Immunology demystified: A guide for transplant hepatologists.

Liver transplantation has become standard practice for treating end-stage liver disease. The success of the procedure relies on effective immunosuppressive medications to control the host's immune response. Despite the liver's inherent capacity to foster tolerance, the early post-transplant period is marked by significant immune reactivity. To ensure favorable outcomes, it is imperative to identify and manage various rejection types, encompassing T-cell-mediated, antibody-mediated, and chronic rejection. However, the approach to prescribing immunosuppressants relies heavily on clinical judgment rather than evidence-based criteria. Given that the majority of patients will require lifelong immuno suppression as the mechanisms underlying operational tolerance are still being investigated, healthcare providers must possess an understanding of immune responses, rejection mechanisms, and the pathways targeted by immunosuppressive drugs. This knowledge enables customization of treatments and improved patient care, even though a consensus on an optimal immunosuppressive regimen remains elusive.

Serum tryptase levels in patients with post-acute COVID-19 syndrome.

OBJECTIVES: to investigate the difference in serum tryptase levels between post-acute COVID-19 syndrome (PACS) patients and controls. BACKGROUND: PACS has been defined as symptoms that persist for more than 3 months after the onset of COVID-19. The pathogenesis is still unknown, but mast cell activation has been proposed as one of the mechanisms, and increased serum tryptase levels have been demonstrated in PACS patients. METHODS: A total number of 133 patients were included: 50 with PACS, 37 asymptomatic COVID-19 convalescents, and 46 controls with a negative history of COVID-19. Serum tryptase levels were determined in all participants. RESULTS: There was no significant difference in serum levels of tryptase among the groups. CONCLUSION: the role of mast cell activation in PACS remains unclear and further research is needed to fill the gaps in understanding the pathogenesis of this complex and heterogeneous disorder (Tab. 2, Ref. 17). Text in PDF www.elis.sk Keywords: post Acute COVID-19 syndrome, tryptase, mast cells, lactate dehydrogenase, ferritin.

The Association Between the Severity of Psoriasis and Obesity Based on the Analysis of Visceral Fat Index and Serum Levels of Tumor Necrosis Factor-α, Interleukin-6, and Resistin.

Aim of this study was to investigate the relationship between the severity of psoriasis and obesity based on the analysis of the visceral fat index and serum levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and resistin. The study included 50 patients with psoriasis and 30 subjects in the control group. The measured parameters were height, weight, waist circumference, visceral fat index, and serum levels of TNF-α, IL-6, and resistin. The severity of the disease was evaluated using the psoriasis area and severity index (PASI). Visceral fat index was measured using the method of bioelectrical impedance analysis. Serum levels of TNF-α, IL-6, and resistin were correlated with visceral fat index, and the relationship of all these parameters with psoriasis severity was also analyzed. Patients with psoriasis have a significantly higher body mass index, waist circumference, and visceral fat index compared with the control group. Elevated serum levels of TNF-α, IL-6, and resistin, as well as a correlation with psoriasis severity and visceral fat index was also found in the patient group. Visceral fat index was a better indicator of the relationship between psoriasis severity and obesity than waist circumference and body mass index. We concluded that serum levels of TNF-α, IL-6, and resistin could be useful in assessing psoriasis activity and optimizing therapeutic strategies. It is suggested that visceral fat index should be evaluated in all patients with psoriasis, especially before the decision on systemic therapy.

Incidence of immediate allergic reactions to mRNA COVID-19 vaccines in adults with drug allergies and other allergic disorders.

