Simple, fast, cost-efficient, reliable, and highly automated DNA content analysis of cells in adherent cultures.

The most commonly used flow cytometric (FCM) analysis of cellular DNA content relies on ethanol fixation followed by RNA digestion and propidium iodide (PI) intercalation into double-stranded DNA. This is a laborious and time-consuming procedure that is subject to systematic errors due to centrifugation and washing steps associated with sample preparation. It can adversely affect the reliability of the results. Here, we present a modified concept of DNA quantification in adherent cell lines by FCM that involves neither ethanol fixation nor any washing and cell transferring steps. Our high throughput assay of adherent cell lines reduces sample-processing time, requires minimal workload, provides a possibility for automation, and, if needed, also allows a significant reduction in the size of individual samples. Working with a well-proven commercial tool-The BD Cycletest™ Plus DNA Reagent Kit-primarily designed for cell cycle analysis and aneuploidy determination in experimental and clinical samples, we suggest a novel, very efficient, and robust approach for DNA research in adherent cell cultures.

Differential diagnosis of increased Erythrocyte Sedimentation rate.

The paper is aimed at differential diagnosis of increased sedimentation rate (ESR) from the point of internal medicine. After the interpretation of the term we describe the technique of the examination and possible errors in pre-analytical as well as analytical phase. The paper includes ranges for conventional FW assessment (analysis of ESR based on Fahraeus-Westergren) and the characteristics of newer methods. We list the overview of the most common causes that affect faster or slower ESR. The stress is put on the assessment of the causes of increased ESR and its persistence from the perspective of clinical practice, we also describe diseases with slower ESR. Attention is drawn to the comparison of the results of the most common acute phase reactants, especially to discordant results of ESR, CRP and procalcitonin in the serum, and to the contribution of the analysis of ESR and CRP in selected diseases. The final part is aimed at the correct diagnostic approach when assessing increased ESR of unknown etiology, underlining the significance of the patient´s history, physical examination and the position of basic as well as complementary laboratory methods and examinations including imaging techniques.

The beneficial effect of csDMARDs co-medication on drug persistence of first-line TNF inhibitor in rheumatoid arthritis patients: data from Czech ATTRA registry.

The study aimed to compare treatment retention for first-line TNF inhibitor (TNFi) in the ATTRA registry patients receiving either combination with conventional synthetic DMARDs or TNFi as monotherapy. A retrospective multicenter study analyzed data of all adult patients with rheumatoid arthritis (n = 3032) starting TNF inhibitor as the first-line biological therapy in combination with csDMARDs or in monotherapy from January 1st 2012 to December 31st 2020. Kaplan-Meier method was employed to calculate drug retentions. Survival curves of treatment retentions were compared through Log-rank test between the studied subgroups. The hazard ratio for drug discontinuation was assessed through univariate cox regression models. In patients who started the first line TNFi therapy, the median treatment retention was 47.7 (42.2; 53.1) months for combination therapy and 22.7 (14.9; 30.6) months for TNFi monotherapy (p < 0.001). Estimated one-year survival was higher in patients on TNFi combined with csDMARDs as compared with TNFi monotherapy (75.3% vs 65.7%); two-year survival rate was 63.2% vs 49.2%, three-year survival rate was 55.4% vs 42.4% and five-year survival 44.9% vs 26.4% of patients. The estimated survival on the first TNFi was higher in patients taking combination therapy with methotrexate than with other csDMARDs (p = 0.003). Use of csDMARDs co-medication was associated with significantly better first TNFi drug survival compared to monotherapy. The combination of TNFi with MTX is more effective than the combination with leflunomide, which did not demonstrate a significant effect.

News in the treatment of axial spondyloarthritis.

Axial spondyloarthritis is a rheumatic disease characterized by inflammation and bone formation causing impaired function of the spine and affected joints. Basic research has highlighted the key role of dysregulation of tumor necrosis factor α, interleukin- 23 and interleukin-17 cytokine production in the etiology of axial spondyloarthritis. Tumor necrosis factor α and interleukin-17 inhibitors have been shown to be effective in clinical trials and are currently approved biological disease-modifying drugs for all disease subgroups. The presumed efficacy of IL-23 blockade has not been confirmed in two clinical trials with anti-IL-23 monoclonal antibodies. Janus kinase inhibitors appear to be a new treatment option.

