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• Cytokine concentration in finger-prick blood VAMS was more variable than in venous. • Room temperature had the most significant changes in concentration over time. • The optimal storage temperature differed for each analyte. • Incomplete extraction of 6 spiked cytokines may indicate cytokine-VAMS interaction. Dried blood spots (DBS) collected on filter paper such as Guthrie cards are stored for years at room temperature. The assumption is that once dried, the samples remain stable and quantifiable indefinitely since the metabolites these were initially designed to measure, are known for their extended stability. The concentration of other blood proteins such as cytokines, however, are known to vary with storage even in liquid samples stored at −80 °C for extended periods of time. We sought to determine if cytokines are stable for up to 5 months when stored as a dried blood sample using volumetric absorptive microsampling (VAMS) devices. To test this, blood was collected from 4 healthy participants, spiked with recombinant cytokines, and collected into 30 µL VAMS devices. These prepared VAMS devices were stored at room temperature, 4 °C, or −20 °C for up to 5 months and matching VAMS liquid extracts were stored at −80 °C for the same period of time. At each timepoint, the samples were extracted from the VAMS devices and the extracts were analysed by Luminex® for quantification of up to 31 cytokines. These methods were also tested in a remote clinical study over a period of up to 8 months. Cytokine analysis revealed that room temperature, the current standard for DBS and VAMS storage, performed the poorest out of all storage temperatures with significant losses in 13/21 analytes compared to 4 °C at 5 months. Storage at 4 °C or colder performed well for the majority of analytes tested, however out of those, the optimal storage temperature differed for each analyte. There were a small number of analytes that performed poorly regardless of storage conditions and for fractalkine, this was found to be caused by inefficient recovery during extraction. Cytokine concentrations from finger-prick samples were also found to be much more variable that those in venous blood samples. Our results highlight the need to understand the stability of analytes of interest before committing to longitudinal collection and storage of samples in VAMS devices. These data give confidence that storage at 4 °C or colder was beneficial for cytokine stability. Wherein 25/31 cytokines were quantifiably stable at −20 °C when stored for 3 months and 17/21 were quantifiably stable after 5 months when stored at 4 °C. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Keratinocyte migration from the wound edge is a crucial step in the reepithelization of cutaneous wounds. Growth factors and cytokines, released from cells that invade the wound matrix, play an important role, and several in vitro assays have been performed to elucidate this. The purposes of this study were to review in vitro human studies on keratinocyte migration to identify those growth factors or cytokines that stimulate keratinocyte migration and whether these assays might serve as a screening procedure prior to testing combinations of growth factors or cytokines to promote wound closure in vivo. Methods: Research papers investigating effect of growth factors and cytokines on human keratinocyte migration in vitro were retrieved from library sources, PubMed databases, reference lists of papers, and searches of relevant journals. Results: Fourteen different growth factors and cytokines enhanced migration in scratch wound assay and HGF together with TGF-β, and IGF-1 with EGF, were more stimulatory than either growth factor alone. HGF with TGF-β1 had a greater chemokinetic effect than either growth factor alone in transmigration assay. TGF-β1, FGF-7, FGF-2 and AGF were chemotactic to keratinocytes. EGF, TGF-α, IL-1α, IGF and MGSA enhanced cell migration on ECM proteins. Conclusion: Many growth factors and cytokines enhanced migration of keratinocytes in vitro, and certain combinations of growth factors were more stimulatory than either alone. These and other combinations that stimulate keratinocyte migration in vitro should be tested for effect on wound closure and repair in vivo. The scratch wound assay provides a useful, inexpensive and easy-to-perform screening method for testing individual or combinations of growth factors or cytokines, or growth factors combined with other modalities such as laser irradiation, prior to performing wound healing studies with laboratory animals. [Copyright &y& Elsevier]

Abstract: Objective: The present paper investigated possible correlations between the clinical presentation of hepatitis B and the TNF-α −308G/A, IFN-γ +874A/T, TGF-beta1 −509C/T, and IL-10 −1081A/G polymorphisms and associated serum levels of these cytokines. Methods: Fifty-three hepatitis patients were selected and divided into two groups: A – inactive (n =30) and B – chronic hepatitis/cirrhosis (n =23). The control group consisted of 100 subjects who were positive for anti-HBc and anti-HBs. The serum concentrations of the cytokines were determined by immunoenzymatic assays. The polymorphisms of the cytokines genes were assessed by PCR and PCR-SSP. Results: The mean serum levels of IFN-γ of the control group were significantly higher than those of groups A and B, whereas the mean levels TGF-beta1 were significantly higher in groups A and B in comparison with the control. In the case of IL-10, the mean serum level recorded in the control group was significantly higher than that of group B. The TNF-α −308AG genotype was considerably more frequent in group B (43.3%) than the control (14.4%). Conclusion: Higher serum levels of IFN-γ and TGF-beta1 were associated with chronic hepatitis B, and lower serum levels of IL-10 were found in patients with the active disease. Furthermore the presence of allele A of the TNF-α −308 polymorphism suggest a risk of the progressive disease. [Copyright &y& Elsevier]

