Identification of reference microRNAs and suitability of archived hemopoietic samples for robust microRNA expression profiling

In many cancers, including neuroblastoma, microRNA (miRNA) expression profiling of peripheral blood (PB) and bone marrow (BM) may increase understanding of the metastatic process and lead to the identification of clinically informative biomarkers. The quality of miRNAs in PB and BM samples archived in PAXgene? blood RNA tubes from large-scale clinical studies and the identity of reference miRNAs for standard reporting of data are to date unknown. In this study, we evaluated the reliability of expression profiling of 377 miRNAs using quantitative polymerase chain reaction (qPCR) in PB and BM samples (n = 90) stored at ?80 °C for up to 5 years in PAXgene? blood RNA tubes. There was no correlation with storage time and variation of expression for any single miRNA (r < 0.50). The profile of miRNAs isolated as small RNAs or co-isolated with small/large RNAs was highly correlated (r = 0.96). The mean expression of all miRNAs and the geNorm program identified miR-26a, miR-28-5p, and miR-24 as the most stable reference miRNAs. This study describes detailed methodologies for reliable miRNA isolation and profiling of PB and BM, including reference miRNAs for qPCR normalization, and demonstrates the suitability of clinical samples archived at ?80 °C into PAXgene? blood RNA tubes for miRNA expression studies.     

Serum microRNA-29a is a promising novel marker for early detection of colorectal liver metastasis

Background: Colorectal cancer (CRC) metastasis occurs in various organs, most frequently in liver. Serological examination including tumor and biochemical markers for liver function evaluation is routinely performed, though its accuracy is not high. MicroRNAs (miRNAs) have been implicated in a variety of human diseases including cancer, and have many characteristics of an ideal biomarker most notably their inherent stability and resilience. Recently, several studies have indicated that circulating miRNAs hold much potential as novel noninvasive biomarkers for cancer and other disease processes. The objective of this study was to investigate the potential of serum miRNAs as novel biomarkers for CRC with liver metastasis. Methods: This study was divided into three phases: (I) 3 candidate serum miRNAs were detected by using real-time RT-PCR, corresponding 38 CRC patients with liver metastasis and 36 CRC patients without metastasis. (II) Marker validation by real-time RT-PCR on a similar cohort of age- and sex-matched CRC patients without (n = 20) and with liver metastasis (n = 20). (III) We examined the correlation between the expressions of candidate serum miRNAs with clinical parameters of CRC patients. Results: Serum miR-29a was significantly higher in colorectal liver metastasis (CRLM) patients than in CRC patients. This marker yielded a receiver operating characteristic curve area of 80.3%. At a cutoff value of 0.155, the sensitivity was 75% and the specificity was 75% in discriminating metastatic from non-metastatic patients. In addition, increased levels of miR-29a expression were also observed in colorectal tumors from CRLM patients compared with CRC patients. No significant difference was observed in the levels of serum miR-92a between metastatic and non-metastatic patients. Conclusions: These findings suggest that serum miR-29a has strong potential as a novel noninvasive biomarker for early detection of CRC with liver metastasis.     

Metabolomic markers of fatigue: Association between circulating metabolome and fatigue in women with chronic widespread pain

Background

Fatigue is a sensation of unbearable tiredness that frequently accompanies chronic widespread musculoskeletal pain (CWP) and inflammatory joint disease. Its mechanisms are poorly understood and there is a lack of effective biomarkers for diagnosis and onset prediction. We studied the circulating metabolome in a population sample characterised for CWP to identify biomarkers showing specificity for fatigue.

Isothermal sensitive detection of microRNA using an autonomous DNA machine recycling output as input

An autonomous DNA machine recycling the output as the input for isothermal, sensitive, and specific detection of miRNAs has been developed. This machine shows considerably high signal amplification efficiency (～1000-fold) and thus a low detection limit (～20 amol). The machine also shows high specificity, discriminating 50 amol of synthetic miRNA from 100-fold larger amounts of its family member and from 100 ng of unrelated total RNAs. Moreover, it is available for practically detecting natural miRNAs in total RNAs.     

