In vitro osteoclastogenesis in autoimmune diseases - Strengths and pitfalls of a tool for studying pathological bone resorption and other disease characteristics.

Osteoclasts play a critical role in bone pathology frequently associated with autoimmune diseases. Studying the etiopathogenesis of these diseases and their clinical manifestations can involve in vitro osteoclastogenesis, an experimental technique that utilizes osteoclast precursors that are relatively easily accessible from peripheral blood or synovial fluid. However, the increasing number of methodical options to study osteoclastogenesis in vitro poses challenges in translating findings to clinical research and practice. This review compares and critically evaluates previous research work based on in vitro differentiation of human osteoclast precursors originating from patients, which aimed to explain autoimmune pathology in rheumatic and enteropathic diseases. The discussion focuses primarily on methodical differences between the studies, including the origin of osteoclast precursors, culture conditions, and methods for identifying osteoclasts and assessing their activity. Additionally, the review examines the clinical significance of the three most commonly used in vitro approaches: induced osteoclastogenesis, spontaneous osteoclastogenesis, and cell co-culture. By analyzing and integrating the gathered information, this review proposes general connections between different studies, even in cases where their results are seemingly contradictory. The derived conclusions and future directions aim to enhance our understanding of a potential and limitations of in vitro osteoclastogenesis and provide a foundation for discussing novel methods (such as osteoclastogenesis dynamic) and standardized approaches (such as spontaneous osteoclastogenesis) for future use in autoimmune disease research.

Comparative analysis of tear cytokines in patients with glaucoma, ocular hypertension, and healthy controls.

PURPOSE: To investigate the ocular surface inflammation in patients with primary open angle glaucoma and ocular hypertension by analyzing tears and to compare findings with healthy controls. METHODS: Observational case-control study. Tear samples were collected by 5 µl microcapillary tube from 24 patients with glaucoma treated by antiglaucoma drops, 9 non-treated patients with ocular hypertension and 45 healthy controls. Tears were analyzed from right eye by multiplex Bio-Plex system for the presence of 6 cytokines: IL1ß, IL10, IL4, IFNγ, MIF and VEGF. RESULTS: Significantly higher concentrations of IL1ß and IL10 (glaucoma or ocular hypertension vs. healthy controls, p < 0.0001), VEGF (glaucoma vs. ocular hypertension, p < 0.05; ocular hypertension vs. healthy controls, p < 0.02) and MIF (glaucoma vs. healthy controls, p < 0.03) were detected in patients' tears. Both patient groups have activated to a significantly lower extent the Th1 pathway represented by IFNγ than Th2 pathway represented by IL10 (p < 0.001) and, at the same time, the IFNγ/IL4 ratio was significantly increased in healthy controls (p < 0.001) and patients with ocular hypertension (p < 0.02) compared to glaucoma individuals. CONCLUSION: This study shows that secretion of inflammation-related cytokines by conjunctival cells is increased in both, glaucoma and ocular hypertension patients and can be detected in their tears. Nevertheless, data indicates stronger ocular surface inflammation in non-treated follow-up patients diagnosed with ocular hypertension than in glaucoma subjects treated by antiglaucoma drops.

Age and sex-specific differences in interleukin 4, interferon gamma, macrophage migration inhibitory factor, and vascular endothelial growth factor levels in the tears of healthy subjects.

OBJECTIVE: To investigate the physiological profile of pro-inflammatory and anti-inflammatory cytokines in tears produced by epithelial cells under the effect of endogenous and exogenous biological factors. Knowing the physiological cytokine profile in tears with its biological characteristics including sex- and age-specific effects is fundamental when tears are analyzed for diagnostic or prognostic purposes in eye diseases. METHODS: Tear samples were collected from right eye of 45 healthy volunteers (24 males, 21 females) by 5 µl microcapillary tube. Cytokines interleukin 1ß, interleukin 10, interleukin 4, interferon gamma, macrophage migration inhibitory factor, and vascular endothelial growth factor were quantified by multiplex Bio-Plex system. RESULTS: The production of macrophage migration inhibitory factor cytokine by epithelial cells on the ocular surface is higher in males compared to females (p = 0.05); actually, most of female tear samples present with undetectable macrophage migration inhibitory factor levels. Our results show the negative correlations between the age and concentrations of interleukin 4 (p < 0.01) and interferon gamma (p < 0.01) in tears, respectively, and positive associations of vascular endothelial growth factor levels with the age above 45 years (p < 0.05). CONCLUSIONS: Data in this study indicate that age and sex may affect the physiological levels of cytokines in tears. Consequently, the impacts of biological factors need to be recognized and taken into consideration before the levels of cytokines in patients' tears are analyzed for medical reasons. Concentrations of interleukin 1ß and interleukin 10 cytokines, however, are very low in healthy tears and do not seem to be influenced by studied biological factors; therefore, they meet the requirements for analytes suitable for medical diagnostic and prognostic purposes.