Abstract: This paper investigates serum levels of interleukin 10 (IL-10) and interleukin 6 (IL-6) in patients with chronic hepatitis C genotype 1 (CHC-GT1), the relation of each with clinical and virological characteristics, how they affect the response to combined therapy and their relation with the IL28B polymorphisms rs12979860. Serum level expression and the polymorphism of IL-10, IL-6 and IL28B were determined in 138 CHC-GT1 patients, treated with pegylated interferon/ribavirin (pegIFN-α/RBV) for 48weeks, in the following samples: baseline, week-12 (during treatment) and week-72 (post-treatment). 77 patients (56%) presented Sustained Virological Response (SVR) and 61 (44%) were non-SVR. Multivariate logistic regression showed that age⩽40years (aOR=3.7, 95%CI=1.5–8.9, P =0.004), low activity of gamma glutamyl transferase (GGT) (aOR=0.9, 95%CI=0.98–0.99, P =0.028), CC genotype of IL28B polymorphim (aOR=2.7, 95%CI=1.0–7.2, P =0.044) and low IL-6 (aOR=0.5, 95%CI=0.3–1.0, P =0.038) were predictor factors of virological response. In all patients, following treatment, IL-6 decreased at week-12 (P =0.004) from baseline and had returned to basal values at week-72. Serum IL-10 concentration was significantly decreased at week-72 only in SVR patients (P ⩽0.001). When patients were stratified by IL28B polymorphisms rs12979860 CC vs non-CC patients, a statistically significant decrease in IL-10 at week-72 in both groups was observed (P =0.003 and P ⩽0.001, respectively). None of the polymorphisms of IL-10 or IL-6 studied were associated with SVR. Conclusions: CC genotype of IL28B and low IL-6 serum concentration are factors associated independently with SVR. Moreover, decreased IL-10 at week-72 is associated with SVR in both CC and non-CC patients, and both factors are important to determine the effectiveness of treatment. [Copyright &y& Elsevier]

Abstract: Aotus and Saimiri are non-human primate models recommended by the World Health Organization for experimental studies in malaria, especially for vaccine pre-clinical trials. However, research using these primates is hindered by the lack of specific reagents to evaluate immune responses to infection or vaccination. As a step toward developing molecular tools for cytokine expression studies in these species, primer pairs for 18 cytokine gene fragments were designed based on human DNA sequences and used to amplify the corresponding genes in Aotus infulatus and Saimiri sciureus genomic DNA samples. IFNγ, TNFα, LTA, IL2, IL3, IL4, IL5, IL6, IL10, IL12, IL13, CSF2 and TGFβ2 gene fragments were amplified and sequenced. Primer pairs for IL8, IL17, IL18, IL27 and MIF failed to generate amplification products. When compared to the available corresponding human and non-human primate sequences, most – except IL3 and IL4 – showed identity degrees above 90%. Small variations in sequence can help to explain the failure to amplify certain genes or the amplification only at lower annealing temperatures as compared to human DNA samples for several primer pairs. The sequences made available provide the basis for designing molecular tools such as primers for real time PCR specific for A. infulatus and/or S. sciureus. The nucleotide sequences reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned accession numbers DQ985386 to DQ985389, DQ989356 to DQ989369, FJ89020 to FJ89024 and FJ89029. [Copyright &y& Elsevier]

Abstract: Few papers have investigated the cytokine profiles of multiple cytokines in cord blood. We obtained cord blood samples from 224 infants admitted to our neonatal intensive care unit. Cytokine profiles of 17 cytokines were investigated using cytometric bead array technology. We found a wide variety of cytokines of various levels which ranged from 0.59pg/ml (in Interleukin (IL)-4) to 222.0pg/ml (in macrophage inflammatory protein-1β. Pro-inflammatory cytokines were highly correlated with each other and with granulocyte-colony stimulating factor and IL-8. On the contrary, IL-5, IL-13, and IL-17 did not show any significant correlation with other cytokines. Several maternal factors were strongly related to several cytokines in cord blood. IL-6, IL-8 and monocyte chemotactic protein-1 were closely related to certain neonatal diseases in preterm neonates. Some cytokines may be regulated independently of each other, while others appear to work as a network affecting physiological and pathological conditions in the fetus. [Copyright &y& Elsevier]