Serum MicroRNAs Related with Chemoradiotherapy Resistance in Advanced-Stage Cervical Squamous Cell Carcinoma

OBJECTIVE: To investigate the serum microRNAs as biomarkers in predicting chemoradiotherapy resistance in advanced-stage cervical squamous cell carcinoma (ACSCC) patients. METHODS: Serum samples were collected from International Federation of Gynecology and Obstetrics (FIGO) stage IIB to IIIB cervical squamous cell carcinoma patients treated with platinum based Concomitant Chemoradiotherapy (CCRT) in our hospital during September 2013 to November 2015. Twenty well-matched samples (10 resistant and 10 sensitive) were chosen to screen the miRNA expression profile using serum samples pooled with microarrays. miRNAs expressed significantly different between two groups were further verified in 131 patients (29 resistant and 102 sensitive) serum samples with TaqMan Real-time PCR. The AUC was used to evaluate the accuracy of the biomarkers for prediction. RESULTS: MiR-136-5, miR-152-3p and miR-206 were expressed significantly different between sensitive and resistant groups. Results of 131 patients verification showed that the levels of miR-206 in sensitive samples and resistant samples were 2.715 ± 0.2115 and 14.64 ± 1.184, respectively, which was significantly different (P < .0001), while miR-136-5p and miR-152-3p could not be tested without pre-amplification reactions. Univariate analysis revealed that miR-206 expression was significantly associated with patients' DFS. Multivariate analysis demonstrated that miR-206 expression, tumor differentiation and pelvic lymph nodes metastasis were the independent prognostic factors associated with DFS in this cohort (P = .008, 0.000, 0.000, respectively). The probability of the prognostic accuracy of miR-206 expression in predicting chemoradiotherapy sensitivity of ACSCC patients was 91.3% (79.3% sensitivity and 92.2% specificity). CONCLUSION: Serum miR-206 is a powerful tool in predicting chemoradiotherapy sensitivity in ACSCC patients.     

Metabolic signatures of esophageal cancer: NMR-based metabolomics and UHPLC-based focused metabolomics of blood serum

Focused metabolic profiling is a powerful tool for the determination of biomarkers. Here, a more global proton nuclear magnetic resonance (¹H NMR)-based metabolomic approach coupled with a relative simple ultra high performance liquid chromatography (UHPLC)-based focused metabolomic approach was developed and compared to characterize the systemic metabolic disturbances underlying esophageal cancer (EC) and identify possible early biomarkers for clinical prognosis. Serum metabolic profiling of patients with EC (n = 25) and healthy controls (n = 25) was performed by using both ¹H NMR and UHPLC, and metabolite identification was achieved by multivariate statistical analysis. Using orthogonal projection to least squares discriminant analysis (OPLS-DA), we could distinguish EC patients from healthy controls. The predictive power of the model derived from the UHPLC-based focused metabolomics performed better in both sensitivity and specificity than the results from the NMR-based metabolomics, suggesting that the focused metabolomic technique may be of advantage in the future for the determination of biomarkers. Moreover, focused metabolic profiling is highly simple, accurate and specific, and should prove equally valuable in metabolomic research applications. A total of nineteen significantly altered metabolites were identified as the potential disease associated biomarkers. Significant changes in lipid metabolism, amino acid metabolism, glycolysis, ketogenesis, tricarboxylic acid (TCA) cycle and energy metabolism were observed in EC patients compared with the healthy controls. These results demonstrated that metabolic profiling of serum could be useful as a screening tool for early EC diagnosis and prognosis, and might enhance our understanding of the mechanisms involved in the tumor progression.     