Concerns have been raised about allergic reactions to messenger ribonucleic acid (mRNA) coronavirus disease 2019 (COVID-19) vaccines. A history of allergic reactions, including anaphylaxis to drugs, has been frequently reported in individuals with anaphylaxis to mRNA vaccines. To estimate the rate of immediate allergic reactions in patients with a history of drug allergy or other allergic disorders. We included adult patients who had received at least 1 dose of an mRNA COVID-19 vaccine at the Special Hospital for Pulmonary Diseases between March 1, 2021, and October 1, 2021, and who reported a history of drug allergy or other allergic diseases (asthma, allergic rhinitis, atopic dermatitis, food or insect venom allergy, mastocytosis, idiopathic anaphylaxis, acute or chronic urticaria, and/or angioedema). Immediate allergic reactions, including anaphylaxis, occurring within 4 hours of vaccination were recorded. Six immediate allergic reactions were noted in the cohort of 1679 patients (0.36%). One patient experienced anaphylaxis (0.06%), which resolved after epinephrine administration, and the other reactions were mild and easily treatable. Most patients with a history of allergies can safely receive an mRNA COVID-19 vaccine, providing adequate observation periods and preparedness to recognize and treat anaphylaxis.

Inhibition of Notch Signaling Stimulates Osteoclastogenesis From the Common Trilineage Progenitor Under Inflammatory Conditions.

Osteoclasts, macrophages and dendritic cells (DCs) can be derived from a common trilineage myeloid progenitor of hematopoietic origin. Progenitor commitment is susceptible to regulation through Notch signaling. Our aim was to determine the effects of Notch modulation on trilineage progenitor commitment and functional properties of differentiated cells under inflammatory conditions. We used the conditional inducible CX3CR1CreERT2 mouse strain to achieve overexpression of the Notch 1 intracellular domain (NICD1) or to inhibit Notch signaling via deletion of the transcription factor RBP-J in a bone marrow population, used as a source of the trilineage progenitor (CD45+Ly6G-CD3-B220-NK1.1-CD11b-/loCD115+). Cre-recombinase, under the control of the CX3CR1 promoter, expressed in the monocyte/macrophage lineage, was induced in vitro by 4-hydroxytamoxifen. Differentiation of osteoclasts was induced by M-CSF/RANKL; macrophages by M-CSF; DCs by IL-4/GM-CSF, and inflammation by LPS. Functionally, DCs were tested for the ability to process and present antigen, macrophages to phagocytose E. coli particles, and osteoclasts to resorb bone and express tartrate-resistant acid phosphatase (TRAP). We found that Notch 1 signal activation suppressed osteoclast formation, whereas disruption of the Notch canonical pathway enhanced osteoclastogenesis, resulting in a higher number and size of osteoclasts. RANK protein and Ctsk gene expression were upregulated in osteoclastogenic cultures from RBP-J+ mice, with the opposing results in NICD1+ mice. Notch modulation did not affect the number of in vitro differentiated macrophages and DCs. However, RBP-J deletion stimulated Il12b and Cd86 expression in macrophages and DCs, respectively. Functional assays under inflammatory conditions confirmed that Notch silencing amplifies TRAP expression by osteoclasts, whereas the enhanced phagocytosis by macrophages was observed in both NICD1+ and RBP-J+ strains. Finally, antigen presentation by LPS-stimulated DCs was significantly downregulated with NICD1 overexpression. This experimental setting allowed us to define a cell-autonomous response to Notch signaling at the trilineage progenitor stage. Although Notch signaling modulation affected the activity of all three lineages, the major effect was observed in osteoclasts, resulting in enhanced differentiation and function with inhibition of canonical Notch signaling. Our results indicate that Notch signaling participates as the negative regulator of osteoclast activity during inflammation, which may be relevant in immune and bone diseases.

Tamoxifen Ameliorates Cholestatic Liver Fibrosis in Mice: Upregulation of TGFß and IL6 Is a Potential Protective Mechanism.