Abstract: Aim: A growing number of mutations mapped in the receptor gene for fibroblast growth factor have been implicated in several cranial development disorders including the Apert and Crouzon syndromes. The present paper investigated cellular mechanisms underlying Apert phenotype, by analyzing the effects of FGF2 in primary cultures of Apert periosteal fibroblasts carrying the FGFR2 Pro253Arg mutation. Results: FGF2 administration significantly decreased extracellular matrix production in mutant cells by stimulating degradative enzymatic activities. Gene expression analysis revealed that decorin and biglycan, two proteoglycans involved in collagen fibrillogenesis, were more expressed in mutant cells and down-regulated by FGF2. FGF2 receptor binding showed little differences in high affinity receptor counts between mutant and wild-type cells, while we showed for the first time that low affinity receptors are significantly fewer in mutant cells. Differences were found in Crouzon syndrome, where both high and low affinity receptor counts were up-regulated. Conclusions: The different mutation and low affinity receptor regulation in mutant receptors support the hypothesis that the impact on the activity of the ligand–receptor complex could allow distinct modes of FGF2 activation in Apert and Crouzon syndromes, which interfere with the FGFR2 signalling cascade. [Copyright &y& Elsevier]

Abstract: Monocyte chemoattractant protein-1 (MCP-1) is a chemokine whose circulating levels have been detected in the lesions of several diseases such as pulmonary fibrosis, rheumatoid arthritis and atherosclerosis. However, the factors involved in the regulation of its production remain largely unknown. The main aim of the present paper was to ascertain the contribution of the familial/genetic factors on the production of MCP-1 in apparently healthy individuals. We also tested the possible relationships between the plasma levels of MCP-1 and other cytokines involved in bone metabolism (receptor activator NF-kB ligand (RANKL), osteoprotegerin (OPG), interleukin-6, macrophage-colony stimulating factor, tumor necrosis factor-α). Using ELISA assays the cytokine levels were measured in 570 apparently healthy individuals belonging to ethnically homogeneous Caucasian families. We found that MCP-1 levels were significantly (P <0.01) correlated with RANKL (in both sexes) and with OPG only in women. The study showed that adjusted for potential covariates, 72% of the MCP-1 variance, was attributable to familial effects. About 49% was due to potential genetic factors and the rest was explained by common environmental sources shared by spouses within each family. In conclusion, our data provide reliable evidence for the substantial role of genetic factors in the determination of the phenotypic variability of MCP-1 plasma levels. The association between the osteoclastogenic cytokines and MCP-1 levels in healthy pedigrees is of special interest and might shed light on MCP-1 involvement in bone remodeling. [Copyright &y& Elsevier]

Abstract: Interleukin-12 (IL-12) is a heterodimeric cytokine produced by macrophages in response to intracellular pathogens. The importance of IL-12 in generation of Th1 response against human pathogens has been characterized. The coactivator p300 is an important histone acetyltransferase (HAT) and has been implicated in the regulation of many genes. Histone deacetylases (HDACs) regulate gene transcription through deacetylation of histones. Whether the reversible histone acetylation/deacetylation modification participates in the regulation of IL-12 p40 transcription expression has not been investigated before. In this study, we analyzed the roles of HDAC1 and p300 in the regulation of human IL-12 p40. Co-transfection studies showed that HDAC1 had a repressing effect on the activity of IL-12 p40 promoter. Contrarily, p300 was able to reinforce the C/EBPβ-mediated activation of IL-12 p40 and it counteracted the HDAC1-mediated repression of the IL-12 promoter. Chromatin immunoprecipitation tests (ChIP) revealed that p300 had a stimulating effect on the acetylation of the histone H3 at IL-12 p40 promoter. In addition, we showed that p300 had a physical interaction with C/EBPβ and can enhance acetylation of C/EBPβ. Data presented in this paper indicate that the reversible histone acetylation/deacetylation modification plays an important role in the transcriptional regulation of IL-12. [Copyright &y& Elsevier]