Metabolomic analysis identifies altered metabolic pathways in Multiple Sclerosis

Multiple sclerosis (MS) is a chronic, demyelinating disease that affects the central nervous system and is characterized by a complex pathogenesis and difficult management. The identification of new biomarkers would be clinically useful for more accurate diagnoses and disease monitoring. Metabolomics, the identification of small endogenous molecules, offers an instantaneous molecular snapshot of the MS phenotype. Here the metabolomic profiles (utilizing plasma from patients with MS) were characterized with a Gas cromatography-mass spectrometry-based platform followed by a multivariate statistical analysis and comparison with a healthy control (HC) population. The obtained partial least square discriminant analysis (PLS-DA) model identified and validated significant metabolic differences between individuals with MS and HC (R2X = 0.223, R2Y = 0.82, Q2 = 0.562; p < 0.001). Among discriminant metabolites phosphate, fructose, myo-inositol, pyroglutamate, threonate, l-leucine, l-asparagine, l-ornithine, l-glutamine, and l-glutamate were correctly identified, and some resulted as unknown. A receiver operating characteristic (ROC) curve with AUC 0.84 (p = 0.01; CI: 0.75–1) generated with the concentrations of the discriminant metabolites, supported the strength of the model. Pathway analysis indicated asparagine and citrulline biosynthesis as the main canonical pathways involved in MS. Changes in the citrulline biosynthesis pathway suggests the involvement of oxidative stress during neuronal damage. The results confirmed metabolomics as a useful approach to better understand the pathogenesis of MS and to provide new biomarkers for the disease to be used together with clinical data.     

Salivary mRNA markers having the potential to detect oral squamous cell carcinoma segregated from oral leukoplakia with dysplasia

BackgroundIn the current study the presence of extracellular IL-1B, IL-8, OAZ and SAT mRNAs in the saliva was evaluated as a tool in the early detection of oral squamous cell carcinoma.Methods34 patients with primary oral squamous cell carcinoma stage T₁N₀M₀/T₂N₀M₀, 20 patients with oral leukoplakia and dysplasia (15 patients with mild dysplasia and 5 with severe dysplasia/in situ carcinoma) and 31 matched healthy-control subjects were included in the study. The presence of IL-1B, IL-8, OAZ and SAT mRNA was evaluated in extracellular RNA isolated from saliva samples using sequence-specific primers and real-time RT-PCR. ROC curve analysis was used to estimate the ability of the biomarkers to detect oral squamous cell carcinoma patients.ResultsThe data reveal that the combination of these four biomarkers provides a good predictive probability of up to 80% (AUC = 0.799, p = 0.002) for patients with oral squamous cell carcinoma but not patients suffering from oral leukoplakia with dysplasia. Moreover, the combination of only the two biomarkers (SAT and IL-8) also raises a high predictive ability of 75.5% (AUC = 0.755, p = 0.007) approximately equal to the four biomarkers suggesting the use of the two biomarkers only in the prediction model for oral squamous cell carcinoma patients limiting the economic and health cost in half.ConclusionSAT and IL-8 mRNAs are present in the saliva in high quality and quantity, with a good discriminatory ability for oral squamous cell carcinoma patients only but not for patients with oral leukoplakia and dysplasia an oral potentially malignant disorder.     

Genome-wide identification of cold-responsive and new microRNAs in Populus tomentosa by high-throughput sequencing

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate the expression of target mRNAs in plant growth, development, abiotic stress responses, and pathogen responses. Cold stress is one of the most common abiotic factors affecting plants, and it adversely affects plant growth, development, and spatial distribution. To understand the roles of miRNAs under cold stress in Populus tomentosa, we constructed two small RNA libraries from plantlets treated or not with cold conditions (4 °C for 8 h). High-throughput sequencing of the two libraries identified 144 conserved miRNAs belonging to 33 miRNA families and 29 new miRNAs (as well as their corresponding miRNA¹s) belonging to 23 miRNA families. Differential expression analysis showed that 21 miRNAs were down-regulated and nine miRNAs were up-regulated in response to cold stress. Among them, 19 cold-responsive miRNAs, two new miRNAs and their corresponding miRNA¹s were validated by qRT-PCR. A total of 101 target genes of the new miRNAs were predicted using a bioinformatics approach. These target genes are involved in growth and resistance to various stresses. The results demonstrated that Populus miRNAs play critical roles in the cold stress response.     