Epigenetic Regulation of Circadian Rhythm and Its Possible Role in Diabetes Mellitus.

This review aims to summarize the knowledge about the relationship between circadian rhythms and their influence on the development of type 2 diabetes mellitus (T2DM) and metabolic syndrome. Circadian rhythms are controlled by internal molecular feedback loops that synchronize the organism with the external environment. These loops are affected by genetic and epigenetic factors. Genetic factors include polymorphisms and mutations of circadian genes. The expression of circadian genes is regulated by epigenetic mechanisms that change from prenatal development to old age. Epigenetic modifications are influenced by the external environment. Most of these modifications are affected by our own life style. Irregular circadian rhythm and low quality of sleep have been shown to increase the risk of developing T2DM and other metabolic disorders. Here, we attempt to provide a wide description of mutual relationships between epigenetic regulation, circadian rhythm, aging process and highlight new evidences that show possible therapeutic advance in the field of chrono-medicine which will be more important in the upcoming years.

Immunoregulatory properties of cell-free DNA in plasma of celiac disease patients - A pilot study.

The elevated plasma cell-free DNA (cfDNA) concentrations were repeatedly reported in association with the process of inflammation. The qualitative and quantitative characteristics of plasma cfDNA in active (newly diagnosed) celiac disease patients (CD) have not yet been studied despite the fact that cfDNA of healthy individuals is able to regulate immune response. We determined the total cfDNA concentration and relative content of telomeric sequences in plasma cfDNA in CD (n = 10) and healthy age- and sex-matched controls (HC, n = 10) by quantitative PCR. To obtain the evidence that the observed biological effects are caused solely by cfDNA molecules, we applied the treatment of paired plasma samples with DNase. Using paired samples of plasma (non-treated/native and treated by DNase), we analyzed the contribution of cfDNA to the activation of TLR9 and TNF-α mRNA expression in THP1 monocytic cell line. There were no significant differences in the quantities of plasma cfDNA and relative contents of telomeric sequences in their pools. When we compared the levels of TNF-α mRNA expression in THP1 cells achieved after stimulation with native CD and HC plasma samples, we found significantly (p = .031) higher expression after stimulation with CD samples. We documented also the ability of cfDNA contained in CD plasma samples to stimulate the production of TLR9 mRNA. The TLR9 mRNA expression levels were significantly (p = .014) lowered after cfDNA removal from CD plasma samples. The design of our experiments allowed us to study the effects of cfDNA without its isolation from plasma. cfDNA contained in CD plasma samples differs significantly in its immunoregulatory capacity from cfDNA in HC plasma. The differences are caused neither by different concentrations of cfDNA in plasma samples nor by different relative abundance of telomeric sequences. Further studies are needed to elucidate the role of plasma cfDNA in celiac disease pathogenesis.

Autoimmunity-Associated PTPN22 Polymorphisms in Latent Autoimmune Diabetes of the Adult Differ from Those of Type 1 Diabetes Patients.

BACKGROUND: A portion of adults with humoral immune changes have clinical diabetes that is initially not insulin-requiring (latent autoimmune diabetes of the adult, LADA). One of the genes strongly associated with autoimmune diabetes is PTPN22. We hypothesized that the manifestation and clinical features of LADA are linked to functional variants of PTPN22. METHODS: We genotyped allelic frequencies of 1 protective and 3 risk-associated PTPN22 variants in 156 Czech LADA patients, 194 type 2 diabetes mellitus patients with LADA-like progression to insulinotherapy and 324 type 1 diabetes mellitus patients, and subsequently examined the associations of PTPN22 variants with the expression of autoantibodies and other clinical features of LADA. RESULTS: We challenged the paradigm that stated that the PTPN22 c.1858T allele serves as a risk allele for LADA, although we confirmed its risk status in the geographically matched T1DM cohort. In contrast, the frequencies of other PTPN22 alleles (c.-1123C, c.788A and c.1970-852C) differed significantly from the healthy controls. We confirmed gender-related differences in the frequency of some PTPN22 polymorphisms (but not c.1858C>T) in LADA. The particular PTPN22 alleles and genotypes were associated with specific clinical features of the examined patients (autoantibodies, HbA1c and age at diagnosis of diabetes). CONCLUSIONS: The variability in PTPN22 haplotypes suggests that the genetic signature of LADA is independent and should not be considered a hybrid form of T1DM and T2DM. Further studies should elucidate the associations with clinical characteristics of the LADA patients and focus on the newly emerging types of diabetes with the disease onset in early to mid-adulthood.

