•Cytokines (CKs) level should be measured immediately after the serum sample collection.•Serum samples storage for 24 h resulted in a decrease of 7 out of 8 tested CKs level.•Up to 2 freeze-thaw ...cycles mainly did not affect serum CKs level.•Six following freeze-thaw cycles affected the stability of all tested CKs.•Storage at −80 °C between freeze-thaw cycles is superior to -20 °C.
Cytokines (CKs) are one of the key components of the molecular network modulating multiple immunological interactions. Within such biological systems, CKs functions are associated with several processes, thus quantification of these analytes in serum samples, as well as a faithful determination of its concentration, are crucial for the translational aspect of many studies.
This study is focused on the evaluation of the effects of storage duration and multiple freeze-thaw cycles on CKs stability.
Serum samples were obtained from 24 healthy participants. Samples were prospectively stored at 4 °C for 1–7 and 30 days, and also underwent multiple freeze-thaw cycles. Afterwards, CK levels were determined by enzyme-linked immunosorbent assay.
Among the 8 examined CKs all of them showed significant degradation (determined with the two-way ANOVA and post-hoc test) after 4 days of sample storage at 4 °C. Serum were affected by freezing at −20 °C and thawing, and 2 of CKs (IL-1β and IL-8) showed significant concentration decrease after following 2 freeze-thaw cycles. It has been also determineded that CKs in serum samples after multiple freeze-thaw cycles had better stability, when samples were stored at −80 °C (compared to storage at −20 °C).
This study demonstrates that long storage at 4 °C, as well as multiple freeze-thaw cycles of serum samples, must be avoided and CK concentrations should be measured immediately after sample collection.
Because accumulation of α-synuclein (αS) in the brain is a hallmark of Parkinson disease (PD) and related disorders, we examined its occurrence in human cerebrospinal fluid (CSF). Following affinity ...enrichment and trypsin digestion of CSF collected from a neurologically healthy donor, we identified several αS-derived peptides by mass spectrometry. The concentration of αS amounted to <0.001% of the CSF proteome. We then built, validated and optimized a sandwich-type, enzyme-linked immunoadsorbent assay (ELISA) to measure total αS levels in unconcentrated CSF. In a cross-sectional study of 100 living donors, we examined cell-free CSF samples from subjects clinically diagnosed with advanced PD, dementia with Lewy bodies (DLB), Alzheimer disease (AD), and a group of non-neurodegenerative disease controls (NCO). In these four groups the CSF αS concentrations ranged from 0.8 to 16.2 pg/μl. Mean CSF αS values were lower in donors with a primary synucleinopathy (PD, DLB: n=57) than in the other two groups (AD, NCO: n=35; p=0.025). By contrast, living Creutzfeldt–Jakob disease patients showed markedly elevated CSF αS levels (n=8; mean, 300 pg/μl; p<0.001). Our results unequivocally confirm the presence of αS in adult human CSF. In a first feasibility study employing a novel ELISA, we found relatively low CSF αS concentrations in subjects with parkinsonism linked to synucleinopathy, PD and DLB. In definite prion disease cases, we recorded a marked rise in total CSF αS resulting from rapid cell death. Our results will likely aid future biomarker explorations in neurodegenerative conditions and facilitate target validation studies.
