NGS analysis of collagen type I genes in Polish patients with Osteogenesis imperfecta: a nationwide multicenter study.

Osteogenesis imperfecta (OI) is a rare genetic disorder of the connective tissue. It presents with a wide spectrum of skeletal and extraskeletal features, and ranges in severity from mild to perinatal lethal. The disease is characterized by a heterogeneous genetic background, where approximately 85%-90% of cases have dominantly inherited heterozygous pathogenic variants located in the COL1A1 and COL1A2 genes. This paper presents the results of the first nationwide study, performed on a large cohort of 197 Polish OI patients. Variants were identified using a next-generation sequencing (NGS) custom gene panel and multiplex ligation probe amplification (MLPA) assay. The following OI types were observed: 1 (42%), 2 (3%), 3 (35%), and 4 (20%). Collagen type I pathogenic variants were reported in 108 families. Alterations were observed in α1 and α2 in 70% and 30% of cases, respectively. The presented paper reports 97 distinct causative variants and expands the OI database with 38 novel pathogenic changes. It also enabled the identification of the first glycine-to-tryptophan substitution in the COL1A1 gene and brought new insights into the clinical severity associated with variants localized in "lethal regions". Our results contribute to a better understanding of the clinical and genetic aspects of OI.

CXCL8, CCL2, and CMV Seropositivity as New Prognostic Factors for a Severe COVID-19 Course.

The exact pathophysiology of severe COVID-19 is not entirely elucidated, but it has been established that hyperinflammatory responses and cytokine storms play important roles. The aim of this study was to examine CMV status, select chemokines, and complement components in COVID-19, and how concentrations of given molecules differ over time at both molecular and proteomic levels. A total of 210 COVID-19 patients (50 ICU and 160 non-ICU patients) and 80 healthy controls were enrolled in this study. Concentrations of select chemokines (CXCL8, CXCL10, CCL2, CCL3, CCR1) and complement factors (C2, C9, CFD, C4BPA, C5AR1, CR1) were examined at mRNA and protein levels with regard to a COVID-19 course (ICU vs. non-ICU group) and CMV status at different time intervals. We detected several significant differences in chemokines and complement profiles between ICU and non-ICU groups. Pro-inflammatory chemokines and the complement system appeared to greatly contribute to the pathogenesis and development of severe COVID-19. Higher concentrations of CXCL8 and CCL2 in the plasma, with reduced mRNA expression presumably through negative feedback mechanisms, as well as CMV-positive status, correlated with more severe courses of COVID-19. Therefore, CXCL8, CCL2, and CMV seropositivity should be considered as new prognostic factors for severe COVID-19 courses. However, more in-depth research is needed.

IL-18 Gene rs187238 and rs1946518 Polymorphisms and Expression in Gingival Tissue in Patients with Periodontitis.

Periodontitis is a chronic disease with disturbed balance between the immune and inflammatory response of the host to bacteria. Many studies have shown that proinflammatory cytokines play a significant role in the pathogenesis of periodontal disease. In this study, we examined the association between the IL-18 gene rs187238 and rs1946518 polymorphisms and periodontitis in non-smoking and smoking patients. This study enrolled 200 patients with periodontitis (130 non-smokers and 70 smokers) and 156 control subjects (124 non-smokers and 32 smokers). There were no statistically significant differences in the distribution of the rs187238 and rs1946518 IL-18 genotypes and alleles between patients with periodontitis and control subjects, between smoking patients with periodontitis and smoking control subjects, and between non-smoking patients with periodontitis and non-smoking control subjects. There were no statistically significant differences in clinical parameters in relation to the IL18 rs187238 genotypes. In patients with the IL18 rs1946518 GG genotype, we observed increased values of bleeding on probing (BoP) and periodontal probing depth (PPD), compared to subjects with the TT genotype. In patients with periodontitis, we observed statistically significant decreased expression of the IL-18 gene in comparison with healthy subjects (0.231 ± 0.163 vs. 0.663 ± 0.197, p = 0.0008). In addition, the IL-18 gene expression in gingival tissue in patients with periodontitis correlated positively with the number of remaining teeth. The results of our study suggest that the IL-18 rs187238 and rs1946518 polymorphisms are not significant risk indicators of periodontitis in our population. However, in patients with the IL18 rs1946518 GG genotype, we observed increased values of BoP and PPD, compared to subjects with the TT genotype. In addition, in gingival tissue of patients with periodontitis, we have detected decreased expression of IL-18. The gingival expression of IL-18 in patients with periodontitis correlated positively with number of remaining teeth. The above results suggest that IL-18, in addition to its pro-inflammatory effects in periodontal disease, may also exhibit protective properties.