The available treatments for cholestatic liver fibrosis are limited, and the disease often progresses to liver cirrhosis. Tamoxifen is a selective modulator of estrogen receptors, commonly used in breast cancer therapy. A recent in vitro study showed that tamoxifen deactivates hepatic stellate cells, suggesting its potential as an antifibrotic therapeutic, but its effects in vivo remain poorly investigated. In the present study, we show that tamoxifen protects against the cholestatic fibrosis induced by a diet supplemented with 0.025% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Mice fed with a DDC-supplemented diet for four weeks and treated with tamoxifen developed a significantly milder degree of liver fibrosis than vehicle-treated mice, as evidenced by a lower percentage of Sirius red-stained area (60.4% decrease in stained area in male and 42% decrease in female mice, p < 0.001 and p < 0.01, respectively) and by lower hydroxyproline content. The finding was further confirmed by qPCR analysis, which showed a lower expression of genes for Col1a1, Acta2, Sox9, Pdgf, and Krt19, indicating the inhibitory effect on hepatic stellate cells, collagen production, and biliary duct proliferation. The degree of protection was similar in male and female mice. Tamoxifen per se, injected into standard-diet-fed mice, increased the expression of genes for Il6 (p < 0.01 and p < 0.001 in male and female mice, respectively) and Tgfß (p < 0.01 for both sexes), and had no adverse effects. We showed that tamoxifen sex-independently protects against cholestatic DDC-induced liver fibrosis. The increased expression of Il6 and Tgfß seems to be a plausible protective mechanism that should be the primary focus of further research.

Vitamin D in liver cancer: novel insights and future perspectives.

Vitamin D has been a focus of attention in liver cancer due to its direct and indirect antineoplastic effects. This review critically evaluates data from recently published basic and clinical studies investigating the role of vitamin D in liver cancer. Basic studies indicate that vitamin D plays an important role in liver cancer development by suppressing the activity of hepatic stellate cells and Kupffer cells. Furthermore, vitamin D has a direct anti-proliferative, anti-angiogenic, proapoptotic, and prodifferentiative effect on liver cancer cells. Recent investigation suggested several interesting mechanisms of these actions, such as inactivation of Notch signaling, p27 accumulation, and tyrosine-protein kinase Met/extracellular signal-regulated kinases inhibition. On the other hand, data from clinical observational studies, although promising, are still inconclusive. Unfortunately, studies on the effect of vitamin D supplementation were generally focused on short-term outcomes of chronic liver diseases (liver enzyme levels or elastographic finding); therefore, there are still no reliable data on the effect of vitamin D supplementation on liver cancer occurrence or survival.

Transcriptome profiling of osteoclast subsets associated with arthritis: A pathogenic role of CCR2hi osteoclast progenitors.

Introduction: The existence of different osteoclast progenitor (OCP) subsets has been confirmed by numerous studies. However, pathological inflammation-induced osteoclastogenesis remains incompletely understood. Detailed characterization of OCP subsets may elucidate the pathophysiology of increased osteoclast activity causing periarticular and systemic bone resorption in arthritis. In our study, we rely on previously defined OCP subsets categorized by the level of CCR2 expression as circulatory-like committed CCR2hi OCPs, which are substantially expanded in arthritis, and marrow-resident CCR2lo OCPs of immature phenotype and behavior. Methods: In order to perform transcriptome characterization of those subsets in the context of collagen-induced arthritis (CIA), we sorted CCR2hi and CCR2lo periarticular bone marrow OCPs of control and arthritic mice, and performed next-generation RNA sequencing (n=4 for each group) to evaluate the differential gene expression profile using gene set enrichment analysis with further validation. Results: A disparity between CCR2hi and CCR2lo subset transcriptomes (863 genes) was detected, with the enrichment of pathways for osteoclast differentiation, chemokine and NOD-like receptor signaling in the CCR2hi OCP subset, and ribosome biogenesis in eukaryotes and ribosome pathways in the CCR2lo OCP subset. The effect of intervention (CIA) within each subset was greater in CCR2hi (92 genes) than in CCR2lo (43 genes) OCPs. Genes associated with the osteoclastogenic pathway (Fcgr1, Socs3), and several genes involved in cell adhesion and migration (F11r, Cd38, Lrg1) identified the CCR2hi subset and distinguish CIA from control group, as validated by qPCR (n=6 for control mice, n=9 for CIA mice). The latter gene set showed a significant positive correlation with arthritis clinical score and frequency of CCR2hi OCPs. Protein-level validation by flow cytometry showed increased proportion of OCPs expressing F11r/CD321, CD38 and Lrg1 in CIA, indicating that they could be used as disease markers. Moreover, osteoclast pathway-identifying genes remained similarly expressed (Fcgr1) or even induced by several fold (Socs3) in preosteoclasts differentiated in vitro from CIA mice compared to pre-cultured levels, suggesting their importance for enhanced osteoclastogenesis of the CCR2hi OCPs in arthritis. Conclusion: Our approach detected differentially expressed genes that could identify distinct subset of OCPs associated with arthritis as well as indicate possible therapeutic targets aimed to modulate osteoclast activity.

