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We report the development of a distance-based paper analytical device combined with a hydrophilic bridge valve (B-dPAD) as a quantitative immunoassay method to monitor human interleukin-6 (IL-6) in human samples. Our device design features (i) a circular sample inlet zone, (ii) a circular capture zone with immobilized anti-IL-6 (anti-Ab

Atherosclerosis (AS) is the most common factor causing many cardiovascular and cerebrovascular diseases and has received considerable attention. The occurrence mechanism of AS is uncertain because it is a choronically pathological process that is influenced by multi-aspects, among which cytokines play the key roles in regulating the processes of the immune system. For example, two key cytokines, namely, IL-10 and MCP-1 (chemokine), which are involved in AS progression with varied levels, can be used for AS status monitoring and early diagnosis of AS-associated diseases. Hence, a new paper-based, surface-enhanced Raman spectroscopy (SERS) sensing platform was established for the detection of these two key cytokines. By combining a nanoporous networking membrane as the substrate and SERS nanotags as the probe for signal reading, together with a sandwich design, sensitive and specific identification and quantification of cytokine targets in human serum were achieved with excellent sensing characteristics. The lowest detectable concentration was determined to be 0.1 pg mL-1 for both IL-10 and MCP-1 in human serum. The assay also exhibits high specificity towards target cytokine detection, with low-nonspecific binding and acceptable cross-reactivity in the presence of other structurally similar targets. Finally, the practicability was validated by performing duplex detection in human serum, which further demonstrates the high specificity of the assay for the detection of target cytokines. Taken together, these promising results illustrate that this developed sensing assay is a candidate for clinical multi-target analysis in real environments.

Although the established ELISA-based sensing platforms have many benefits, the importance of cytokine and cancer biomarkers detection for point-of-care diagnostics has propelled the search for more specific, sensitive, simple, accessible, yet economical sensor. Paper-based biosensor holds promise for future in-situ applications and can provide rapid analysis and data without the need to conduct in a laboratory. Electrochemical detection plays a vital role in interpreting results obtained from qualitative assessment to quantitative determination. In this review, various factors affecting the design of an electrochemical paper-based biosensor are highlighted and discussed in depth. Different detection methods, along with the latest development in utilizing them in cytokine and cancer biomarkers detection, are reviewed. Lastly, the fabrication of portable electrochemical paper-based biosensor is ideal in deliberating positive societal implications in developing countries with limited resources and accessibility to healthcare services. Ministry of Education (MOE) Published version The authors would like to acknowledge the financial support for this work funded by Singapore Ministry of Education Academic Research Fund Tier 1 (MOE-2018-T1-001-004).

The study was focused on assessment of potential health risks of paper-based food contact materials (FCMs) in a step-wise approach using three toxicological bioassays in vitro and chemical analyses of migrating contaminants. 3T3 NRU cytotoxicity test showed high sensitivity to detect basal toxicity of FCMs extracts and served as a first-line test for selection of samples for further testing. The reconstructed human intestine model EpiIntestinal showed more realistic tissue response than cell culture monolayer and higher resistance despite prolonged exposure to the selected 6 samples, i.e. negligible decrease of viability and intestinal penetration, nevertheless an increase of IL-8 after exposure to black printed sample extract. Yeast based assays identified weak agonistic/antagonostic activity to human androgen receptor of the black printed sample. In accordance with the biological effects, the targeted LC and GC analytical methods confirmed the presence of high amounts of phthalates, photoinitiators and PAHs that could justify the hazard of the black printed sample. Heavily printed uncoated FCMs are recognized not to be suitable for direct contact with food. The selected bioassays and chemical analyses might be useful tools to detect targeted biological effects of xenobiotics suspected to contribute to human exposure from food.