Regulation of lipid metabolism in the snow alga Chlamydomonas nivalis in response to NaCl stress: An integrated analysis by cytomic and lipidomic approaches

The cultures of the snow alga Chlamydomonas nivalis in the exponential phage were stressed by NaCl (up to 1.5%) for 0～48 h, followed by Nile Red staining-based cytomic analysis (flow cytometry and confocal laser scanning microscopy). The fluorescent intensities of total lipids, and neutral and polar lipids increased to the maximum within 7 h in the NaCl stressed cells with the highest increase in total lipids by 2-fold (0.75%-NaCl for 7 h), the highest increase in neutral lipids by 68-fold (1%-NaCl for 7 h) and the highest increase in polar lipids by 10-fold (1.25%-NaCl for 5 h), respectively. Seven types and 22 kinds of polar lipid molecules were selected and identified as biomarkers by UPLC/Q-TOF-MS-based lipidomic analysis, which demonstrated differences in total lipids between the stress group (0.75%-NaCl for 7 h) and the control. The biological roles of the biomarkers in the alga under NaCl stress were discussed. The integrated approach based on “omics” technologies developed in the present work is validated as a powerful tool to successfully reveal the regulation of lipid metabolism in microalgae in response to stress stimulation.     

Sphingomyelin synthase 2 over-expression induces expression of aortic inflammatory biomarkers and decreases circulating EPCs in ApoE KO mice

AimsThis study sought to assess the effect of sphingomyelin synthase 2 (SMS2) over-expression on plaque component and endothelial dysfunction in atherosclerosis.Main methodsWe generated recombinant adenovirus vectors containing human SMS2 cDNA (AdV-SMS2) or control gene GFP cDNA (AdV-GFP). Both AdVs were injected (i.v.) into ApoE KO mice to establish SMS2 over-expressing and control mice models, respectively. The mice were fed a high fat diet for 30 days. We then examined their plasma lipid levels, expression levels of aortic inflammatory biomarkers critical for the plaque's stability, and numbers of peripheral endothelial progenitor cells (EPC).Key findingsCompared with the control mice, SMS2 over-expression had significantly (1) increased aortic matrix metalloproteinase-2 (MMP-2), monocyte chemoattractant protein-1 (MCP-1), tissue factor (TF) and cyclooxygenase-2 (COX-2) mRNA levels (1.9-fold, 2.2-fold, 2.6-fold and 3.2-fold, respectively, P < 0.01) and protein levels (2.2-fold, 1.9-fold, 1.9-fold and 2.1-fold, respectively, P < 0.01); (2) increased MMP-2, COX-2 in situ expression in aortic root (2.6-fold and 2.3-fold, respectively, P < 0.01); (3) decreased aortic COX-1 mRNA levels (65%, P < 0.01) and protein levels (64%, P < 0.01); and (4) decreased CD34/KDR-positive cells (33%, P < 0.01), circulating angiogenic cells (CACs) (50%, P < 0.05), and colony forming units (CFUs) (40%, P < 0.05) in circulation.SignificanceSMS2 over-expression was probably associated with increased expression of aortic inflammatory biomarkers, as well as decreased numbers of CD34/KDR-positive cells, CACs and CFUs in circulation. Therefore, SMS2 over-expression might correlate with endothelial dysfunction and aggravate atherosclerotic plaque instability in ApoE KO mice.     

Pre-storage centrifugation conditions have significant impact on measured microRNA levels in biobanked EDTA plasma samples