Obsessive-compulsive disorder (OCD) is a highly prevalent neuropsychiatric disorder. Recently, there has been a growing interest in investigating the association between pro-inflammatory cytokines ...and the pathogenesis of OCD. However, studies targeting interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) in OCD are limited. Therefore, the present study aimed to explore the potential role of pro-inflammatory cytokines IL-1beta and IL-6 in the pathophysiology and development of OCD. This study recruited 58 OCD patients and 30 age-sex-matched healthy controls (HCs). A qualified psychiatrist diagnosed OCD patients and assessed HCs based on the Diagnostic and Statistical Manual for Mental Health Disorders, 5th edition (DSM-5) criteria. We measured the severity of OCD using the Yale-Brown Obsessive Compulsive Scale (Y-BOCS). Serum IL-1beta and IL-6 levels were measured using ELISA kits following the appropriate methods. The results showed that serum IL-1beta levels were significantly elevated in OCD patients compared to HCs (23.68±1.65 pg/ml vs. 15.75±1.02 pg/ml; p = 0.002). Similarly, OCD patients exhibited significantly higher serum IL-6 levels than HCs (44.97±0.73 pg/ml vs. 37.04±0.35 pg/ml; p<0.001). We observed both cytokines were positively correlated with the Y-BOCS scores in OCD patients (IL-1beta: r = 0.380, p = 0.015; IL-6: r = 0.324, p = 0.026) which indicates their role in disease pathophysiology. These results suggest that serum IL-1beta and IL-6 levels may be associated with the pathophysiology of OCD. Also, these cytokines levels in blood samples can serve as early risk assessment tools for the development of OCD. We recommend further studies in a large and homogeneous population to support these findings.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Photoprotective properties of 1,25-dihydroxyvitamin Dsub.3 (1,25(OH)sub.2Dsub.3) to reduce UV-induced DNA damage have been established in several studies. UV-induced DNA damage in skin such as single ...or double strand breaks is known to initiate several cellular mechanisms including activation of poly(ADP-ribose) (pADPr) polymerase-1 (PARP-1). DNA damage from UV also increases extracellular signal-related kinase (ERK) phosphorylation, which further increases PARP activity. PARP-1 functions by using cellular nicotinamide adenine dinucleotide (NAD+) to synthesise pADPr moieties and attach these to target proteins involved in DNA repair. Excessive PARP-1 activation following cellular stress such as UV irradiation may result in excessive levels of cellular pADPr. This can also have deleterious effects on cellular energy levels due to depletion of NAD+ to suboptimal levels. Since our previous work indicated that 1,25(OH)sub.2Dsub.3 reduced UV-induced DNA damage in part through increased repair via increased energy availability, the current study investigated the effect of 1,25(OH)sub.2Dsub.3 on UV-induced PARP-1 activity using a novel whole-cell enzyme- linked immunosorbent assay (ELISA) which quantified levels of the enzymatic product of PARP-1, pADPr. This whole cell assay used around 5000 cells per replicate measurement, which represents a 200–400-fold decrease in cell requirement compared to current commercial assays that measure in vitro pADPr levels. Using our assay, we observed that UV exposure significantly increased pADPr levels in human keratinocytes, while 1,25(OH)sub.2Dsub.3 significantly reduced levels of UV-induced pADPr in primary human keratinocytes to a similar extent as a known PARP-1 inhibitor, 3-aminobenzamide (3AB). Further, both 1,25(OH)sub.2Dsub.3 and 3AB as well as a peptide inhibitor of ERK-phosphorylation significantly reduced DNA damage in UV-exposed keratinocytes. The current findings support the proposal that reduction in pADPr levels may be critical for the function of 1,25(OH)sub.2Dsub.3 in skin to reduce UV-induced DNA damage.
The control of brucellosis across sub-Saharan Africa is hampered by the lack of standardized testing and the use of tests with poor performance. This study evaluated the performance and costs of ...serological assays for human brucellosis in a pastoralist community in northern Tanzania. Serum collected from 218 febrile hospital patients was used to evaluate the performance of seven index tests, selected based on international recommendation or current use. We evaluated the Rose Bengal test (RBT) using two protocols, four commercial agglutination tests and a competitive enzyme-linked immunosorbent assay (cELISA). The sensitivity, specificity, positive predictive value, negative predictive value, Youden's index, diagnostic accuracy, and per-sample cost of each index test were estimated. The diagnostic accuracy estimates ranged from 95.9 to 97.7% for the RBT, 55.0 to 72.0% for the commercial plate tests, and 89.4% for the cELISA. The per-sample cost range was $0.69-$0.79 for the RBT, $1.03-$1.14 for the commercial plate tests, and $2.51 for the cELISA. The widely used commercial plate tests performed poorly and cost more than the RBT. These findings provide evidence for the public health value of discontinuing the use of commercial agglutination tests for human brucellosis in Tanzania.