Investigation of cellular phosphorylation and signaling pathway has recently gained much attention for the study of pathogenesis of cancer. Related conventional bioanalytical operations for this study including cell culture and Western blotting are time-consuming and labor-intensive. In this work, a paper-based microreactor has been developed to integrate cell culture and subsequent immunoassay on a single paper. The paper-based microreactor was a filter paper with an array of circular zones for running multiple cell cultures and subsequent immunoassays. Cancer cells were directly seeded in the circular zones without hydrogel encapsulation and cultured for 1 day. Subsequently, protein expressions including structural, functional, and phosphorylated proteins of the cells could be detected by their specific antibodies, respectively. Study of the activation level of phosphorylated Stat3 of liver cancer cells stimulated by IL-6 cytokine was demonstrated by the paper-based microreactor. This technique can highly reduce tedious bioanalytical operation and sample and reagent consumption. Also, the time required by the entire process can be shortened. This work provides a simple and rapid screening tool for the investigation of cellular phosphorylation and signaling pathway for understanding the pathogenesis of cancer. In addition, the operation of the paper-based microreactor is compatible to the molecular biological training, and therefore, it has the potential to be developed for routine protocol for various research areas in conventional bioanalytical laboratories.

Psychological stress is an important factor in susceptibility to many diseases. Our laboratory has been investigating the impact of stress on the susceptibility to Theiler’s virus-induced demyelination (TVID), a mouse model of multiple sclerosis. Using immunodominant viral peptides specific for identification of either CD4+ or CD8+ T cells, stress reduced IFN-γ-producing virus-specific CD4+ and CD8+ T cells in the spleen and CD8+ T cells in the CNS. Expression of mRNA for the Th1 transcription factor T-bet and the Th2 transcription factor GATA-3 were decreased in spleen cells isolated from stressed mice. Cytokine production by cells isolated from the CNS or spleens following stimulation with virus indicated that stress decreased both type 1 and type 2 responses. The adverse effects of stress were partially reversed by concurrent RU486 administration but mimicked by dexamethasone, indicating a major role for glucocorticoids. Global stress-induced immunosuppression resulted in higher levels of virus replication and dissemination. The higher viral load subsequently led to an earlier disease onset and more severe clinical and histological signs of demyelinating disease. Our results have important implications for understanding the pathogenesis of MS, and suggest that stressful events during early infection with an agent capable of inducing demyelination may result in immunosuppression and failure to eliminate the pathogen, which in turn may lead to the development of MS.

Objective: To investigate the relation of endothelial cell activation with delayed cerebral ischaemia (DCI) and outcome after subarachnoid haemorrhage (SAH).Methods: Concentrations of soluble (s) intercellular adhesion molecule-1, sE-selectin, sP-selectin, ED1-fibronectin, von Willebrand Factor (vWf), and vWf propeptide were measured within three days of SAH onset. The associations with poor outcome were investigated at three months in 106 patients. In 90 patients in whom the occurrence of cerebral ischaemia could be dated accurately, two analyses were undertaken: one for all ischaemic events (n = 32), including those related to treatment, and another for spontaneous DCI ( n = 11). Concentrations of markers were dichotomised at their medians. The associations of endothelial cell activation markers with outcome were expressed as odds ratios ( OR) from logistic regression and those with ischaemic events as hazard ratios (HR) derived from Cox regression.Results: Early vWf concentrations were associated with poor outcome ( crude OR = 4.6 (95% CI, 2.0 to 10.9; adjusted OR = 3.3 (1.1 to 9.8). Early levels of vWf were also positively related to occurrence of all ischaemic events ( crude HR = 2.3 ( 1.1 to 4.9); adjusted HR = 1.8 ( 0.8 to 3.9) and with occurrence of spontaneous DCI ( crude HR = 3.5 (0.9 to 13.1); adjusted HR = 2.2 (0.5 to 9.8). None of the other markers showed any associations.Conclusions: Concentrations of sICAM-1, sP-selectin, sE- selectin, and ED1-fibronectin do not predict the occurrence of DCI or outcome. The positive associations of raised early vWf concentrations with ischaemic events and poor outcome after SAH may reflect a predisposition to further ischaemic injury through formation of microthrombi in the cerebral circulation.

We examined the utility of dried blood on filter paper for the source of cytokine messenger RNA (mRNA). Total RNA was isolated from the dried blood of mice infected with Plasmodium yoelii, and cDNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR). As a reference, we extracted total RNA from peripheral blood collected at the same time as the preparation for dried blood. There was no difference in cytokine mRNA expression between the two sources; the dried blood on filter paper and the peripheral blood. Th1 cells, Th2 cells, and monocytes/macrophages derived cytokine mRNAs in the dried blood from infected mice were detected, and the increase of some of the cytokines mRNAs after infection was also observed. These results suggested that the dried blood on filter paper is satisfactory RNA source for immunological examination in field-based studies.

