Treatment cessation in HBeAg-negative chronic hepatitis B: clinical response is associated with increase in specific proinflammatory cytokines.

Patients with HBeAg-negative chronic hepatitis B may experience an immune response after stopping nucleos(t)ide analogue (NA)therapy, which may potentially trigger HBsAg loss or off-therapy sustained viral control. The immunological mechanisms determining clinical response remain poorly understood. To identify inflammatory signatures associated with defined outcomes, we analysed plasma cytokines and chemokines from 57 HBeAg-negative patients enrolled in the Nuc-Stop Study at baseline and 12 weeks after NA cessation. Clinical response at 12 weeks was classified into four groups: immune control, viral relapse, evolving clinical relapse, and resolving clinical relapse. Twelve weeks after treatment cessation 17 patients (30%) experienced immune control, 19 (33%) viral relapse, 6 (11%) evolving clinical relapse, and 15 (26%) resolving clinical relapse. There was a significant increase in interferon-γ-induced protein 10 (IP-10; p = 0.012) and tumor necrosis factor (TNF; p = 0.032) in patients with evolving clinical relapse. Sparse partial least-squares multivariate analyses (sPLS-DA) showed higher first component values for the clinical relapse group compared to the other groups, separation was driven mainly by IP-10, TNF, IL-9, IFN-γ, MIP-1ß, and IL-12. Our results demonstrate that evolving clinical relapse after NA cessation is associated with a systemic increase in the proinflammatory cytokines IP-10 and TNF.Clinical trial registration: ClinicalTrials.gov, Identifier: NCT03681132.

Cytokine Levels in Saliva Are Associated with Salivary Gland Fibrosis and Hyposalivation in Mice after Fractionated Radiotherapy of the Head and Neck.

Cytokines are mediators of inflammation that could lead to fibrosis. The aim was to monitor cytokine levels in saliva and serum after locally fractionated radiotherapy of the head and neck in mice and investigate associations with salivary gland fibrosis and hyposalivation. C57BL/6 mice were randomized to sham or X-ray irradiation of 66 Gy in 10 fractions over 5 days. Blood and saliva were collected on days -7, 5, 35, 80, and 105 following cytokine analysis. The harvested submandibular salivary gland was assessed for the presence of fibrosis. Decision tree regression analysis was used to investigate whether cytokine levels could predict late endpoints in terms of hyposalivation or fibrosis. Significant formation of fibrosis in gland tissue and reduced saliva production was found after irradiation. The pro-inflammatory cytokines IL-1α, TNF, TIMP1, G-CSF, KC, and MIP-1α showed increased levels in saliva in irradiated mice and a strong correlation with late endpoints. The decision tree analysis largely separated controls from irradiated animals, with IL-1α being the strongest predictor. Pro-inflammatory cytokines in saliva, but not in serum, were associated with late endpoints. This indicates that cytokine expression in saliva is a good biomarker for local salivary gland damage with IL-1α as the strongest single predictor.

Association of pro-inflammatory cytokines with trauma and post-traumatic stress disorder visiting a tertiary care hospital in Kathmandu.

Post-traumatic stress disorder (PTSD) is a mental disorder that can occur after trauma. Although inflammatory markers such as cytokines are found altered in trauma and PTSD, there is no consensus regarding which can be considered as biomarkers. Studies from South Asia region is also rare. We studied cytokines among trauma affected patients and matched healthy controls. Fifty patients (cases) with trauma, visiting the University hospital in Kathmandu and thirty-nine healthy controls were selected, and the levels of cytokines were determined using a Luminex IS 200. We compared the levels of the cytokines in thirty-four age and gender matched pairs of case and control among three groups: healthy volunteers, cases diagnosed as PTSD, and cases without PTSD. Among the 34 pair-matched cases and controls, IL-6 was significantly higher in both PTSD positive cases [2.43 (0.00-14.54) pg/ml; p = 0.004] and PTSD negative cases [3.00 (0.92-3.86) pg/ml; p = 0.005], than in controls [0.39 (0.00-11.38) pg/ml]. IL-1ß was significantly higher in PTSD positive cases [0.17 (0.00-5.27) pg/ml; p = 0.011] than in controls 0.00 (0.00-0.12) pg/ml. Other cytokines did not show significant differences. IL-6 was higher in both the trauma affected groups and IL-1ß was higher in the trauma affected group with PTSD when compared to healthy controls. This supports the immune system activation hypothesis after trauma.