BackgroundIn the past few years, an increasing number of studies have reported the potential use of microRNAs (miRNA) as circulating biomarkers for diagnosis or prognosis of a wide variety of diseases. There is, however, a lack of reproducibility between studies. Due to the high miRNA content in platelets this may partly be explained by residual platelets in the plasma samples used. When collecting fresh plasma samples, it is possible to produce cell-free/platelet-poor plasma by centrifugation. In this study, we systematically investigated whether biobanked EDTA plasma samples could be processed to be suitable for miRNA analysis.Materials and methodsBlood samples were collected from ten healthy volunteers and centrifuged to produce platelet-poor-plasma (PPP) and standard biobank plasma. After one week at ?80 °C the biobanked EDTA plasma was re-centrifuged by different steps to remove residual platelets. Using RT-qPCR the levels of 14 miRNAs in the different plasma preparations were compared to that of PPP.ResultsWe were able to remove residual platelets from biobanked EDTA plasma by re-centrifugation of the thawed samples. Nevertheless, for most of the investigated miRNAs, the miRNA level was significantly higher in the re-centrifuged biobanked plasma compared to PPP, even when the platelet count was reduced to 0–1×10⁹/L.ConclusionWe found, that pre-storage centrifugation conditions have a significant impact on the measured EDTA plasma level of miRNAs known to be present in platelets. Even for the miRNAs found to be less effected, we showed that a 1.5–3 fold change in plasma levels may possible be caused by or easily overseen due to sample preparation and/or storage.     

Dietary copper and human health: Current evidence and unresolved issues

Although copper (Cu) is recognized as an essential trace element, uncertainties remain regarding Cu reference values for humans, as illustrated by discrepancies between recommendations issued by different national authorities. This review examines human studies published since 1990 on relationships between Cu intake, Cu balance, biomarkers of Cu status, and health. It points out several gaps and unresolved issues which make it difficult to assess Cu requirements. Results from balance studies suggest that daily intakes below 0.8 mg/day lead to net Cu losses, while net gains are consistently observed above 2.4 mg/day. However, because of an incomplete collection of losses in all studies, a precise estimation of Cu requirements cannot be derived from available data. Data regarding the relationship between Cu intake and potential biomarkers are either too preliminary or inconclusive because of low specificity or low sensitivity to change in dietary Cu over a wide range of intakes. Results from observation and intervention studies do not support a link between Cu and a risk of cardiovascular disease, cognitive decline, arthritis or cancer for intakes ranging from 0.6 to 3 mg/day, and limited evidence exists for impaired immune function in healthy subjects with a very low (0.38 mg/day) Cu intake. However, data from observation studies should be regarded with caution because of uncertainties regarding Cu concentration in various foods and water. Further studies that accurately evaluate Cu exposure based on reliable biomarkers of Cu status are needed.     

Neutrophil CD64 expression and serum IL-8: Sensitive early markers of severity and outcome in sepsis

The aim of the present study was to investigate which biomarker/s reliably assess severity and mortality early in the sepsis process. In 47 critically-ill patients within the 24 h of septic onset, Interleukins (IL)-8, -1β, -6, -10, and -12p70, tumor necrosis factor-α (TNF-α), procalcitonin (PCT) and C-reactive protein (CRP) were measured in serum. Additionally, CD64 expression was measured in neutrophils. In early sepsis, neutrophil CD64 expression and IL-8 levels are the only biomarkers that increased with sepsis severity, differentiating disease stages: sepsis, severe sepsis and septic shock (p < 0.001). The biomarkers that best evaluate the severity of sepsis (via APACHE II) were CD64, IL-8 and IL-6 (p < 0.01), and the severity of organ failure (via SOFA) were CD64 and IL-8 (p < 0.01). CD64 expression and IL-8 levels were associated with mortality within 28-days (OR = 1.3, p = 0.01 for CD64 and OR = 1.26, p = 0.024 for IL-8 by logistic regression analysis) and ROC curve analysis showed high sensitivity and specificity for predicting sepsis stages and the 28 day mortality. We conclude that there is an early increase of neutrophil CD64 expression and IL-8 levels during sepsis. Based on this single measurement it is possible to reliably assess the stage, detect the severity and predict the 28-day mortality of sepsis.     