Preventive CCL2/CCR2 Axis Blockade Suppresses Osteoclast Activity in a Mouse Model of Rheumatoid Arthritis by Reducing Homing of CCR2hi Osteoclast Progenitors to the Affected Bone.

Detailed characterization of medullary and extramedullary reservoirs of osteoclast progenitors (OCPs) is required to understand the pathophysiology of increased periarticular and systemic bone resorption in arthritis. In this study, we focused on identifying the OCP population specifically induced by arthritis and the role of circulatory OCPs in inflammatory bone loss. In addition, we determined the relevant chemokine axis responsible for their migration, and targeted the attraction signal to reduce bone resorption in murine collagen-induced arthritis (CIA). OCPs were expanded in periarticular as well as circulatory compartment of arthritic mice, particularly the CCR2hi subset. This subset demonstrated enhanced osteoclastogenic activity in arthritis, whereas its migratory potential was susceptible to CCR2 blockade in vitro. Intravascular compartment of the periarticular area contained increased frequency of OCPs with the ability to home to the arthritic bone, as demonstrated in vivo by intravascular staining and adoptive transfer of splenic LysMcre/Ai9 tdTomato-expressing cells. Simultaneously, CCL2 levels were increased locally and systemically in arthritic mice. Mouse cohorts were treated with the small-molecule inhibitor (SMI) of CCR2 alone or in combination with methotrexate (MTX). Preventive CCR2/CCL2 axis blockade in vivo reduced bone resorption and OCP frequency, whereas combining with MTX treatment also decreased disease clinical score, number of active osteoclasts, and OCP differentiation potential. In conclusion, our study characterized the functional properties of two distinct OCP subsets in CIA, based on their CCR2 expression levels, implying that the CCR2hi circulatory-like subset is specifically induced by arthritis. Signaling through the CCL2/CCR2 axis contributes to OCP homing in the inflamed joints and to their increased osteoclastogenic potential. Therefore, addition of CCL2/CCR2 blockade early in the course of arthritis is a promising approach to reduce bone pathology.

NOTCH3 rs1043996 Polymorphism Is Associated with the Occurrence of Alcoholic Liver Cirrhosis Independently of PNPLA3 and TM6SF2 Polymorphisms.

Alcoholic liver cirrhosis (ALC) is the most common indication for liver transplantation (LT) in Croatia and presents a risk factor for the development of hepatocellular carcinoma (HCC). However, genetic susceptibility has not yet been systematically studied. We aimed to investigate the contribution of the risk polymorphisms PNPLA3 rs738409, EGF rs4444903, TM6SF2 rs58542926, MTHFR rs1801133, previously identified in other populations and, additionally, the contribution of Notch-related polymorphisms (NOTCH1 rs3124591, NOTCH3 rs1043996 and rs1044116, NOTCH4 rs422951). The study included 401 patients. The ALC group consisted of 260 LT candidates, 128 of whom had histopathologically confirmed HCC, and 132 of whom were without HCC. The control group included 141 patients without liver disease. Genotyping was performed by PCR using Taqman assays. The patients' susceptibility to ALC was significantly associated with PNPLA3 rs738409, TM6SF2 rs58542926, and NOTCH3 rs1043996 polymorphisms. These polymorphisms remained significantly associated with ALC occurrence in a logistic regression model, even after additional model adjustment for sex and age. Cirrhotic patients with the PNPLA3 GG genotype demonstrated higher activity of ALT aminotransferases than patients with CC or CG genotypes. The susceptibility to the development of HCC in ALC was significantly associated with PNPLA3 rs738409 and EGF rs4444903 polymorphisms, and logistic regression confirmed these polymorphisms as independent predictors.

