead, we found full-length IL-6R on circulating microvesicles, establishing microvesicle release as a novel mechanism for sIL-6R generation.     

Cerebrospinal fluid and serum protein levels of tumour necrosis factor-alpha (TNF-alpha) interleukin-6 (IL-6) and soluble interleukin-6 receptor (sIL-6R gp80) in multiple sclerosis patients

The aim of this study was to evaluate soluble proteins of tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and IL-6 receptor subunit gp80 (sIL-6R gp80), as markers of multiple sclerosis (MS). Paired cerebrospinal fluid (CSF) and serum samples of 20 MS patients and 15 controls suffering from non-inflammatory neurological diseases have been assayed retrospectively using monoclonal antibodies-based ELISAs. While TNF-alpha could not be detected in CSF, it was measurable in 20% of total sera. Interleukin-6 was measurable in 5% of total CSF and in 10% of total sera only. However, soluble IL-6R gp80 protein subunit was readily measurable, showing sera concentration (pg/mL) about 34 times higher and specific content (pg/mg total protein) around five times lower than those in paired CSF, similarly for both group of patients. No significant difference of sIL-6R gp80 level, which could be disease-, gender- or age-related, and no correlation of CSF sIL-6R gp80 content with that of paired serum or with routine clinical data for CSF, have been observed. We have concluded that soluble proteins of TNF-alpha, IL-6 and sIL-6R gp80 assayed by monoclonal antibodies-based ELISAs could not serve as markers of the MS activity.     

The interleukin-6 receptor Asp358Ala single nucleotide polymorphism rs2228145 confers increased proteolytic conversion rates by ADAM proteases

The pleiotropic activities of Interleukin (IL-)6 are controlled by membrane-bound and soluble forms of the IL-6 receptor (IL-6R) in processes called classic and trans-signaling, respectively. The coding single nucleotide polymorphism (SNP) rs2228145 of the Interleukin 6 receptor (IL-6R Asp358Ala variant) is associated with a 2-fold increase in soluble IL-6R (sIL-6R) serum levels resulting in reduced IL-6-induced C-reactive protein (CRP) production and a reduced risk for coronary heart disease. It was suggested that the increased sIL-6R level leads to decreased IL-6 classic or increased IL-6 trans-signaling. Irrespective of the functional outcome of increased sIL-6R serum level, it is still under debate, whether the increased sIL-6R serum levels emerged from differential splicing or ectodomain shedding. Here we show that increased proteolytic ectodomain shedding mediated by the A Disintegrin and metalloproteinase domain (ADAM) proteases ADAM10 and ADAM17 caused increased sIL-6R serum level in vitro as well as in healthy volunteers homozygous for the IL-6R Asp358Ala allele. Differential splicing of the IL-6R appears to have only a minor effect on sIL-6R level. Increased ectodomain shedding resulted in reduced cell-surface expression of the IL-6R Asp358Ala variant compared to the common IL-6R variant. In conclusion, increased IL-6R ectodomain shedding is a mechanistic explanation for the increased serum IL-6R levels found in persons homozygous for the rs2228145 IL-6R Asp358Ala variant.     

Storage effects on genomic DNA in rolled and mature coca leaves

Rolled and mature leaf tissue was harvested from Erythroxylum coca var. coca Lam. (coca) to determine a method for storage that would maintain DNA with high quality and content up to 50 days. Harvesting coca leaf tissue under Andean field conditions often requires storage from 3 to 10 days before extraction where tissue integrity is lost. All samples of rolled and mature coca leaf tissue were harvested and separately stored fresh in RNAlater for 50 days at 4 degrees, -20 degrees, and 23 degrees C, while similar samples were air-dried for 72 h at 23 degrees C or oven-dried for 72 h at 40 degrees C after storage, before extraction. Triplicate samples of each tissue type were extracted for DNA at 10-day intervals and showed that DNA integrity and content were preserved in leaf tissue stored at 4 degrees and -20 degrees C for 50 days. Rolled and mature leaf tissue stored at 4 degrees, -20 degrees, and 23 degrees C showed insignificant degradation of DNA after 10 days, and by day 50, only leaf tissue stored at 4 degrees and -20 degrees C had not significantly degraded. All air- and oven-dried leaf tissue extracts showed degradation upon drying (day 0) and continuous degradation up to day 50, despite storage conditions. Amplified fragment length polymorphism analysis of DNA from rolled and mature leaf tissue of coca stored at 4 degrees and -20 degrees C for 0, 10, and 50 days showed that DNA integrity and content were preserved. We recommend that freshly harvested rolled or mature coca leaf tissue be stored at 4 degrees, -20 degrees, and 23 degrees C for 10 days after harvest, and if a longer storage is required, then store at 4 degrees or -20 degrees C.     

The soluble interleukin 6 receptor: mechanisms of production and implications in disease.