Zoledronic acid increases the circulating soluble RANKL level in mice,with a further increase in lymphocyte-derived soluble RANKL in zoledronic acid- and glucocorticoid-treated mice stimulated with bacterial lipopolysaccharide

The nitrogen-containing bisphosphonate (BP) zoledronic acid (ZA) is a potent antiresorptive drug used in conjunction with standard cancer therapy to treat osteolysis or hypercalcemia due to malignancy. However, it is unclear how ZA influences the circulating levels of bone remodeling factors. The aim of this study was to evaluate the effects of ZA on the serum levels of soluble receptor activator of NF-kB ligand (sRANKL) and osteoprotegerin (OPG). The following four groups of C57BL/6 mice were used (five mice per group): (1) the placebo + phosphate-buffered saline (PBS) group, in which placebo-treated mice were injected once weekly with PBS for 4 weeks; (2) the placebo + ZA group, in which placebo-treated mice were injected once weekly with ZA for 4 weeks; (3) the prednisolone (PSL) + PBS group, in which PSL-treated mice were injected once weekly with PBS for 4 weeks; and (4) the PSL + ZA group, in which PSL-treated mice were injected once weekly with ZA for 4 weeks. At the 3-week time point, all mice were subjected to oral inflammatory stimulation with bacterial lipopolysaccharide (LPS). The sera of these mice were obtained every week and the levels of sRANKL and OPG were measured using enzyme-linked immunosorbent assay. At the time of sacrifice, femurs were prepared for micro-computed tomography (micro-CT), histological, and histomorphometric analyses. Our data indicated that ZA administration remarkably reduced bone turnover and significantly increased the basal level of sRANKL. Interestingly, the PSL + ZA group showed a dramatically elevated sRANKL level after LPS stimulation. In contrast, the PSL + ZA group in nonobese diabetic mice with severe combined immunodeficiency disease (NOD-SCID mice), which are characterized by the absence of functional T- and B-lymphocytes, showed no increase in the sRANKL level. Our data suggest that, particularly with combination treatment of ZA and glucocorticoids, surviving lymphocytes might be the source of inflammation-induced sRANKL. Thus, circulating sRANKL levels might be modulated by ZA.     

A Validated Prognostic Biomarker Score for Adult Patients with Nonmetastatic Soft Tissue Sarcomas of the Trunk and Extremities

BACKGROUND: The prognostic value of serum biomarkers in soft tissue sarcoma (STS) is limited, and its clinical applicability is compromised by a common inability to adjust for important confounders. The aim of this study was to determine the prognostic value of pretreatment biomarkers on disease-specific survival (DSS) adjusted for confounders. METHODS: The study included 818 patients with localized STS. Pretreatment levels of albumin, C-reactive protein, hemoglobin, neutrophils, and lymphocytes were tested individually and combined in prognostic scores: neutrophil/lymphocyte ratio (NLR), Glasgow Prognostic Score (GPS), and Aarhus Composite Biomarker Score (ACBS) which includes all five biomarkers. Patients were randomly split into a test cohort and a validation cohort. The prognostic value of biomarkers on DSS was estimated using crude and adjusted Cox proportional hazard models. The different biomarker scores were compared using Akaike's information criteria. RESULTS: In the test cohort of 403 patients, all biomarkers except lymphocyte count were significant prognostic factors for DSS also after adjusting for confounders. NLR, GPS, and ACBS were independently associated with decreased survival; however, ACBS was significantly superior to NLR (P = .02) and GPS (P = .002). These findings were validated in the randomly assigned validation cohort of 415 patients. In the pooled data of 818 patients, the ACBS performed better than GPS and NLR. ACBS 2 was independently associated with decreased DSS compared to ACBS 0, hazard ratio 2.3[95% confidence interval: 1.5-3.5], P < .001. CONCLUSION: Patients with abnormal values in more than one serum biomarkers had a significant additional risk of dying compared to patients with only one abnormal value. ACBS was validated as an independent prognostic factor that is superior to both NLR and GPS.     