A Cease in Shift Work Reverses Arterial Stiffness but Increases Weight and Glycosylated Hemoglobin A 5-Month Follow-Up in Industry.

BACKGROUND: Literature suggests an association between shift work and cardiovascular disease (CVD). Limited evidence is available on how a cessation of shift work affects CVD risk factors. AIM: We investigated whether a five-month plant shutdown affected CVD risk factors in 30 industrial shift workers. METHODS: We collected demographic data, self-reported data on physical activity (PA) and medical history by questionnaire. Pre- and post-plant shutdown, we measured blood pressure (BP), heart rate, lipids, glycosylated hemoglobin (HbA1c) and C-reactive protein (CRP). Additionally, we collected markers of inflammation, Matrix metalloproteinase-9 (MMP-9), Interleukin-6 (IL-6), Monocyte chemoattractant protein-1 (MCP-1), Tumor necrosis factor-alpha (TNF-α), P-selectin, Interleukin-1 beta (IL-1ß), and Interleukin-23 (IL-23). We also examined arterial stiffness (central blood pressure, augmentation pressure, and pulse wave velocity) by means of SphygmoCor® (AtCor Medical Pty Ltd., Sydney, Australia). We monitored sleep by actigraphy prior to and after plant shutdown, with additional registration of sleep quality and assessment of insomnia symptoms. RESULTS: After five months of plant shutdown, we found that HbA1c increased by 1.9 mmol/mol, weight by 1 kg and MCP-1 by 27.3 pg/mL, all unexpectedly. The other markers of inflammation did not change during shutdown, but CRP decreased close to significant levels. There were no changes in lipids during follow-up. Pulse-wave velocity (PWV) was reduced from 8.1 m/s (SD = 1.5) to 7.6 m/s (SD = 1.5), p = 0.03. The workers reported fewer signs of insomnia after shutdown. CONCLUSIONS: Our findings suggest that a five-month cessation in shift work increases weight and HbA1c, but also improves insomnia symptoms and reverses arterial stiffening.

Serum Amyloidogenic Nanoplaques and Cytokines in Alzheimer's Disease: Pilot Study in a Small Naturalistic Memory Clinic Cohort.

BACKGROUND: Neuroinflammation is a central component of Alzheimer's disease (AD) and correlates closely with amyloid pathology. Markers of inflammation such as cytokines, and amyloidogenic aggregates, so-called nanoplaques, are both promising biomarker candidates for AD. We have previously shown that there is a relationship between the levels of nanoplaques and cytokines in cerebrospinal fluid, but it is unknown whether this association extends to serum. OBJECTIVE: Investigate in a naturalistic memory clinic cohort whether the associations between nanoplaques and cytokines in the cerebrospinal fluid extends to serum. METHODS: We collected serum from 49 patients assessed for cognitive complaints at the Oslo University Hospital Memory Clinic (15 with clinical AD). We assessed the levels of serum nanoplaques with the novel Thioflavin-T fluorescence correlation spectroscopy (ThT-FCS) assay. Serum levels of nine cytokines (eotaxin-1, granulocyte colony-stimulating factor [G-CSF], interleukin [IL]-6, IL-7, IL-8, monocyte chemoattractant protein-1 (MCP-1), gamma induced protein 10 (IP-10), macrophage inflammatory protein [MIP]-1α, and MIP-1ß) were quantified with a multiplex assay and read on a Luminex IS 200 instrument. RESULTS: Serum nanoplaques were not increased in clinical AD patients compared to non-AD memory clinic patients and nanoplaques were not associated with any cytokines. The cytokines IL-8 and G-CSF were increased in patients with clinical AD compared to non-AD patients. CONCLUSION: In this small pilot study, serum nanoplaques were not associated with serum cytokines. Nanoplaque levels could not be used to separate clinical AD patients from non-AD patients in this unselected memory clinic cohort.

Macrophage migration inhibitory factor: a potential biomarker for chronic low back pain in patients with Modic changes.