CD13 is a critical regulator of cell-cell fusion in osteoclastogenesis.

The transmembrane aminopeptidase CD13 is highly expressed in cells of the myeloid lineage, regulates dynamin-dependent receptor endocytosis and recycling and is a necessary component of actin cytoskeletal organization. Here, we show that CD13-deficient mice present a low bone density phenotype with increased numbers of osteoclasts per bone surface, but display a normal distribution of osteoclast progenitor populations in the bone marrow and periphery. In addition, the bone formation and mineral apposition rates are similar between genotypes, indicating a defect in osteoclast-specific function in vivo. Lack of CD13 led to exaggerated in vitro osteoclastogenesis as indicated by significantly enhanced fusion of bone marrow-derived multinucleated osteoclasts in the presence of M-CSF and RANKL, resulting in abnormally large cells containing remarkably high numbers of nuclei. Mechanistically, while expression levels of the fusion-regulatory proteins dynamin and DC-STAMP1 must be downregulated for fusion to proceed, these are aberrantly sustained at high levels even in CD13-deficient mature multi-nucleated osteoclasts. Further, the stability of fusion-promoting proteins is maintained in the absence of CD13, implicating CD13 in protein turnover mechanisms. Together, we conclude that CD13 may regulate cell-cell fusion by controlling the expression and localization of key fusion regulatory proteins that are critical for osteoclast fusion.

Pre- and intraoperative predictors of acute kidney injury after liver transplantation.

BACKGROUND: Acute kidney injury (AKI) after liver transplantation (LT) is a frequent and multifactorial event related to increased morbidity and mortality. Risk factors for AKI after LT still need to be clarified. AIM: To identify the predictors of acute kidney injury after liver transplantation. METHODS: The frequency and pre- and intraoperative predictors of AKI within the first 7 d after LT were evaluated in adult liver transplant candidates in a single LT center in Croatia. AKI was defined according to the Kidney Disease: Improving Global Outcomes criteria. RESULTS: Out of 205 patients (mean age 57 ± 10 years; 73.7% males, 52.7% with alcohol-related liver disease) 93 (45.36%) developed AKI, and the majority of them (58.06%) had stage 1. Only 5.38% of patients required renal replacement therapy after LT. The majority of patients (82.8%) developed AKI within the first two days after the procedure. Multivariate logistic regression identified pre-LT body mass index (OR = 1.1, 95%CI: 1.05-1.24) and red blood cell transfusion (OR = 1.66, 95%CI: 1.09-2.53) as independent predictors of early post-LT AKI occurrence. 30-d survival after LT was significantly better for patients without AKI (P = 0.01). CONCLUSION: Early AKI after LT is a frequent event that negatively impacts short-term survival. The pathogenesis of AKI is multifactorial, but pre-LT BMI and intraoperative volume shifts are major contributors.

Concentrations of Selected Cytokines and Vascular Endothelial Growth Factor in Aqueous Humor and Serum of Diabetic Patients.

Purpose: To investigate the aqueous humor and serum levels of selected cytokines and vascular endothelial growth factor (VEGF) in diabetic patients, implicating their role in the pathogenesis of diabetic eye complications.Materials and methods:   Atotal of 65 patients (27 males and 38 females) who underwent cataract surgery were recruited into the study. The study group consisted of 30 cataract patients with type 2 diabetes mellitus, and this group was divided into two subgroups: 14 patients with diabetic retinopathy (DR group) and 16 patients without DR (NDR group). The control group consisted of 35 non-diabetic cataract subjects.Results: Patients in the DR group had significantly higher aqueous humor concentrations of interleukin (IL)-1ß, IL-6, IL-8, IL-10, monocyte chemotactic protein (MCP-1) and VEGF. Likewise, serum concentrations of IL-1ß, IL-6, IL-8, IL-12, TNF-α and IFN-γ were significantly higher in the DR group as compared to the controls. Aqueous humor concentrations of IL-1ß, IL-8, MCP-1 and VEGF were significantly higher in the DR group as compared with the NDR group.Conclusion: Our findings support the hypothesis that chronic inflammation and a disturbance of the immune system play important roles in the pathogenesis of diabetic cataract and DR.