Acute kidney injury due to antimicrobial therapy.

One of the common causes of acute kidney injury (AKI) is drug nephrotoxicity. A large group of drugs associated with AKI includes a considerable number of antimicrobials. Clinical manifestations range from mild forms of tubular damage to significant deterioration of renal function requiring renal replacement therapy. Several mechanisms have been described, although the most common are acute interstitial nephritis, acute tubular necrosis, crystalic nephropathy or proximal/distal tubulopathy with electrolyte abnormalities. General risk factors for antimicrobial-induced AKI include pre-existing chronic kidney disease and concomitant use of drugs with nephrotoxic potential. Prevention and early recognition of AKI are the standard approach to mitigate AKI and avoid morbidity.

Treatment of giant cell arteritis - current approach and new possibilities.

Giant Cell Arteritis (GCA) is an autoimmune mediated systemic vasculitis affecting large arteries - the aorta and its branches. It has the highest incidence of all systemic vasculitides and manifests nearly exclusively in patients aged 50 or older. Amongst its non-specific and specific symptoms are headaches, mastication claudication or signs of rheumatic polymyalgia, a relatively common and immediate treatment requiring condition being acute vision loss due to optic ischemia. A GCA diagnosis is based on clinical and paraclinical findings and imaging techniques including PET/CT; with an important role still being played by histological verification from temporal artery biopsy. Treatment is based on immunosuppressive agents - systemic glucocorticoids, with adjunct therapy options being methotrexate and tocilizumab. Currently, there are also several clinical trials examining the efficacy of other modern biological agents in GCA.

Early diagnosis of systemic scleroderma.

Systemic scleroderma (SSc) is a systemic immune-mediated connective tissue disease characterized by fibroproductive changes in connective tissue and microvascular disorders. The disease affects the skin, musculoskeletal system and internal organs. It is a disease with a significant rate of morbidity and mortality, significantly worsening the quality of life of patients. Early initiation of therapy is necessary to prevent disease progression. This review article discusses the current possibilities of early diagnosis of systemic scleroderma.

Diffuse alveolar hemorrhage as a life threatening manifestation of newly diagnosed granulomatosis with polyangiitis following COVID-19 infection - a case report.

A case report of a patient with newly diagnosed granulomatosis with polyangiitis (GPA) after undergoing COVID-19 (Coronavirus Disease 2019) is discussed. GPA is one of the ANCA-associated vasculitis, which is characterized by the presence of autoantibodies against cytoplasmic enzymes neutrophils (Anti Neutrophil Cytoplasmatic Antibodies). It is a vasculitis that mainly affects small blood vessels, leading to damage to the kidneys, lungs, and upper respiratory tract, including the paranasal sinuses and orbits. This disease can result in an acute life-threatening condition. Such complications include diffuse alveolar hemorrhage (DAH), a condition characterized by blood leakage from the pulmonary vessels into the alveoli, often leading to acute vital signs and even respiratory failure. DAH can have many causes - autoimmune diseases including vasculitides as well as non-immunological causes. Early and adequate comprehensive therapy including immunosuppressive treatment (cyclophosphamide/rituximab and glucocorticoids) can be life-saving.

Overlap syndrome SLE - ANCA associated vasculitis.

Incidence of ANCA antibodies in patients with systemic lupus erythematosus (SLE) is described in 24-31 %, but they are not related to the distribution and severity of organ involvement in SLE; the routine monitoring is not recommended. Overlap syndrome of systemic lupus erythematosus and ANCA associated vasculitis (AAV) is rare. The difficult diagnosis and treatment of this syndrome is described in this case report of the patient with SLE and severe kidney involvement resulting from AAV.

Revealed heterogeneity in rheumatoid arthritis based on multivariate innate signature analysis.