Interleukin 6 (IL-6) performs a prominent role during disease and has been described as both a pro- and anti-inflammatory cytokine. A key feature in the regulation of IL-6 responses has been the identification of a soluble interleukin 6 receptor (sIL-6R), which forms a ligand-receptor complex with IL-6 that is capable of stimulating a variety of cellular responses including proliferation, differentiation and activation of inflammatory processes. Elevated sIL-6R levels have been documented in numerous clinical conditions indicating that its production is coordinated as part of a disease response. Thus, sIL-6R has the potential to regulate both local and systemic IL-6-mediated events. This review will outline the central role of sIL-6R in the coordination of IL-6 responses. Details relating to the mechanisms of sIL-6R production will be provided, while the potential significance of sIL-6R during the development of clinical conditions will be emphasized. We want to convey, therefore, that when thinking about the inflammatory capability of IL-6, it is essential to consider not only the action of IL-6 itself, but also the effect sIL-6R may have on cellular processes.     

Analysis of soluble interleukin 6 receptor in cerebrospinal fluid in inflammatory and non-inflammatory conditions

The objective of this study was to investigate the pathophysiological roles of soluble interleukin 6 receptor (sIL-6R) in cerebrospinal fluid (CSF). CSF was obtained from patients suspected with meningitis. Eight patients without any meningeal signs or symptoms were enrolled as controls. An additional 34 CSF samples were collected to measure both biologically active and immunoreactive sIL-6R. All CSF samples were proven to be aseptic. IL-6 and sIL-6R were measured using specific ELISAs. Patients were divided into three groups on the basis of cell number in CSF; inflammatory group (cell number >5 microl, mean 241+/-363.1, n=61); non-inflammatory group (cell number < or =5 microl, mean=2.1+/-1.7, n=12) and controls (cell number < or =5 microl, mean=0.3+1.7, n=8). Among these three groups, the differences in protein (F (2,78)=8.274, P<0.0001) and IL-6 concentration (F (2,78)=6.475, P<0.001) were statistically significant but those of sIL-6R concentration were not. There were only weak correlations between log (sIL-6R) versus log (cell number) (r=0.23, P=0.0375), log (protein) (r=0.239, P=0.0358) and log (IL-6) (r=0.27, P=0.0167). Amounts of immunoreactive and biologically active sIL-6R were closely correlated (r=0.62, n=34, P<0.005). It was concluded that sIL-6R is present constitutively in CSF and its level may not increase significantly in inflammatory conditions; infiltrating cells in CSF are not the main source of sIL-6R; and sIL-6R in CSF can bind IL-6.     

Stability of cytokines,chemokines and soluble activation markers in unprocessed blood stored under different conditions

BackgroundBiomarkers such as cytokines, chemokines, and soluble activation markers can be unstable when processing of blood is delayed. The stability of various biomarkers in serum and plasma was investigated when unprocessed blood samples were stored for up to 24 h at room and refrigerator temperature.MethodsBlood was collected from 16 healthy volunteers. Unprocessed serum, EDTA and heparinized blood was stored at room (20–25 °C) and refrigerator temperature (4–8 °C) for 0.5, 2, 4, 6, 8, and 24 h after collection before centrifugation and separation of serum and plasma. Samples were batch tested for various biomarkers using commercially available immunoassays. Statistically significant changes were determined using the generalized estimating equation.ResultsIFN-γ, sIL-2Rα, sTNF-RII and β₂-microglobulin were stable in unprocessed serum, EDTA and heparinized blood samples stored at either room or refrigerator temperature for up to 24 h. IL-6, TNF-α, MIP-1β and RANTES were unstable in heparinized blood at room temperature; TNF-α, and MIP-1β were unstable in unprocessed serum at room temperature; IL-12 was unstable in unprocessed serum at refrigerator temperature; and neopterin was unstable in unprocessed EDTA blood at room temperature. IL-1ra was stable only in unprocessed serum at room temperature.ConclusionAll the biomarkers studied, with the exception of IL-1ra, were stable in unprocessed EDTA blood stored at refrigerator temperature for 24 h. This indicates that blood for these biomarkers should be collected in EDTA and if delays in processing are anticipated the unseparated blood should be stored at refrigerator temperature until processing.     

The effect of repeated endurance exercise on IL-6 and sIL-6R and their relationship with sensations of fatigue at rest

Strenuous, prolonged exercise increases interleukin-6 (IL-6) release. The effect of IL-6 is dependent on the availability of IL-6 receptors. Few studies have addressed the impact of exercise on IL-6 receptor levels or procalcitonin (PCT), an indicator of systemic inflammation. Changes in these molecules may give insight into cytokine-related mechanisms underlying exercise-related fatigue. Thirteen trained male subjects partook in the study. They cycled a total distance of 468 km over 6 days. Blood samples were obtained prior to and immediately following Day 1 of the study and then each morning prior to exercise. Blood samples were analysed for plasma IL-6, soluble IL-6 receptor (sIL-6R), C-reactive protein (CRP), PCT, creatine kinase (CK) and cortisol concentrations. Subjects also completed mood state questionnaires each day prior to exercise. IL-6 was elevated immediately post-exercise on Day 1 but was unchanged at rest for the duration of the event. In contrast, sIL-6R, CRP, PCT and CK concentrations were unchanged immediately post-exercise on Day 1 but were significantly elevated at rest over the duration of the event compared with pre-event baseline. sIL-6R was highly correlated to CRP. Cortisol concentrations remained unchanged at all time points. In conclusion, strenuous, prolonged exercise stimulated an acute phase response which was maintained throughout the 6-day event. sIL-6R increase is associated with CRP and may affect subjective sensations of post-exercise fatigue at rest.     