Plasma Levels of Interleukins 36α, 36ß, and 37 in Patients with Psoriasis and Their Correlation with Disease Activity Parameters.

Psoriasis is a chronic, proliferative, inflammatory skin disease characterised by skin lesions and systemic symptoms. Numerous cytokines are produced in psoriasis as a result of inflammation. The aim of this study was to examine the plasma concentrations of IL-36α, IL-36ß, and IL-37 in psoriasis and their correlations with disease activity parameters. This study recruited 84 individuals, 53 with plaque-type psoriasis and 31 healthy controls. The plaque type of psoriasis is the most common type and is typically characterized by circular-to-oval red plaques distributed over body surfaces of the extremities and scalp. In patients with psoriasis, we observed statistically significantly decreased plasma concentrations of IL-36ß and IL-37. The concentrations of IL-36α were increased in comparison with control group. The plasma concentrations of IL-36α and IL-36ß were statistically significantly correlated with all tested parameters of disease activity: the Psoriasis Activity Severity Index, Dermatology Life Quality Index, and Body Surface Area Index. There were no statistically significant correlations between plasma levels of IL-37 and the tested parameters of disease activity. These results indicate a role of IL36α, IL-36ß, and IL-37 in the pathogenesis of psoriasis.

Global Gene Expression of Cultured Human Dermal Fibroblasts: Focus on Cell Cycle and Proliferation Status in Improving the Condition of Face Skin.

Chronological skin ageing is an inevitable physiological process that results in thin and sagging skin, fine wrinkles, and gradual dermal atrophy. The main therapeutic approaches to soft tissue augmentation involve using dermal fillers, where natural fillers, such as autologous fibroblasts, are involved in generating dermal matrix proteins. The aim of this study was to determine the global transcriptome profile of three passages of dermal autologous fibroblasts from a male volunteer, focusing on the processes of the cell cycle and cell proliferation status to estimate the optimal passage of the tested cells with respect to their reimplantation. We performed K-means clustering and validation of the expression of the selected mRNA by qRT-PCR. Ten genes were selected (ANLN, BUB1, CDC20, CCNA2, DLGAP5, MKI67, PLK1, PRC1, SPAG5, and TPX2) from the top five processes annotated to cluster 5. Detailed microarray analysis of the fibroblast genes indicated that the cell population of the third passage exhibited the highest number of upregulated genes involved in the cell cycle and cell proliferation. In all cases, the results of qRT-PCR confirmed the differences in expression of the selected mRNAs between fibroblasts from the primary culture (C0) and from the first (C1), second (C2), and third (C3) cell passage. Our results thus suggest that these cells might be useful for increasing fibroblast numbers after reimplantation into a recipient's skin, and the method used in this study seems to be an excellent tool for autologous transplantation allowing the rejuvenation of aging skin.

Comparative Analysis of Global Gene Expression and Complement Components Levels in Umbilical Cord Blood from Preterm and Term Neonates: Implications for Significant Downregulation of Immune Response Pathways related to Prematurity.