Serum concentrations of fibroblast growth factor 21 are elevated in patients with congenital or acquired lipodystrophy

ObjectivePatients with lipodystrophy (LD) suffer from loss of subcutaneous adipose tissue accompanied by dysregulation of several adipocyte-secreted factors. However, regulation of adipocyte-expressed fibroblast growth factor (FGF) 21 which acts in an insulin-mimetic, lipid-lowering, and anti-atherogenic manner has not been investigated in non-human immunodeficiency virus (HIV) LD.Material and methodsCirculating serum FGF21 levels were quantified in 37 patients with non-HIV LD and 37 controls matched for age, gender, and body mass index. Moreover, FGF21 plasma levels and mRNA expression were measured in LD mice and control animals. Additionally, serum FGF21 levels were assessed in 10 LD patients before and during metreleptin therapy.ResultsMedian FGF21 serum concentrations were significantly higher in LD patients (381.2 ng/l) as compared to the control group (231.2 ng/l; p = 0.023). There was an independent and positive association between circulating FGF21 and serum triglycerides (TG), as well as fibrate treatment, in multiple linear regression analysis. LD mice showed significantly upregulated FGF21 plasma levels (4.5-fold), as well as mRNA expression in various adipose tissue depots and liver as compared to controls (p < 0.05). Metreleptin treatment did not significantly alter circulating FGF21 levels in human subjects.ConclusionsSerum concentrations of FGF21 are elevated in patients with non-HIV LD with adipose tissue and liver being potential sources of increased production. TG and fibrate treatment are independent positive predictors of circulating FGF21.     

Association of serum amyloid A levels with adipocyte size and serum levels of adipokines: Differences between men and women

The aim of this study was to characterize the association between adipocyte enlargement and circulating levels of serum amyloid A (SAA). Furthermore, we wanted to search for possible associations with measures of glycemic control and levels of circulating adipokines and/or inflammatory markers in men and women with a large range in body mass index. The study cohort consisted of 167 subjects, 114 non-diabetic and 53 with Type 2 diabetes. Adipocyte diameter as well as circulating levels of SAA, C-reactive protein (CRP), adiponectin, leptin, interleukin-6, tumor necrosis factor alpha, glucose and insulin were measured. Women had higher serum levels of SAA than men (p = 0.044). SAA levels were weakly but positively correlated with BMI (p = 0.043) and % body fat (p = 0.027) in all subjects as well as subcutaneous adipocyte diameter (p = 0.034) in women. Furthermore, in all subjects we found correlations between SAA levels and levels of CRP (p < 0.001), interleukin-6 (p < 0.001), leptin (p = 0.003), insulin (p = 0.006), HbA1c (p = 0.02) and HOMA-IR (p = 0.002). A majority of the correlations were strongest in women. In conclusion, serum levels of SAA are strongly correlated with serum levels of inflammatory markers as well as measures of glycemic control. There seems to be large sex differences in these associations suggesting that sex-specific factors need to be considered when analyzing SAA levels in relation to metabolic disease.     

Aging is associated with circulating cytokine dysregulation

PurposeAging is accompanied by a progressive increase in pro-inflammatory cytokine status. However, little is known about the development of age-dependent modifications in other circulating cytokines. The aim of this study was to investigate in vivo the influence of age on circulating cytokine production in healthy subjects (HC).MethodsCirculating cytokines were measured by CBA and ELISA in 73 HC. Intracellular cytokine production was assessed in CD3+ and CD14+ cells by flow cytometry. Production of cytokines in cell culture supernatants was also studied after polyclonal stimulation.ResultsSubjects were divided into three different groups according to age: 28 young HC (<30 years, 26.2 ± 2.4), 24 middle age HC (30–60 years, 44.7 ± 8.4) and 21 elderly HC (>60 years, 70.6 ± 7.9). Age was positively correlated with the circulating levels of IL-12p70, IL-1β, TNFα, IL-6, and IL-10. Age had a negative correlation with circulating levels of IL-17. Besides, age was positively correlated with spontaneous intracellular expression of proinflammatory cytokines in circulating monocytes. No correlation was found with other intracellular cytokine expression or with the production of cytokines in cell culture supernatants after in vitro stimulation. Gender had a marginal effect on the circulating cytokine profile.ConclusionAging has a significant impact on the production of circulating cytokines in healthy individuals. The circulating cytokine milieu may contribute to the development of age-restricted conditions.