Serum levels of IL-6 type cytokines and soluble IL-6 receptors in active B-cell chronic lymphocytic leukemia and in cladribine induced remission

We have investigated the serum concentrations of interleukin-6 (IL-6) and two IL-6 family cytokines-oncostatin M (OSM) and leukemia inhibitory factor (LIF)-in 63 patients with B-cell chronic lymphocytic leukemia (B-CLL) and 17 healthy controls using the enzyme-linked immunosorbent assay (ELISA) method. Simultaneously, we measured the serum levels of the soluble forms of two subunits of the IL-6 receptor complex-ligand binding glycoprotein 80 (sIL-6R) and glycoprotein 130 (sgp130). The cytokines and receptors were evaluated in 25 untreated patients and 38 patients treated with cladribine (2-CdA), as well as in 17 healthy controls. We have correlated the serum levels of these proteins with Rai's clinical stage of the disease, the response to 2-CdA treatment and some hematological parameters. We have also evaluated the correlation of the IL-6 serum level with the concentration of OSM and IL-6 soluble receptors. IL-6 was measurable in 62/63 (98.4%), OSM in 20/25 (80%) of untreated and 14/38 (37.8%) of the treated patients. sIL-6R and sgp130 were detectable in all 63 patients and LIF in none of the CLL patients. IL-6 serum level in untreated patients was not significantly different as compared to its concentration in the control group (P>0.05). However, in the patients treated with 2-CdA the IL-6 level was significantly lower (P<0.02), and the lowest concentration was found in the patients with complete remission (CR; median 1.4pg/ml; P<0.02). The concentration of sIL-6R was significantly higher in untreated (median 61.8 ng/ml) and treated (median 50.1 ng/ml) CLL patients when compared to normal persons (median 41.2 ng/ml; P=0.04; P<0.001, respectively). There was no difference between the sIL-6R levels in the patients with CR and the healthy controls. In non-responders sIL-6R concentration was the highest and similar to its level in the untreated patients. OSM level was higher in the untreated patients (median 1.8pg/ml) than in the normal controls (median 0.0pg/ml; P<0.001) and in the CR patients (median 0.0pg/ml; P<0.03). The serum concentration of sgp130 was similar in the untreated (median 480 pg/ml) and treated (median 470 pg/ml) patients, as well as in the healthy persons (median 420 pg/ml; P>0.05). We have found significant positive correlation between the levels of sIL-6R and the lymphocytes count in CLL patients (p=0.423; P<0.001). In addition, sIL-6R and OSM serum concentrations correlated also with CLL Rai stage. In conclusion, the serum level of IL-6, OSM and sIL-6R, but not LIF and sgp130, are useful indicators of CLL activity.     

Interleukin-6 concentrations in the placenta and blood in normal pregnancies and preeclampsia.

AIM: To evaluate whether IL-6 concentrations in the placenta and blood from women with preeclampsia differed from those in normal pregnancies. METHODS: This study involved 41 pregnant women carrying single fetuses. Of these pregnancies, 23 were normal pregnant and 18 were preeclamptic patients. The average gestational age at entry was 37-38 weeks of gestation. Blood was collected before the onset of labor. Serum was separated and stored at -20 degrees C. A tissue segment of the placenta was cut and chilled in liquid nitrogen immediately after delivery and stored at -80 degrees C. The frozen tissue was added to phosphate-buffered saline and fully homogenized. After centrifugation, the separated supernatant was stored at -80 degrees C. IL-6 levels in separated serum and IL-6 and total protein (TP) levels in separated supernatant were measured. The presence of IL-6 in the placenta was evaluated by immunohistochemistry in five preeclamptic and five normal pregnant patients. RESULTS: Neither IL-6/TP levels in the placenta nor IL-6 levels in blood differed significantly between the two groups. IL-6 immunostaining on trophoblastic cells in the placenta was weak in one and absent in four in normal pregnancies, and absent in all patients with preeclampsia. There was no strong immunostaining for IL-6 in preeclampsia by immunohistochemistry. CONCLUSIONS: Our findings suggest that IL-6 in the placenta and blood does not play a significant role in the induction of an immunologic imbalance, which may contribute to the etiological mechanism leading to preeclampsia.