Abstract: Several proinflammatory cytokines can induce periodontal tissue destruction and are thought to be useful indicators or diagnostic markers for periodontitis. Here, we aimed to investigate whether oncostatin M (OSM) was present in gingival crevicular fluid (GCF) and to clarify the correlation of GCF OSM and interleukin-6 (IL-6) levels with the severity of periodontitis. Sixty-two sites in 14 patients were divided into 4 groups based on probing depth (PD) and bleeding on probing (BOP). GCF was collected using paper strips from clinically health sites (PD≤3mm, CAL: 1–3mm, without BOP, n =31), mildly diseased sites (PD≤3mm, CAL: 3–5mm, with BOP, n =11), moderately diseased sites (PD=4–6mm, CAL: 5–8mm, with BOP, n =11), and severely diseased sites (PD>6mm, CAL: 8–12mm, with BOP, n =9). IL-6 and OSM in GCF were quantified by enzyme-linked immunosorbent assay and are expressed as concentrations (pg/ml) and total amounts (pg/site). Correlations of OSM and IL-6 levels with the severity of periodontitis in all groups were determined using Spearman rank correlation (r s). Our results showed that OSM and IL-6 were detected in most GCF samples. The total amounts of OSM and IL-6 were significantly positive correlated with severity of diseased sites (OSM: r s =0.526, p &#60;0.01; IL-6: r s =0.729, p &#60;0.01). No correlations of OSM or IL-6 concentration in GCF were found with disease severity. OSM and IL-6 levels in GCF were positively correlated to each other when expressed as either concentrations or total amounts (concentrations: r =0.485, p &#60;0.01; total amounts r =0.490, p &#60;0.01). In conclusion, our findings suggest that IL-6 and OSM may play a role in modulating the inflammatory cascade of chronic periodontitis. [Copyright &y& Elsevier]

• IFN-α-2b decreases lipid levels in hepatocytes from rats with hepatic preneoplasia. • IFN-α-2b induces redistribution of cholesterol in the different cellular organelles. • IFN-α-2b treatment affects HMGCR gene and protein expressions and activity. • IFN-α-2b-induced action on HMGCR impacts on de novo synthesis of cholesterol. IFN-α administration to patients has been long discouraged and pushed back by new and apparently better drugs; however the adverse secondary effect, the high costs and the lack of specific action, make these new drugs hard to be used and put IFN-α again in the eye of the researchers. IFN-α-2b was demonstrated to induce apoptosis and modulation of lipid metabolism and the mechanisms are still unknown. Here, we sought to find the link between these features using a model of early stage cancer development. Using in vitro and in vivo approaches, we evaluated apoptosis and lipid metabolism. IFN-α-2b induced changes in hepatic cholesterol mass due to decreased synthesis and increased secretion. Interestingly, the drop in cellular cholesterol levels was necessary for IFN-α-2b to induce apoptosis. Results presented in this paper show the complexity of the action of IFN-α-2b on the early stages of liver cancer development. We show for the first time an interrelationship between cholesterol, apoptosis and IFN-α-2b. This makes clear the need for a reevaluation of IFN-α-2b action in order to develop softer, safer and more bearable derivatives. In this regard, knowing the molecular mechanisms by which IFN-α exerts its cellular actions is of crucial importance, and it is the main condition for therapy success for classical and new malignancies. [ABSTRACT FROM AUTHOR]

PML, also known as TRIM19, belongs to the family of tripartite motif (TRIM) proteins. Like some other TRIM proteins, PML exerts a broad antiviral activity. PML is mainly expressed in the nucleus, where it forms dynamic structures known as PML nuclear bodies that recruit many proteins, such as Sp100 and Daxx. It was reported that HIV-1 infection induces the translocation of PML from the nucleus to the cytoplasm (Turelli et al., 2001). This paper also suggested that PML could interfere with HIV-1 infection but this was later contradicted (Berthoux et al., 2003). We sought to re-evaluate the implication of PML during HIV-1 infection. We show that infection by HIV-1 but also other retroviruses triggers the formation of PML cytoplasmic bodies, as early as 30 min post-infection. PML re-localization was blocked by reverse-transcription inhibitors and was not observed following infection with unrelated viruses, suggesting it is specifically triggered by retroviral reverse-transcription. Furthermore, we show that PML interferes with an early step of retroviral infection since PML knockdown dramatically increases RT efficiency. However, the effect of PML on retroviral infection was found to be indirect and to operate through the stabilization of one of its well-characterized partners, Daxx. In the presence of PML, Daxx inhibits an early step of reverse-transcription thereby interfering with HIV-1 infection. Like PML, Daxx is an interferon-inducible protein that provides intrinsic immunity against various viruses. Our study unravels for the first time its capacity to inhibit an early step of HIV-1 replication. [ABSTRACT FROM AUTHOR]