The development of neutralizing antibodies (NAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) following infection or vaccination is likely to be critical for the development ...of sufficient population immunity to drive cessation of the coronavirus disease of 2019 (COVID-19) pandemic. A large number of serologic tests, platforms, and methodologies are being employed to determine seroprevalence in populations to select convalescent plasma samples for therapeutic trials and to guide policies about reopening. However, the tests have substantial variations in sensitivity and specificity, and their ability to quantitatively predict levels of NAbs is unknown. We collected 370 unique donors enrolled in the New York Blood Center Convalescent Plasma Program between April and May of 2020. We measured levels of antibodies in convalescent plasma samples using commercially available SARS-CoV-2 detection tests and in-house enzyme-linked immunosorbent assays (ELISAs) and correlated serological measurements with NAb activity measured using pseudotyped virus particles, which offer the most informative assessment of antiviral activity of patient sera against viral infection. Our data show that a large proportion of convalescent plasma samples have modest antibody levels and that commercially available tests have various degrees of accuracy in predicting NAb activity. We found that the Ortho anti-SARS-CoV-2 total Ig and IgG high-throughput serological assays (HTSAs) and the Abbott SARS-CoV-2 IgG assay quantify levels of antibodies that strongly correlate with the results of NAb assays and are consistent with gold standard ELISA results. These findings provide immediate clinical relevance to serology results that can be equated to NAb activity and could serve as a valuable roadmap to guide the choice and interpretation of serological tests for SARS-CoV-2.
The efficacy of omalizumab (anti-IgE) and increased IgE levels in patients with chronic spontaneous urticaria (CSU) suggest autoallergic mechanisms.
We sought to identify autoallergic targets of IgE ...in patients with CSU.
Serum samples of patients with CSU together with those of patients with idiopathic anaphylaxis and healthy control subjects (7 of each) were screened for IgE autoantibodies by using an array of more than 9000 proteins. Sera of 1062 patients with CSU and 482 healthy control subjects were used in an IgE-anti–IL-24–specific ELISA to investigate the association of IgE-anti-IL-24 and CSU.
By using array analyses, more than 200 IgE autoantigens were found in patients with CSU that were not found in control subjects. Of the 31 IgE autoantigens detected in more than 70% of patients, 8 were soluble or membrane bound and expressed in the skin. Of these, only IgE autoantibodies to IL-24 were found in all patients with CSU. In vitro studies showed IL-24 to release histamine from human mast cells sensitized with purified IgE of patients with CSU but not control subjects. By using ELISA, mean ± SD levels of IgE-anti–IL-24 were 0.52 ± 0.24 IU/mL in patients with CSU and 0.27 ± 0.08 IU/mL in control subjects, with 80% of patients with CSU but only 20% of control subjects having levels greater than 0.33 IU/mL (P < .0001). IgE-anti–IL-24 showed acceptable predictive properties for CSU, with a likelihood ratio of 3.9. Clinically, IgE-anti–IL-24 levels showed an association with disease activity, as assessed by the urticaria activity score and with reduced basophil counts.
Our findings show that patients with CSU frequently exhibit IgE autoantibodies against many autoantigens and that IL-24 is a common, specific, and functional autoantigen of IgE antibodies in patients with CSU.