BACKGROUND: Low back pain (LBP) is a leading cause of disability worldwide, but the aetiology remains poorly understood. Finding relevant biomarkers may lead to better understanding of disease mechanisms. Patients with vertebral endplate bone marrow lesions visualised on MRI as Modic changes (MCs) have been proposed as a distinct LBP phenotype, and inflammatory mediators may be involved in the development of MCs. OBJECTIVES: To identify possible serum biomarkers for LBP in patients with MCs. METHODS: In this case control study serum levels of 40 cytokines were compared between patients with LBP and MC type 1 (n=46) or type 2 (n=37) and healthy controls (n=50). RESULTS: Analyses identified significantly higher levels of six out of 40 cytokines in the MC type 1 group (MC1), and five in the MC type 2 group (MC2) compared with healthy controls. Six cytokines were moderately correlated with pain. Principal component analyses revealed clustering and separation of patients with LBP and controls, capturing 40.8% of the total variance, with 10 cytokines contributing to the separation. Macrophage migration inhibitory factor (MIF) alone accounted for 92% of the total contribution. Further, receiver operating characteristics analysis revealed that MIF showed an acceptable ability to distinguish between patients and controls (area under the curve=0.79). CONCLUSIONS: These results suggest that cytokines may play a role in LBP with MCs. The clinical significance of the findings is unknown. MIF strongly contributed to clustering of patients with LBP with MCs and controls, and might be a biomarker for MCs. Ultimately, these results may guide future research on novel treatments for this patient group.

The Silk Protein Sericin Promotes Viability of ARPE-19 and Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelial Cells in vitro.

PURPOSE: Maintaining mature and viable retinal pigment epithelial cells (RPE) in vitro has proven challenging. Investigating compounds that can promote RPE-viability and maturation is motivated by RPE transplantation research, the quest to understand RPE physiology, and a desire to modulate RPE in pathological states. We have previously reported that the silk protein sericin promotes viability, maturation, and pigmentation of human fetal RPE. In the present study, our aim was to uncover whether these effects can be seen in adult retinal pigment epithelial cell line-19 (ARPE-19) and induced pluripotent stem cell-derived RPE (iPSC-RPE). METHODS: ARPE-19 and iPSC-RPE were cultured with or without 10 mg/mL sericin. After 7 days, viability was assessed with calcein-acetoxymethyl ester (CAM) and ethidium homodimer-1 (EH-1) assays, flow cytometry, and morphometric analysis. Expression levels of RPE65, tyrosinase, and Pmel17 were quantified to compare maturation between the sericin-treated and control cultures. Light microscopy and staining of the tight junction protein zonula occludens protein 1 (ZO-1) were employed to study sericin's effects on RPE morphology. We also measured culture medium pH, glucose, lactate, and extracellular ion content. RESULTS: Sericin-supplemented RPE cultures demonstrated significantly better viability compared to control cultures. Sericin appeared to improve ARPE-19 maturation and morphology in vitro. No effects were seen on RPE pigmentation with the concentration of sericin and duration of cell culture herein reported. CONCLUSIONS: This is the first study to demonstrate that supplementing the culture media with sericin promotes the viability of iPSC-RPE and ARPE-19. Sericin's viability-promoting effects may have important implications for retinal therapeutics and regenerative medicine research.

Cytokines Explored in Saliva and Tears from Radiated Cancer Patients Correlate with Clinical Manifestations, Influencing Important Immunoregulatory Cellular Pathways.

Although radiotherapy is a common form of treatment for head and neck cancer, it may lead to tissue damage in the salivary and lacrimal glands, possibly affecting cytokine expression in the gland fluid of treated individuals. Cytokine profiles in saliva and tear fluid of 29 radiated head and neck cancer patients and 20 controls were screened using a multiplex assay. Correlations between cytokine expression and clinical oral and ocular manifestations were examined, and cellular pathways influenced by these cytokines were assessed using the Functional Enrichment Analysis Tool. Significantly elevated cytokines identified in patient saliva were CCL21, IL-4, CX3CL1, CCL2, CXCL1 and CCL15. Many of these cytokines correlated positively with objective signs of oral dryness, and reduced saliva production in the patients. Although CCL21 and IL-4 levels were significantly lower in patient tear fluid, they correlated with subjective ocular symptoms. These increased salivary cytokines affected pro-inflammatory and apoptotic cellular pathways, including T cell signalling, several interleukin signalling pathways, TNF and TGF-ß receptor signalling, and the apoptotic p53 pathway. In conclusion, the upregulated salivary cytokines identified suggest an interplay between innate and adaptive immunity, affecting immunoregulatory cellular pathways. Whether this is due to late effects of radiotherapy or tissue repair remains to be investigated.

Efficient extracellular vesicle isolation by combining cell media modifications, ultrafiltration, and size-exclusion chromatography.