Background: Preterm birth is the most frequent cause of neonatal death, but its aetiology remains unclear. It has been suggested that the imbalance of immunological mechanisms responsible for maintaining pregnancy is contributing to preterm birth pathogenesis. We aimed to investigate global gene expression and the levels of several complement system components in umbilical cord blood samples from preterm neonates and compare them to term newborns. We sought to examine how differentially expressed genes could affect various immune-related pathways that are believed to be crucial factors in preterm birth. Material and methods: We enrolled 27 preterm infants (<37 weeks GA) and 52 term infants (>37 weeks GA), from which umbilical cord blood samples were collected. From these samples, peripheral blood mononuclear cells were isolated and subsequent RNA isolation was performed. We used Affymetrix Human Gene 2.1 ST Array Strip for microarray experiment and DAVID resources for bioinformatics analysis of the obtained data. Concentrations of C2, C3a, C5/C5a, C9, FactorD, Properdin were measured in umbilical cord blood plasma samples using multiplex fluorescent bead-based immunoassays using Luminex technology. Results: The levels of C3a and C5/5a were significantly elevated in preterm neonates compared to term babies, whereas C9 concentration was evidently increased in term infants. The expression of 250 genes was upregulated at least 2-fold and 3781 genes were downregulated at least 2-fold in preterm neonates in comparison with term infants. Functional annotation analysis revealed that in preterm infants in comparison to term babies there was a significant downregulation of genes encoding several Toll-like receptors, interleukins and genes involved in major signalling pathways (e.g. NF-κB, MAPK, TNF, Notch, JAK) and vital cellular processes (e.g. intracellular signal transduction, protein ubiquitination, protein transport, RNA splicing, DNA-templated transcription). Conclusions: Preterm birth results in immediate and long-term complications. Our results indicate that infants born prematurely show significant differences in complement components concentration and a downregulation of over 3,000 genes, involved mainly in various immune-related pathways, including innate immune response, phagocytosis and TLR function, when compared to full-term babies. Further studies on larger cohorts are needed to elucidate the role of immunity in prematurity.

Differential Secretion of Angiopoietic Factors and Expression of MicroRNA in Umbilical Cord Blood from Healthy Appropriate-For-Gestational-Age Preterm and Term Newborns-in Search of Biomarkers of Angiogenesis-Related Processes in Preterm Birth.

Objectives: Premature birth, defined as less than 37 weeks gestation, affects approximately 12% of all live births around the world. Advances in neonatal care have resulted in the increased survival of infants born prematurely. Although prematurity is a known risk factor for different cardiovascular diseases, little is known about the pathophysiology of vasculature during premature gestation and angiopoietic factors network during premature birth. Aims: The objective of this study was to determine whether the profile of several pro-angiogenic and anti-angiogenic factors in umbilical cord blood (UCB) is different in healthy appropriate-for-gestational-age preterm newborns and normal term babies. The second aim of this study was to investigate the microRNA (miRNAs) expression profile in UCB from preterm labor and to detect miRNAs potentially taking part in control of angogenesis-related processes (Angio-MiRs). Methods: Using an immunobead Luminex assay, we simultaneously measured the concentration of Angiogenin, Angiopoietin-1, FGF-acidic, FGF-basic, PDGF-aa, PlGF, VEGF, VEGF-D, Endostatin, Thrombospondin-2, NGF, BDNF, GDNF, and NT-4 in UCB samples collected from the preterm (n = 27) and term (n = 52) delivery. In addition, the global microRNA expression in peripheral blood mononuclear cells (PBMCs) circulating in such UCB samples was examined in this study using microarray MiRNA technique. Results: The concentrations of five from eight measured pro-angiogenic factors (VEGF, Angiopoietin-1, PDGF-AA, FGF-a, and FGF-b) were significantly lower in UCB from preterm newborns. On the contrary, two angiostatic factors (Endostatin and Thrombospondin-2) were significantly up-regulated in preterm UCB. Among analyzed neurotrophins in preterm newborns, the elevated UCB concentration was found only in the case of GDNF, whereas BDNF was significantly reduced. Moreover, two angiopoietic factors, VEGF-D and PlGF, and two neurotrophins, NT4 and NGF, did not differ in concentration in preterm and term babies. We also discovered that among the significantly down-regulated miRNAs, there were several classical Angio-MiRs (inter alia MiR-125, MiR-126, MiR-145, MiR-150, or MiR155), which are involved in angiogenesis regulation in newborn after preterm delivery. Conclusions: This is the first report of simultaneous measurements of several angiopoietic factors in UCB collected from infants during preterm and term labor. Here, we observed that several pro-angiogenic factors were at lower concentration in UCB collected from preterm newborns than term babies. In contrast, the two measured angiostatic factors, Endostatin and Thrombospondin-2, were significantly higher in UCB from preterm babies. This can suggest that distinct pathophysiological contributions from differentially expressed various angiopoietic factors may determine the clinical outcomes after preterm birth. Especially, our angiogenesis-related molecules analysis indicates that preterm birth of healthy, appropriate-for-gestational-age newborns is an "anti-angiogenic state" that may provide an increased risk for improper development and function of cardiovascular system in the adulthood. This work also contributes to a better understanding of the role of miRNAs potentially involved in angiogenesis control in preterm newborns.