The present study aims to investigate the specific protective effects and underlying mechanisms of Ganshuang granule (GSG) on dimethylnitrosamine (DMN)-induced hepatic fibrosis in rat models. Hepatic ...fibrosis was experimentally evoked in rats by DMN administration, and varying dosages of GSG were employed as an intervention. Hepatocellular damage was assessed by measuring serum levels of aminotransferase and bilirubin, accompanied by histopathological examinations of hepatic tissue. The hepatic concentrations of platelet-derived growth factor (PDGF) and transforming growth factor-beta1 (TGF-beta1) were quantitated via enzyme-linked immunosorbent assay (ELISA). The expression of alpha-smooth muscle actin (alpha-SMA) within hepatic tissue was evaluated using immunohistochemical techniques. The levels of hepatic interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and a spectrum of interleukins (IL-2, IL-4, IL-6, IL-10) were quantified by quantitative real-time PCR (qRT-PCR). Additionally, hepatic stellate cells (HSCs) were cultured in vitro and exposed to TNF-alpha in the presence of naringin, a principal component of GSG. The gene expression levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metallopeptidase-1 (MMP-1) in these cells were also quantified by qRT-PCR. Proliferative activity of HSCs was evaluated by the Cell Counting Kit-8 assay. Finally, alterations in Smad protein expression were analyzed through Western blotting. Administration of GSG in rats with fibrosis resulted in reduced levels of serum aminotransferases and bilirubin, along with alleviation of histopathological liver injury. Furthermore, the fibrosis rats treated with GSG exhibited significant downregulation of hepatic TGF-beta1, PDGF, and TNF-alpha levels. Additionally, GSG treatment led to increased mRNA levels of IFN-gamma, IL-2, and IL-4, as well as decreased expression of alpha-SMA in the liver. Furthermore, treatment with naringin, a pivotal extract of GSG, resulted in elevated expression of MMP-1 and decreased levels of TIMP-1 in TNF-alpha-stimulated HSCs when compared to the control group. Additionally, naringin administration led to a reduction in Smad expression within the HSCs. GSG has the potential to mitigate fibrosis induced by DMN in rat models through the regulation of inflammatory and fibrosis factors. Notably, naringin, the primary extract of GSG, may exert a pivotal role in modulating the TGF-beta-Smad signaling pathway.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Elucidation of the chain of disease transmission and identification of the source of coronavirus disease 2019 (COVID-19) infections are crucial for effective disease containment. We describe an ...epidemiological investigation that, with use of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological assays, established links between three clusters of COVID-19.
In Singapore, active case-finding and contact tracing were undertaken for all COVID-19 cases. Diagnosis for acute disease was confirmed with RT-PCR testing. When epidemiological information suggested that people might have been nodes of disease transmission but had recovered from illness, SARS-CoV-2 IgG serology testing was used to establish past infection.
Three clusters of COVID-19, comprising 28 locally transmitted cases, were identified in Singapore; these clusters were from two churches (Church A and Church B) and a family gathering. The clusters in Church A and Church B were linked by an individual from Church A (A2), who transmitted SARS-CoV-2 infection to the primary case from Church B (F1) at a family gathering they both attended on Jan 25, 2020. All cases were confirmed by RT-PCR testing because they had active disease, except for A2, who at the time of testing had recovered from their illness and tested negative. This individual was eventually diagnosed with past infection by serological testing. ELISA assays showed an optical density of more than 1·4 for SARS-CoV-2 nucleoprotein and receptor binding domain antigens in titres up to 1/400, and viral neutralisation was noted in titres up to 1/320.
Development and application of a serological assay has helped to establish connections between COVID-19 clusters in Singapore. Serological testing can have a crucial role in identifying convalescent cases or people with milder disease who might have been missed by other surveillance methods.
National Research Foundation (Singapore), National Natural Science Foundation (China), and National Medical Research Council (Singapore).
Viral respiratory tract infections are the main causative agents of the onset of infection-induced asthma and asthma exacerbations that remain mechanistically unexplained. Here we found that ...deficiency in signaling via type I interferon receptor led to deregulated activation of group 2 innate lymphoid cells (ILC2 cells) and infection-associated type 2 immunopathology. Type I interferons directly and negatively regulated mouse and human ILC2 cells in a manner dependent on the transcriptional activator ISGF3 that led to altered cytokine production, cell proliferation and increased cell death. In addition, interferon-γ (IFN-γ) and interleukin 27 (IL-27) altered ILC2 function dependent on the transcription factor STAT1. These results demonstrate that type I and type II interferons, together with IL-27, regulate ILC2 cells to restrict type 2 immunopathology.
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