Extracellular vesicles (EVs) are a heterogeneous population of biological particles released by cells. They represent an attractive source of potential biomarkers for early detection of diseases such as cancer. However, it is critical that sufficient amounts of EVs can be isolated and purified in a robust and reproducible manner. Several isolation methods that seem to produce distinct populations of vesicles exist, making data comparability difficult. While some methods induce cellular stress that may affect both the quantity and function of the EVs produced, others involve expensive reagents or equipment unavailable for many laboratories. Thus, there is a need for a standardized, feasible and cost-effective method for isolation of EVs from cell culture supernatants. Here we present the most common obstacles in the production and isolation of small EVs, and we suggest a combination of relatively simple strategies to avoid these. Three distinct cell lines were used (human oral squamous cell carcinoma (PE/CA-PJ49/E10)), pancreatic adenocarcinoma (BxPC3), and a human melanoma brain metastasis (H3). The addition of 1% exosome-depleted FBS to Advanced culture media enabled for reduced presence of contaminating bovine EVs while still ensuring an acceptable cell proliferation and low cellular stress. Cells were gradually adapted to these new media. Furthermore, using the Integra CELLine AD1000 culture flask we increased the number of cells and thereby EVs in 3D-culture. A combination of ultrafiltration with different molecular weight cut-offs and size-exclusion chromatography was further used for the isolation of a heterogeneous population of small EVs with low protein contamination. The EVs were characterized by nanoparticle tracking analysis, immunoaffinity capture, flow cytometry, Western blot and transmission electron microscopy. We successfully isolated a significant amount of small EVs compatible with exosomes from three distinct cell lines in order to demonstrate reproducibility with cell lines of different origin. The EVs were characterized as CD9 positive with a size between 60-140 nm. We conclude that this new combination of methods is a robust and improved strategy for the isolation of EVs, and in particular small EVs compatible with exosomes, from cell culture media without the use of specialized equipment such as an ultracentrifuge.

Cell type specific DNA methylation in cord blood: A 450K-reference data set and cell count-based validation of estimated cell type composition.

Epigenome-wide association studies of prenatal exposure to different environmental factors are becoming increasingly common. These studies are usually performed in umbilical cord blood. Since blood comprises multiple cell types with specific DNA methylation patterns, confounding caused by cellular heterogeneity is a major concern. This can be adjusted for using reference data consisting of DNA methylation signatures in cell types isolated from blood. However, the most commonly used reference data set is based on blood samples from adult males and is not representative of the cell type composition in neonatal cord blood. The aim of this study was to generate a reference data set from cord blood to enable correct adjustment of the cell type composition in samples collected at birth. The purity of the isolated cell types was very high for all samples (>97.1%), and clustering analyses showed distinct grouping of the cell types according to hematopoietic lineage. We explored whether this cord blood and the adult peripheral blood reference data sets impact the estimation of cell type composition in cord blood samples from an independent birth cohort (MoBa, n = 1092). This revealed significant differences for all cell types. Importantly, comparison of the cell type estimates against matched cell counts both in the cord blood reference samples (n = 11) and in another independent birth cohort (Generation R, n = 195), demonstrated moderate to high correlation of the data. This is the first cord blood reference data set with a comprehensive examination of the downstream application of the data through validation of estimated cell types against matched cell counts.

Effect of Storage Temperature on the Phenotype of Cultured Epidermal Cells Stored in Xenobiotic-Free Medium.

PURPOSE: Cultured epidermal cell sheets (CECS) are used in the treatment of large area burns to the body and have potential to treat limbal stem cell deficiency (LSCD) as shown in animal studies. Despite widespread use, storage options for CECS are limited. Short-term storage allows flexibility in scheduling surgery, quality control and improved transportation to clinics worldwide. Recent evidence points to the phenotype of cultured epithelial cells as a critical predictor of post-operative success following transplantation of CECS in burns and in transplantation of cultured epithelial cells in patients with LSCD. This study, therefore assessed the effect of a range of temperatures, spanning 4-37 °C, on the phenotype of CECS stored over a 2-week period in a xenobiotic-free system. MATERIALS AND METHODS: Progenitor cell (p63, ΔNp63α and ABCG2) and differentiation (C/EBPδ and CK10) associated marker expression was assessed using immunocytochemistry. Immunohistochemistry staining of normal skin for the markers p63, ABCG2 and C/EBPδ was also carried out. Assessment of progenitor cell side population (SP) was performed using JC1 dye by flow cytometry. RESULTS: P63 expression remained relatively constant throughout the temperature range but was significantly lower compared to control between 20 and 28 °C (p < 0.05). High C/EBPδ together with low p63 suggested more differentiation beginning at 20 °C and above. Lower CK10 and C/EBPδ expression most similar to control was seen at 12 °C. The percentage of ABCG2 positive cells was most similar to control between 8 and 24 °C. Between 4 and 24 °C, the SP fluctuated, but was not significantly different compared to control. Results were supported by staining patterns indicating differentiation status associated with markers in normal skin sections. CONCLUSIONS: Lower storage temperatures, and in particular 12 °C, merit further investigation as optimal storage temperature for maintenance of undifferentiated phenotype in CECS.