Transcriptome Profile of Human Fibroblasts in an Ex Vivo Culture.

Implantation of autologous fibroblasts is a method used to correct age-related changes in facial skin. The aim of this study was to establish the optimal population of cultured human fibroblasts according to the organization of the extracellular matrix in the dermis. Transcriptome profile analysis of cells derived from three consecutive passages indicated that fibroblasts after the second passage were the population with the greatest number of upregulated genes encoding the critical biological processes responsible for skin regeneration, such as extracellular matrix organization, collagen fibril organization, and cell adhesion. Furthermore, genes encoding proteinases responsible for the degradation of dermal extracellular matrix proteins were noticeably downregulated at this stage of culture. Autologous fibroblasts seem to be an optimal and safe biological filler for the renewal of all skin structures.

Expression of selected angiogenesis-related small microRNAs in patients with abnormally increased secretion of glucocorticoids.

INTRODUCTION: Higher cortisol levels are associated with cardiovascular morbidity and mortality in the elderly, partially resulting from biologic effects of glucocorticoids (GCs) on endothelial cells observed in an experimental setting. These features are replicated in patients with endogenous GC excess (Cushing's syndrome) or with exogenous hypercortisolism due to excessive pharmacological application of GCs. Both groups present also an increased cardiovascular disease event rate. GCs may also adversely influence recovery after myocardial infarction. Recently it was proposed that microRNAs (miRNAs) - small noncoding RNAs functioning as antisense regulators of gene expression by targeting mRNA - may have a central role in regulating endothelial function through multiple mechanisms. Thus, the purpose of this study was to evaluate the effects of chronic GC excess on the expression of selected endothelium-controlling miRNAs expressed in nucleated cells circulating in peripheral blood (PBNCs) of patients with endogenous hypercortisolism either due to corticotrophin-independent or corticotrophin-dependent Cushing's syndrome (CS). MATERIAL AND METHODS: Peripheral blood nuclear cells were collected from 35 healthy subjects and 31 patients with endogenous hypercortisolism as a source of miRNAs. A self-validated individual quantitative RT-PCR study was then performed to evaluate the expression levels of selected miRNAs in PBNCs. Additionally, endothelin-1 (ET-1) expression in peripheral blood was assessed with respect to endothelial dysfunction using Western blotting. RESULTS: The ET-1 expression levels in CS were higher than in controls, confirming endothelial dysfunction in the CS group. Furthermore, miRNA analysis revealed a significantly decreased intracellular expression of selected endothelium-related miRNAs in patients with endogenous hypercortisolism, including miRNA-17-5p, miRNA-126-3p, and miRNA-126-5p, compared to controls. In contrast, two other angiogenic miRNAs, miRNA-150-5p and miRNA-223-3p, were significantly upregulated compared to controls. CONCLUSIONS: Cardiovascular events related to hypercortisolism remain a challenging problem in medical practice. This study has demonstrated that the chronic excess of GCs in endogenous CS might induce significant dysregulation of selected miRNAs involved in the control of endothelium biology. However, the lack of knowledge about specific miRNA expression postpones the full understanding of the biological roles of such miRNAs in hypercortisolism. Moreover, dysregulated miRNAs seem to be promising targets for further research, especially to search for potential therapies for several GC-induced cardiovascular complications.