Background: In vitro stimulation of whole blood or isolated blood cells with specific antigens is used for several purposes. Immediately following incubation with antigens, samples have to be centrifuged to stop the reactions by remaining cells and the supernatant refrigerated or analysed directly to preserve the analytes of interest, which makes samples difficult to prepare outside laboratories. We have tested whether spotting whole blood on filter paper after activation can be used in one of the tests for Mycobacterium tuberculosis infection (MTI), the QuantiFERON®-TB Gold In Tube test (QFT), where the spotting technique can make it suitable for use in locations without facilities like a centrifuge and a refrigerator. Materials and methods: Samples from 22 individuals undergoing screening for MTI and 10 healthy controls were incubated, centrifuged and IFN-γ measured by Enzyme-linked immunosorbent assay (ELISA), as described in the kit insert. In parallel, activated blood was spotted on filter paper (Schleicher & Schuell) and dried. The dried blood spot samples were analysed for 21 inflammatory markers with an in-house assay based on Luminex technology. Results: Our multiplex measurements of inflammatory markers in samples from suspected MTI patients confirmed the IFN-γ findings in the QFT. IL-2, GM-CSF, IL-5, and IL-1β were also found as useful markers for MTI. We were not able to distinguish between active tuberculosis and latent MTI. Conclusion: Applying blood on filter paper after incubation makes in vitro stimulation tests feasible in locations where heat and electricity is unavailable.

The analysis of samplings from periodontal pockets is important in the diagnosis and therapy of periodontitis. In this study, three different sampling techniques were compared to determine whether one method yielded samples suitable for the reproducible and simultaneous determination of bacterial load, cytokines, neutrophil elastase, and arginine-specific gingipains (Rgps). Rgps are an important virulence factor of Porphyromonas gingivalis, the exact concentration of which in gingival crevicular fluid (GCF) has not been quantified.GCF was sampled from four sites per patient (one sample per quadrant using two samples per method) in 36 patients with chronic periodontitis. One week later, the procedure was repeated with alternative methods. Variables determined were loads of Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) and P. gingivalis, levels of interleukin-6 and -8, activity of neutrophil elastase, and level of Rgps.The detected cytokine levels were higher using paper strips compared to paper points. Bacteria were found in similar loads from paper strips and paper points. Rgps were only detectable in high quantities by washing the periodontal pocket. The level of Rgps correlated with the load of P. gingivalis.The use of paper strips was suitable for the simultaneous determination of microbial and immunologic parameters. Obtaining GCF by washing can be useful for special purposes. The gingipain concentration in periodontal pockets was directly determined to be ≤1.5 μM. This value indicated that most of the substrates of these proteases by in vitro assays identified until now can be easily degraded in P. gingivalis-infected sites.