OBJECTIVES: A growing body of evidence highlights the persistent activation of the innate immune system and type I interferon (IFN) signature in the pathogenesis of rheumatoid arthritis (RA) and its association with disease activity. Since the recent study revealed heterogeneity in the IFN signature in RA, we investigated for the first time the heterogeneity in innate signature in RA. METHODS: The innate gene expression signature (10 TLRs, 7 IL1/IL1R family members, and CXCL8/IL8) was assessed in peripheral blood mononuclear cells from RA patients (n=67), both with active (DAS28≥3.2, n=32) and inactive disease (DAS28<3.2, n=35), and in healthy control subjects (n=55). RESULTS: Of the 13 deregulated innate genes (TLR2, TLR3, TLR4, TLR5, TLR8, TLR10, IL1B, IL1RN, IL18, IL18R1, IL1RAP, and SIGIRR/IL1R8) associated with RA, TLR10 and IL1RAP are being reported for the first time. Multivariate analysis based on utilising patient similarity networks revealed the existence of four patient's subsets (clusters) based on different TLR8 and IL1RN expression profiles, two in active and two in inactive RA. Moreover, neural network analysis identified two main gene sets describing active RA within an activity-related innate signature (TLR1, TLR2, TLR3, TLR7, TLR8, CXCL8/IL8, IL1RN, IL18R1). When comparing active and inactive RA, upregulated TLR2, TLR4, TLR6, and TLR8 and downregulated TLR10 (P<0.04) expression was associated with the disease activity. CONCLUSIONS: Our study on the comprehensive innate gene profiling together with multivariate analysis revealed a certain heterogeneity in innate signature within RA patients. Whether the heterogeneity of RA elucidated from diversity in innate signatures may impact the disease course and treatment response deserves future investigations.

Vitamin D deficiency and its health effects.

Vitamin D3 cholecalciferol is produced from its cholesterol precursors in the skin under the influence of ultraviolet calc. Its subsequent hydroxylation in the liver and kidneys leads to the formation of its most active metabolite calcitriol, which plays one of the key roles in the management of calcium phosphate metabolism. However, it also has the ability to regulate the function of a number of cells and tissues that express the vitamin D receptor. The most widespread method to evaluate the status of vitamin D in the body is to measure serum levels of its meta-bolite 25 hydroxyvitamin D - 25 (OH) D. Optimal values range between 75-125 nmoll / l. Its deficit is widespread in the human population worldwide and has a significant impact on the prevalence of metabolic bone diseases. Its deficiency may support the dysfunction of many other body systems. Ensuring optimal levels of vitamin D in the popula-tion is a challenge not only for health care and especially for government administration.

Inducibly decreased MITF levels do not affect proliferation and phenotype switching but reduce differentiation of melanoma cells.

Melanoma arises from neural crest-derived melanocytes which reside mostly in the skin in an adult organism. Epithelial-mesenchymal transition (EMT) is a tumorigenic programme through which cells acquire mesenchymal, more pro-oncogenic phenotype. The reversible phenotype switching is an event still not completely understood in melanoma. The EMT features and increased invasiveness are associated with lower levels of the pivotal lineage identity maintaining and melanoma-specific transcription factor MITF (microphthalmia-associated transcription factor), whereas increased proliferation is linked to higher MITF levels. However, the precise role of MITF in phenotype switching is still loosely characterized. To exclude the changes occurring upstream of MITF during MITF regulation in vivo, we employed a model whereby MITF expression was inducibly regulated by shRNA in melanoma cell lines. We found that the decrease in MITF caused only moderate attenuation of proliferation of the whole cell line population. Proliferation was decreased in five of 15 isolated clones, in three of them profoundly. Reduction in MITF levels alone did not generally produce EMT-like characteristics. The stem cell marker levels also did not change appreciably, only a sharp increase in SOX2 accompanied MITF down-regulation. Oppositely, the downstream differentiation markers and the MITF transcriptional targets melastatin and tyrosinase were profoundly decreased, as well as the downstream target livin. Surprisingly, after the MITF decline, invasiveness was not appreciably affected, independently of proliferation. The results suggest that low levels of MITF may still maintain relatively high proliferation and might reflect, rather than cause, the EMT-like changes occurring in melanoma.

Widespread Expression of Hedgehog Pathway Components in a Large Panel of Human Tumor Cells and Inhibition of Tumor Growth by GANT61: Implications for Cancer Therapy.