Notch receptors and ligands in inflammatory arthritis - a systematic review.

BACKGROUND: Notch pathway is highly conserved across species and is involved in the regulation of cell differentiation and activity both in embryonic development and adult life. Notch signaling has an important role in the development of hematopoietic stem cells and their differentiation to committed lineages, as well as in the regulation of several non-hematopoietic cell lines. OBJECTIVE: As Notch signaling has been implicated in various inflammatory and autoimmune diseases, it is of interest to elucidate what role do Notch receptors and ligands have in inflammatory arthritides. METHODS: We performed a search on the role of Notch receptors (1-4) and Notch ligands Delta-like (DLL) 1, 3, 4 and Jagged (Jag) 1 and 2 in animal models of inflammatory arthritis and most common types of human inflammatory arthritis (rheumatoid arthritis, psoriatic arthritis or ankylosing spondylitis). The initial search identified 135 unique articles, of which 24 were ultimately deemed relevant and included in this systematic review. RESULTS: Overall, identified articles describe roles for Notch ligands and receptors in inflammatory arthritis, with Notch activation resulting in enhanced Th1/17 polarization, osteoclast differentiation, macrophage activation and fibroblast-like synoviocyte proliferation. However, the inhibitory role of Notch signaling, especially by Jag1 is also described. CONCLUSION: There is evidence that Notch pathway activation affects multiple cell lineages present within the arthritic environment, therefore potentially acting as one of the drivers of disease pathogenesis. Since cell lineage-selective transgenic mouse models and specific Notch receptor inhibitors are becoming increasingly available, it can be expected that future research will evaluate whether Notch signaling components initiate crucial pathogenic impulses and, therefore, present viable therapeutic targets in inflammatory arthritis.

Importance of genetic polymorphisms in liver transplantation outcomes.

Although, liver transplantation serves as the only curative treatment for patients with end-stage liver diseases, it is burdened with complications, which affect survival rates. In addition to clinical risk factors, contribution of recipient and donor genetic prognostic markers has been extensively studied in order to reduce the burden and improve the outcomes. Determination of single nucleotide polymorphisms (SNPs) is one of the most important tools in development of personalized transplant approach. To provide a better insight in recent developments, we review the studies published in the last three years that investigated an association of recipient or donor SNPs with most common issues in liver transplantation: Acute cellular rejection, development of new-onset diabetes mellitus and non-alcoholic fatty liver disease, hepatocellular carcinoma recurrence, and tacrolimus concentration variability. Reviewed studies confirmed previously established SNP prognostic factors, such as PNPLA3 rs738409 for non-alcoholic fatty liver disease development, or the role of CYP3A5 rs776746 in tacrolimus concentration variability. They also identified several novel SNPs, with a reasonably strong association, which have the potential to become useful predictors of post-transplant complications. However, as the studies were typically conducted in one center on relatively low-to-moderate number of patients, verification of the results in other centers is warranted to resolve these limitations. Furthermore, of 29 reviewed studies, 28 used gene candidate approach and only one implemented a genome wide association approach. Genome wide association multicentric studies are needed to facilitate the development of personalized transplant medicine.

LPS-induced inflammation desensitizes hepatocytes to Fas-induced apoptosis through Stat3 activation-The effect can be reversed by ruxolitinib.