Human Peripheral Blood Cells mRNA Levels are Highly Sensitive to Duration of Ex Vivo Post-Sampling Conditions Prior to RNA Isolation.

BACKGROUND: This study aimed to evaluate an effect of the time period between drawing the peripheral blood and specimen processing on the stability of mRNA levels of 7 selected genes. METHODS: Blood samples derived from 15 healthy volunteers were always processed at five consecutive time points 0.5, 1.5, 2, 3, and 9 hours; mRNA was quantified by real-time PCR. RESULTS: Anti-inflammatory genes CCL2 and IL10 showed a significant rise of expression between the 3rd and 9th hour after blood collection (p ≤ 0.5). Significant decrease of mRNA levels in relation to time lag was observed for TLR4 and MYC genes (p ≤ 0.5). Interestingly, the initial two hours after drawing the blood revealed a high interindividual variability in cellular response to stress connected with blood drawing and ex vivo post-sampling condition. CONCLUSIONS: These results point out the need for a strict standardization of handling the blood specimen with regards to peripheral blood sample processing time between phlebotomy and RNA isolation.

Increased Expression of TLR4 and TLR7 but Not Prolactin mRNA by Peripheral Blood Monocytes in Active Celiac Disease.

BACKGROUND: Celiac disease (CD) is an organ-specific autoimmune disease, and both adaptive and innate immunity are involved in its development. OBJECTIVES: The aim of the study was to determine whether the markers of intestinal mucosal inflammation in CD can be detected in peripheral blood monocytes (PBMs), and whether the immune properties of PBMs change as the clinical signs and symptoms of CD improve after the introduction of a gluten-free diet (GFD). The focus was on changes in mRNA expression of selected toll-like receptors (TLR2, TLR4, TLR7), stress cytokine prolactin (PRL), and proand anti-inflammatory cytokines (TNF-α, IL-6, IL-12, IL-10) in PBMs. MATERIAL AND METHODS: The study involved 20 CD patients diagnosed according to the European Society for Pediatric Gastroenterology, Hepatology and Nutrition criteria and Marsh criteria: 10 recently-diagnosed cases (rCD) and 10 on a GFD for a minimum of one year. The control group comprised 10 ageand sex-matched healthy volunteers. PBMs from peripheral blood specimens were separated using immunomagnetic CD14+ beads. Total RNA was isolated using a standard commercial kit. Cytokine and TLR mRNA levels were quantified by relative qPCR with PGK1 as a reference gene. RESULTS: Significantly higher expression of TLR4 and TLR7 mRNA was observed in PBMs from rCD patients compared to the healthy controls (1.63 times higher; p < 0.05). TLR7 mRNA levels in rCDs were also significantly elevated in comparison to the CD-GFD patients (2.11 times higher; p < 0.01). TNF-α mRNA expression tended to be higher in both groups of patients; by contrast, in IL-6 mRNA, a trend to a fourfold decrease was detected in PBMs from the CD-GFD subjects. IL-10, IL-12 and PRL levels did not differ among the groups. CONCLUSIONS: The data suggest that the inflammatory process in rCD intestinal mucosa and submucosa reflecting enterocyte damage can be detected in PBMs in peripheral blood. Further, the cytokine and TLR expression profile in PBMs alters after one year of GFD treatment.

Ethylenediaminetetraacetic Acid, Sodium Citrate, Heparin and Citrate Phosphate Dextrose-Adenine Anticoagulants Differentially Affect Cytokine mRNA Expression in Blood Leukocytes.

BACKGROUND: The aim of this study is to determine whether and how the anticoagulant agents EDTA, sodium citrate, and heparin present in vacutainers and CPDA located in transfusion bags modify peripheral blood cell behavior. METHODS: We compared the effect of four anticoagulants within an expression study quantifying mRNA by realtime PCR in 60 blood samples. RESULTS: We observed a significant increase of TNFα and CCL2 mRNA expression in leukocytes placed in EDTA anticoagulant compared to leukocytes collected with citrate or heparin (p ≤ 0.5). Compared to blood cells influenced by one of the three anticoagulant agents present in vacuum tubes, cells from transfusion bags affected by CPDA displayed significantly lower levels of CCL2 and MYC mRNA, whereas levels of IL-10 and TNFα mRNA were much higher (p ≤ 0.5). CONCLUSIONS: The anticoagulant EDTA significantly increases proinflammatory cytokine mRNA in freshly isolated PBMC. Anticoagulant CPDA significantly alters levels of some of the tested mRNAs in comparison to blood cells drawn into one of the three anticoagulant agents present in vacutainers.