The sonic Hedgehog/GLI signaling pathway (HH) is critical for maintaining tissue polarity in development and contributes to tumor stemness. Transcription factors GLI1⁻3 are the downstream effectors of HH and activate oncogenic targets. To explore the completeness of the expression of HH components in tumor cells, we performed a screen for all HH proteins in a wide spectrum of 56 tumor cell lines of various origin using Western blot analysis. Generally, all HH proteins were expressed. Important factors GLI1 and GLI2 were always expressed, only exceptionally one of them was lowered, suggesting the functionality of HH in all tumors tested. We determined the effect of a GLI inhibitor GANT61 on proliferation in 16 chosen cell lines. More than half of tumor cells were sensitive to GANT61 to various extents. GANT61 killed the sensitive cells through apoptosis. The inhibition of reporter activity containing 12xGLI consensus sites by GANT61 and cyclopamine roughly correlated with cell proliferation influenced by GANT61. Our results recognize the sensitivity of tumor cell types to GANT61 in cell culture and support a critical role for GLI factors in tumor progression through restraining apoptosis. The use of GANT61 in combined targeted therapy of sensitive tumors, such as melanomas, seems to be immensely helpful.

[Life threatening manifestations of lupus and antiphospholipid syndrome in internal medicine]. / Zivot ohrozující projevy systémového lupusu a antifosfolipidového syndromu ve vnitrním lékarství.

The clinical picture of systemic lupus and antiphospholipid syndrome is remarkably varied and disease manifestations are commonly very heterogeneous. Relatively often both diseases are associated with severe, acute and life threatening manifestations, which places demands on the knowledge of differential diagnostics and experience of the physicians. This article deals with the serious and mostly acute impairment of cardiovascular, respiratory, renal, gastrointestinal, hematopoietic or nervous systems, briefly discusses the acute pregnancy complication and summarizes the basic therapeutic option. It emphasizes the role of both, sometimes inseparable, diseases in differential diagnosis of acute symptoms in internal medicine.Key words: clinical symptoms - diagnostics - live threatening manifestations - lupus erythematosus - systemic antiphospholipid syndrome - therapy.

Serum protein pattern associated with organ damage and lupus nephritis in systemic lupus erythematosus revealed by PEA immunoassay.

BACKGROUND: Systemic lupus erythematosus (SLE) is a remarkably heterogeneous autoimmune disease. Despite tremendous efforts, our knowledge of serum protein patterns in severe SLE phenotypes is still limited. We investigated the serum protein pattern of SLE, with special emphasis on irreversible organ damage and active lupus nephritis (LN) as assessed by renal Systemic Lupus Erythematosus Disease Activity Index. METHODS: We used proximity extension immunoassay (PEA, Proseek Multiplex, Olink) to assess the serum levels of ninety-two inflammation-related proteins in Czech patients with SLE (n = 75) and age-matched healthy control subjects (n = 23). Subgroup analysis was carried out on the basis of organ damage (with/without, 42/33) and biopsy-proven LN (with/without, 27/48; active LN, n = 13; inactive LN, n = 14). RESULTS: Of thirty deregulated proteins between SLE and the healthy controls (Pcorr  < 0.05), the top upregulated proteins in SLE were sirtuin 2, interleukin 18 (IL18), and caspase 8 (Pcorr  < 0.0006). Of these, sirtuin 2 and caspase 8 had not yet been reported with SLE. Elevated levels of IL8, CCL2/MCP1, CCL11, and MMP10 (Pcorr  < 0.05) were detected in patients with organ damage for which the serum levels of CCL11 and MMP10 were particularly informative in organ damage prediction. Comparing patients based on LN, elevated levels of CSF1, sIL15RA, sCD40, sCX3CL1, caspase 8, sIL18R1, bNGF, and GDNF (Pcorr  < 0.05) were detected in active LN. Except GDNF, all LN-associated markers showed usefulness in prediction of active renal disease. CONCLUSIONS: This highly sensitive PEA analysis identified the serum pattern of SLE, organ damage, and active LN, with many novel candidate proteins detected. Their exact role and suitability as biomarkers in SLE deserve further investigation.

Porcine EEF1A1 and EEF1A2 genes: genomic structure, polymorphism, mapping and expression.