Objectives: It has been suggested that autoimmunity to peripheral myelin proteins is involved in the pathogenesis of Guillain-Barre syndrome (GBS) and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). We aimed to compare reactivity of peripheral blood mononuclear cells (PBMC) to antigens of peripheral myelin proteins in patients with GBS and patients with CIDP with that of healthy controls and patients with other non-immune mediated neuropathies (ON). Methods: We prepared PBMC from blood from 83 healthy controls and from 64 patients with GBS, 54 with CIDP, and 62 with ON. PBMC were tested in antigen specific proliferation assays against peptides from myelin proteins P0, P2, PMP22, and myelin basic protein (MBP), which is identical to myelin P1, and against whole human MBP. Interferon-gamma (IFN-γ) and interleukin (IL)-5 enzyme linked immunospot (ELISPOT) assays were also performed in some subjects to assess spontaneous and peripheral myelin antigen specific PBMC cytokine secretion. Results: Antigen specific PBMC proliferation assays showed no significant elevation of peptide specific T cell responsiveness in patients with GBS or CIDP compared with healthy controls or patients with ON. Levels of spontaneous ELISPOT IFN-γ secretion were increased in patients with GBS and significantly increased in those with CIDP compared with healthy controls and patients with ON. No convincing differences in antigen specific ELISPOT IFN-γ secretion levels to individual peptides were detectable in patients with GBS. The proportion of patients with CIDP with an increased number of PBMC producing IFN-γ in response to peptide PMP-22 51–64 was significantly increased compared with healthy controls and patients with ON. No significant differences in antigen specific ELISPOT IL-5 secretion levels were detectable in patients with GBS or CIDP compared with controls, but levels of spontaneous IL-5 secretion were significantly higher in patients with CIDP than in healthy controls or patients with ON. Conclusions: Although the lack of significantly increased antigen specific PBMC proliferation in GBS and CIDP does not support a role for T cells, the more sensitive ELISPOT technique detected increased numbers of PBMC secreting IFN-γ spontaneously in 25% of patients with GBS, providing further evidence for a role of T cells in the immunopathology of GBS. Increased numbers of spontaneous IFN-γ and IL-5 secreting cells, and increased IFN-γ secretion in response to PMP-22 51–64 , in patients with CIDP provide further evidence for a role of myelin specific T cells in CIDP.

In normal conditions, human gut mucosa is infiltrated with a large number of mononuclear cells. This is a reflection of the fact that human intestine is continuously subjected to a massive stimulation by luminal antigens. This state of "physiological" inflammation is a tightly controlled phenomenon, as several mucosal cells interact to generate and maintain an appropriate local immune response. Changes in cell type number and/or function, including the release of soluble mediators, have been associated with the development of chronic inflammatory diseases, such as Crohn's disease (CD) and ulcerative colitis (UC), the two major forms of inflammatory bowel disease. Evidence also indicates that the type of inflammatory response occurring in the intestine of patients with CD differs from that in UC, and this probably reflects distinct pathways of immune activation. In CD mucosa, a Th1 response with high IL-12 and IFNgamma production prevails, while in UC a humoral immunity appears to be predominant. Despite this, CD and UC share downstream inflammatory events, characterised by high levels of inflammatory cytokines, free radicals, matrix-degrading enzymes and growth factors.

Objective: To review the clinical and pathological features of parvovirus B19 meningoencephalitis and its sequelae in 12 previously published cases, and to perform additional tests to determine the pathogenesis of the disease. Methods: Cases were reviewed and available serum and cerebrospinal fluid (CSF) tested for antiganglioside antibodies and a range of cytokines. In situ hybridisation for parvovirus B19 DNA was performed on postmortem brain tissue in two cases. HLA-DRB1 typing was undertaken on genomic DNA extracted from peripheral blood leucocytes. Results: Cerebellar involvement was suggested either clinically or pathologically in four cases. In the two cases with postmortem histology, there was marked atrophy of the molecular and granular layers of the cerebellum with focal loss of Purkinje cells. Brain scanning by MRI or CT was done in six cases during the acute phase. Three were abnormal with evidence of demyelination. Three had markedly enlarged ventricles, in two of which there was high signal intensity from the white matter on both T1 and T2 weighted images. The three cases with abnormal brain scans had long term neurological sequelae (mental retardation, personality change, altered affect). In situ hybridisation on available postmortem brain tissue was negative in the two cases tested. All cases in which HLA-DR alleles were determined carried at least one of the following alleles: HLA-DRB1*01, *04, *07, *09, *15, *16. Available serum and CSF was tested for antiganglioside antibodies (all negative) and for a panel of cytokines, which had a similar profile in both serum (n = 5) and CSF (n = 1) during the acute phase. Cytokines that were consistently detectable were IL-6 (mean 726.20 pg/ml), TNFα (50.64 pg/ml), IFNγ (39.64 pg/ml), GM-CSF (216.12 pg/ml), and MCP-1 (154.43 pg/ml); IL-1β, IL-5, and IL-13 were undetectable. Conclusions: HLA-DR associations, an increased cytokine response, and benefit from immunomodulatory treatment (in one case) support a role for the immune response in the pathogenesis of parvovirus B19 meningoencephalitis.