Recent studies have established a concept of tumour necrosis factor-α (TNF-α)/Fas signalling crosstalk, highlighting TNF-α as a critical cytokine in sensitizing hepatocytes to death induced by Fas activation. However, in the exact inflammatory response, besides TNF-α, many other mediators, that might modulate apoptotic response differentially, are released. To resolve the issue, we studied the effects of lipopolysaccharide (LPS), one of the crucial inductors of inflammation in the liver, on apoptotic outcome. We show that LPS-induced inflammation diminishes the sensitivity of hepatocytes to Fas stimulus in vivo at caspase-8 level. Analysis of molecular mechanisms revealed an increased expression of various pro-inflammatory cytokines in non-parenchymal liver cells and hepatocyte-specific increase in Bcl-xL, associated with signal transducer and activator of transcription 3 (Stat3) phosphorylation. Pre-treatment with ruxolitinib, a selective Janus kinase (JAK) 1/2 inhibitor, prevented the LPS-induced Stat3 phosphorylation and restored the sensitivity of hepatocytes to Fas-mediated apoptosis. Furthermore, ruxolitinib pre-treatment diminished the LPS-induced Bcl-xL up-regulation without an inhibitory effect on LPS-induced expression of pro-inflammatory cytokines. In summary, although the reports are showing that the effects of isolated pro-inflammatory mediators, such as TNF-α or neutrophils, are pro-apoptotic, the overall effect of inflammatory milieu on hepatocytes in vivo is Stat3-dependent desensitization to Fas-mediated apoptosis.

FasL (rs763110) gene polymorphism is not associated with susceptibility to rheumatoid arthritis in Croatian population.

AIM: To investigate the association of FasL gene polymorphism (rs763110) with rheumatoid arthritis occurrence, disease activity, and tumor necrosis factor-α (TNF-α) plasma concentration in Croatian patients, and to conduct an updated meta-analysis. METHODS: This cross-sectional study enrolled 81 patients with rheumatoid arthritis and 94 control patients. After the assessment of the Disease Activity Score (DAS)-28, blood was taken for analysis. DNA was isolated from the whole blood to determine FasL polymorphism (rs763110) by polymerase chain reaction. Protein levels of TNF-α were determined with ELISA. After a detailed literature search, we conducted an updated meta-analysis using the Review Manager 5 software. RESULTS: Rheumatoid arthritis patients had significantly higher TNF-α concentration in plasma (1.65 [1.2-2.42] pg/mL) than controls (0.99 [0.77-1.35] pg/mL, P<0.001). The FasL rs763110 polymorphism was not associated with rheumatoid arthritis occurrence in either codominant, dominant, recessive, overdominant, or log additive model. Furthermore, the rs763110 genotype was not associated with DAS 28 score or TNF-α concentration. After we added our results to an updated meta-analysis, the significant association previously reported for Western Eurasians was abolished. CONCLUSION: Our data suggest that the association between FasL rs763110 polymorphism and RA susceptibility in Western Eurasians observed in previous studies might be overestimated and should be limited to the population of Southwestern Asia until further investigations are performed.

Combined manual and automated immunophenotypisation identified disease-specific peripheral blood immune subpopulations in rheumatoid arthritis, ankylosing spondylitis and psoriatic arthritis.