Eukaryotic translation elongation factor 1 alpha (EEF1A) plays a key role in protein synthesis. In higher vertebrates EEF1A occurs in two isoforms, EEF1A1 and EEF1A2, encoded by distinct genes. The purpose of this study was to compare the two porcine genes as for the genomic sequence, gene organization and mRNA expression in different tissues, as well as to search for polymorphism and chromosomal assignment. Standard methods of DNA and mRNA analysis were used. We determined the complete genomic sequence of the porcine EEF1A1 and EEF1A2 genes. The two genes differ in the lengths of transcription units (3102 and 8588 bp, respectively), but have similar genomic organization and their coding sequences are highly similar (78% identity of coding sequences and 92.4% identity of amino acid sequences). Several polymorphisms in the two genes were detected. EEF1A1 and EEF1A2 were mapped to SSC1p11.1 and SSC17q23.3, respectively. mRNA of EEF1A1 was expressed in all studied tissues (the highest expression was in 44-day fetal muscle and low expression in adult liver and brain), while EEF1A2 was expressed only in skeletal-muscle, tongue, heart, diaphragm and brain tissues. EEF1A2 was not expressed in fetal muscle tissue (44 days). In this paper results are provided on genomic sequences, genomic organization, polymorphism, chromosomal assignment and spatial and temporal expressions of the porcine EEF1A1 and EEF1A2 genes. Novel polymorphisms were described in both genes. Porcine EEF1A2 was studied for the first time.

[Adult-onset Stills disease - a difficult path to diagnosis through fever and effusions of unknown origin]. / Stillova nemoc dospelých - obtízná cesta k diagnóze pres horecku a výpotky nejasné etiologie.

Fever of unknown origin, pleural and pericardial effusions can be caused by a variety of independent agents. On the other hand, we can identify a common causative condition in other cases. Infectious diseases, malignancies and autoimmune diseases are the most common etiological factors. Considering the pleural and pericardial effusion, we also have to think of cardiovascular and pulmonary diseases. The basis of every diagnostic process is thorough medical history and detailed clinical examination followed by laboratory and imaging methods. In spite of that, the right diagnosis sometimes stays long time hidden. One of such conditions is Adult-onset Stills disease (AOSD). It is a rare inflammatory, potentially life-threatening disease with unclear pathogenesis and heterogeneous symptoms. It has some features similar to systemic form of juvenile idiopathic arthritis. Diagnosis is established so called per exclusionem by fulfilling a set of clinical criteria and ruling out other diseases with similar symptomatology. In our article, we present an example of such an arduous diagnostic journey to final diagnosis.

Acute kidney injury due to gentamicin nephrotoxicity and specific miRNAs as biomarkers.

Acute kidney injury (AKI) due to gentamicin nephrotoxicity is a significant concern in clinical medicine, particularly in patients receiving prolonged or high-dose gentamicin therapy. Gentamicin is an aminoglycoside antibiotic frequently used in the treatment of a range of bacterial infections. However, its use is associated with nephrotoxicity which can manifest as AKI. Due to this, it is crucial to diagnose promptly and manage treatment effectively. Ongoing studies are therefore focusing on non-protein-coding RNAs as potential biomarkers for AKI. Numerous microRNAs (miRNAs) have been implicated in gentamicin-induced nephrotoxicity and AKI. They participate in pathways associated with inflammation, cell death, and oxidative stress and each of these factors play critical roles in the development of gentamicin-induced kidney injury. Research studies have demonstrated changes in the expression levels of these miRNAs in response to gentamicin exposure both in vitro and in in vivo models, as well as in human clinical trials involving patients receiving gentamicin therapy. The dysregulation of these miRNAs correlates with the severity of kidney injury and may serve as sensitive biomarkers for early detection and monitoring of AKI induced by gentamicin.

Antigenicity and immunogenicity of different morphological forms of Borrelia burgdorferi sensu lato spirochetes.

Borrelia burgdorferi sensu lato is a species complex of pleomorphic spirochetes, including species that cause Lyme disease (LD) in humans. In addition to classic spiral forms, these bacteria are capable of creating morphological forms referred to as round bodies and aggregates. The subject of discussion is their possible contribution to the persistence of infection or post-infection symptoms in LD. This study investigates the immunological properties of these forms by monitoring reactivity with early (n = 30) and late stage (n = 30) LD patient sera and evaluating the immune response induced by vaccination of mice. In patient sera, we found a quantitative difference in reactivity with individual morphotypes, when aggregates were recognized most intensively, but the difference was statistically significant in only half of the tested strains. In post-vaccination mouse sera, we observed a statistically significant higher reactivity with antigens p83 and p25 (OspC) in mice vaccinated with aggregates compared to mice vaccinated with spiral forms. The importance of the particulate nature of the antigen for the induction of a Th1-directed response has also been demonstrated. In any of morphological forms, the possibility of inducing antibodies cross-reacting with human nuclear and myositis specific/associated autoantigens was not confirmed by vaccination of mice.