Fluorescent particles in the antibody solution result in false TF- and CD14-positive microparticles in flow cytometric analysis.

Tissue factor (TF)-positive microparticles (MPs) are highly procoagulant, and linked to thrombosis in sepsis and cancer. MP-associated TF may be assayed by immunological or functional methods. Several reports have demonstrated discrepancies between TF-protein and TF-activity, which have been explained by antibody binding to "encrypted" or degraded forms of inactive TF-protein. Our goal was to evaluate the possible interference of fluorescent antibody aggregates in solutions containing antibodies against TF and CD14 in flow cytometric analysis. Using monocyte-derived microparticles (MPs) released from human monocytes, incubated with or without lipopolysaccharides (LPS) in vitro, we measured MP-associated TF-protein (flow cytometry) and TF-activity (clot formation assay). MPs released from monocytes exposed to LPS (1 ng mL(-1) ) had ∼14 times higher TF-activity than MPs originated from monocytes exposed to only culture medium. However, using untreated anti-TF antibodies (American Diagnostica and BD) in the flow cytometric analysis, MPs released from unstimulated monocytes had a similar number of TF-positive events as MPs secernated from LPS-stimulated monocytes [∼45,000 events mL(-1) (American Diagnostica); ∼15,000 events mL(-1) (BD)]. These TF-positive events did not exert any TF-activity, and centrifugation (17,000g, 30 min, 4°C) of the antibody solutions prior to use effectively removed the interfering fluorescent events. Removal of fluorescent interference, probably in the form of fluorescent antibody aggregates, from the antibody solutions by centrifugation is essential to prevent the occurrence of false positive flow cytometric events. The events can be mistaken as MP-associated TF-protein, and interpreted as a discrepancy between TF-protein and TF-activity.

Chromosomal 20q gain in the DNA diploid component of aneuploid colorectal carcinomas.

The order of appearance of different genetic aberrations during the shift from diploidy/near-diploidy to aneuploidy in colorectal cancers is not yet clear. We studied genetic alterations in flow cytometrically-sorted DNA diploid and corresponding aneuploid epithelial cell populations from each of 20 colorectal tumors using comparative genomic hybridization, FISH, and PCR. Analysis of the 19 cases in which aberrations were found in the flow-sorted diploid population indicated that large-scale aneuploidization in colorectal cancer was preceded by amplification of oncogene(s) localized to chromosome 20q13.2 and by KRAS mutations, but not by TP53 deletions or losses of large chromosomal regions such as 4q, 8p and 18q.

Are keratoacanthomas variants of squamous cell carcinomas? A comparison of chromosomal aberrations by comparative genomic hybridization.

Keratoacanthoma (KA) is a benign keratinocytic neoplasm that usually presents as a solitary nodule on sun-exposed areas, develops within 6-8 weeks and spontaneously regresses after 3-6 months. KAs share features such as infiltration and cytological atypia with squamous cell carcinomas (SCCs). Furthermore, there are reports of KAs that have metastasized, invoking the question of whether or not KA is a variant of SCC. To date no reported criteria are sensitive enough to discriminate reliably between KA and SCC, and consequently there is a clinical need for discriminating markers. We screened fresh frozen material from 132 KAs and 37 SCCs for gross chromosomal aberrations by using comparative genomic hybridization (CGH). Forty-nine KAs (37.1%) and 31 SCCs (83.7%) showed genomic aberrations, indicating a higher degree of chromosomal instability in SCCs. Gains of chromosomal material from 1p, 14q, 16q, 20q, and losses from 4p were seen significantly more frequently in SCCs compared with KAs (P-values 0.0033, 0.0198, 0.0301, 0.0017, and 0.0070), whereas loss from 9p was seen significantly more frequently in KAs (P-value 0.0434). The patterns of recurrent aberrations were also different in the two types of neoplasms, pointing to different genetic mechanisms involved in their developments.