OBJECTIVES: Rheumatoid arthritis (RA), ankylosing spondylitis (AS) and psoriatic arthritis (PsA) are associated with abnormal immune cell functions. We combined manual and automated profiling in subpopulations of T-cells, B-cells and monocytes, in parallel to functional testing and clinical correlation. METHODS: Using flow cytometry, we analysed the expression of CCR4, CCR6 and CXCR5 on helper and cyotoxic T-cells, CD32B and CD86 on naïve and memory B-cells, and CCR1, CCR2, CCR4 and CXCR4 on monocytes in chronic high-disease activity patients to identify peripheral blood subpopulations. Cell activation, proliferative capability and osteoclastogenic effects were tested in vitro. Comparison with synovial compartment, clinical data and anti-TNF treatment were added to peripheral blood analysis. RESULTS: PsA had lower double-negative T-cell frequency, while RA had lower double-positive T-cell frequency and expanded Th1-like and cytotoxic T-cell subsets. CD32B expression was increased on naïve and memory B-cells in AS and associated with disease activity. CCR6+ and CXCR5+ cytotoxic T-cells and CD32B+ naïve and memory B-cells were highly enriched within the synovial compartment. T-cells and B-cells from AS exhibited enhanced activation and proliferation in vitro, whereas T-cell conditioned medium from RA produced an increased osteoclastogenic effect. CCR1 and CXCR4 were upregulated on osteoclastogenic monocyte subsets of RA, AS and PsA patients. Bioinformatic Citrus analysis identified additional T-cell, B-cell and monocyte clusters specifically associated with each disease. CONCLUSIONS: By combining manual and automated data analysis, our study revealed several disease-specific immune cell subpopulations, particularly cytotoxic T-cell subsets in RA and memory B-cell subsets in AS, which may serve as an indicator of active disease or possible therapeutic target.

SONOELASTOGRAPHIC FEATURES OF HIGH-RISK BREAST LESIONS AND DUCTAL CARCINOMA IN SITU - A PILOT STUDY.

The aim of this study was to evaluate the quantitative sonoelastographic values recorded on shear-wave sonoelastography (SWE) of high-risk breast lesions and ductal carcinoma in situ (DCIS). We retrospectively analyzed histopathologic and SWE data (quantitative maximum, minimum and mean stiffness, lesion-to-fat ratio (E-ratio), lesion size) of 228 women referred to our Department for core needle breast biopsy during a four-year period. Among 230 lesions, histopathologic findings showed 34 high-risk breast lesions and 29 DCIS, which were compared with 167 ductal invasive carcinomas. High-risk lesions had lower values of all sonoelastographic features than ductal in situ and invasive carcinoma, however, only E-ratio showed a statistically significant difference in comparison to DCIS (3.7 vs. 6, p<0.001). All sonoelastographic features showed significant difference between in situ and invasive carcinoma. There was a significant correlation between lesion size and stiffness (r=0.36; p<0.001). Stiffness measured by SWE is an effective predictor of the histopathologic severity of sonographically detectable breast lesions. Elasticity values of high-risk lesions are significantly lower than those of malignant lesions. Furthermore, we showed that along with the sonographic appearance, which in most cases shows typical microcalcifications, DCIS had significantly different elasticity parameters than invasive carcinoma.

Higher Post-Operative Serum Vitamin D Level is Associated with Better Survival Outcome in Colorectal Cancer Patients.

25-Hydroxyvitamin D (25-OHD) may have a prognostic value in colorectal cancer (CRC) patients. However, as 25-OHD concentration is strongly impacted by surgery, it is uncertain what is the most reliable time-point for 25-OHD assessment, pre- or post-operative. Therefore, we examined 515 CRC patients (AJCC I-III) who underwent surgery. Blood samples were collected either pre-operatively (n = 286; median = 1 day before surgery) or post-operatively (n = 229; median = 8 days). Serum 25-OHD concentration was determined by liquid chromatography-tandem mass spectrometry. Association between 25-OHD and survival was tested in the whole cohort, followed by stratified analyses in pre- and post-operatively sampled. Median 25-OHD in the cohort was 36.7 nmol/L and median follow-up time was 5.9 years. There were no differences between pre- and post-operative cohort in age, sex, 25-OHD, AJCC stage, or localization of tumor. After adjustment, higher 25-OHD (>50 nmol/L) was associated with better overall survival only in post-operative (HR = 0.53; 95% CI: 0.33-0.84; P = 0.006), but not in pre-operative cohort (HR = 1.13; 95% CI: 0.77-1.65; P = 0.53). In conclusion, higher post-operative 25-OHD levels were associated with better survival outcome in CRC patients, while no such association was found for pre-operative levels. Time-point of blood collection should be addressed carefully in future research as it might affect the prognostic value of 25-OHD in CRC.