The Impact of Growth Hormone Therapy on the Apoptosis Assessment in CD34+ Hematopoietic Cells from Children with Growth Hormone Deficiency.

Growth hormone (GH) modulates hematopoietic cell homeostasis and is associated with apoptosis control, but with limited mechanistic insights. Aim of the study was to determine whether GH therapeutic supplementation (GH-TS) could affect apoptosis of CD34+ cells enriched in hematopoietic progenitor cells of GH deficient (GHD) children. CD34+ cells from peripheral blood of 40 GHD children were collected before and in 3rd and 6th month of GH-TS and compared to 60 controls adjusted for bone age, sex, and pubertal development. Next, apoptosis assessment via different molecular techniques was performed. Finally, to comprehensively characterize apoptosis process, global gene expression profile was determined using genome-wide RNA microarray technology. Results showed that GH-TS significantly reduced spontaneous apoptosis in CD34+ cells (p < 0.01) and results obtained using different methods to detect early and late apoptosis in analyzed cells population were consistent. GH-TS was also associated with significant downregulation of several members of TNF-alpha superfamily and other genes associated with apoptosis and stress response. Moreover, the significant overexpression of cyto-protective and cell cycle-associated genes was detected. These findings suggest that recombinant human GH has a direct anti-apoptotic activity in hematopoietic CD34+ cells derived from GHD subjects in course of GH-TS.

[The role of ophthalmological examination in the diagnosis of gangliosidosis GM1]. / Znaczenie badania okulistycznego w rozpoznawaniu gangliozydozy GM1.

Gangliosidosis GM1 belongs to a group of lysosomal storage diseases and results from the deficiency of acidic beta-galactosidase activity. The enzyme is essential for the degradation of ganglioside GM1 and its derivatives. The disease causes multi-organ injury, however accumulation of ganglioside GM1 mainly in the brain white and gray matter results in predomination of neurological symptoms. Based on the actual knowledge--the condition is untreatable and especially in the very severe infantile form, the duration of the survival is very short. One of the characteristic symptoms of some lysosomal storage diseases, including gangliosidosis GM1, is "cherry-red" spot found in the fundus of the eye. In the publication the clinical course of gangliosidosis GM1 in two infants is presented. The value of an ophthalmological examination in the diagnosis of this rare condition has been emphasized.

Hypoglycemic potency of novel trivalent chromium in hyperglycemic insulin-deficient rats.

Two sources of chromium III, "chromium 454" and "chromium picolinate," were tested in insulin-deficient Streptozocin-treated diabetic rats. This model was selected in order to evaluate the possibility of any hypoglycemic potency of chromium in a relative absence of blood insulin concentration. Three weeks of the treatment with CRC454 and CrP resulted in a 38% and 11% reduction of blood glucose levels, respectively. Body weight gains were equally improved by both treatments. Blood levels of CK, ALT and AST were significantly reduced by CRC454 and CrP. These results might suggest that any hypoglycemic effect of trivalent chromium under insulin-deficient conditions could be largely dependent upon the type of chromium agent and associated characteristics such as solubility and bioavalibility. In contrast, improvement of body weight gains and blood levels of CK, AST and ALT seems to be less dependent on the type of chromium compound under these experimental conditions. In conclusion, CRC454 showed significant reduction of hyperglycemia under insulin-deficient conditions.