Influence of Clinical and Genetic Factors on the Progression of Age-Related Macular Degeneration: A 3-Year Follow-Up.

The aim of the present study was to analyze the relationship of age-related macular degeneration (AMD) progression with clinical characteristics, demographic, and environmental risk factors that would affect disease development. In addition, the influence of three genetic AMD polymorphisms (CFH Y402H, ARMS2 A69S, and PRPH2 c.582-67T>A) on AMD progression was investigated. In total, 94 participants with previously diagnosed early or intermediate AMD in at least one eye were recalled for an updated re-evaluation after 3 years. The initial visual outcomes, medical history, retinal imaging data, and choroidal imaging data were collected to characterize the AMD disease status. Among the AMD patients, 48 demonstrated AMD progression, and 46 showed no disease worsening at 3 years. Disease progression was significantly associated with worse initial visual acuity (OR = 6.74, 95% CI = 1.24-36.79, p = 0.03) and the presence of the wet AMD subtype in fellow eyes (OR = 3.79, 95%CI = 0.94-15.2, p = 0.05). In addition, a higher risk of AMD progression appeared in the patients with active thyroxine supplementation (OR = 4.77, CI = 1.25-18.25, p = 0.002). The CC variant of CFH Y402H was associated with AMD advancement compared to the TC+TT phenotype (OR = 2.76, 95% CI: 0.98-7.79, p = 0.05). Identifying risk factors of AMD progression may lead to earlier intervention and better outcomes, preventing the expansion of the late stage of the disease.

Effectiveness of Soluble CTLA-4-Fc in the Inhibition of Bone Marrow T-Cell Activation in Context of Indoleamine 2.3-Dioxygenase (IDO) and CD4+Foxp3+ Treg Induction.

Background: Rheumatoid arthritis (RA) is a chronic autoimmune disease with systemic inflammation finally resulting in damaged joints. One of the RA development models suggests bone marrow (BM) as a place of inflammation development further leading to disease progression. We aimed to investigate the potential of CTLA-4-Fc molecule in inducing tolerogenic milieu in BM measured as indoleamine 2,3-dioxygenase (IDO) expression, CD4+Foxp3+ Treg induction, and T cell activation control. The expression of IDO-pathway genes was also examined in monocytes to estimate the tolerogenic potential in the periphery. Methods: Bone marrow mononuclear cells (BMMC) were stimulated by pro-inflammatory cytokines and CTLA-4-Fc. Next IDO expression, CD4+CD69+ and CD4+Foxp3+ percentage were estimated by PCR and FACS staining, respectively. Enzymatic activity of IDO was confirmed by HPLC in BM plasma and blood plasma. Genes expressed in IDO-pathway were analyzed by NGS in peripheral monocytes isolated from RA patients and healthy controls. Results: We found that CTLA-4-Fc and IFN-γ stimulation results in IDO production by BMMC. CTLA-4-Fc induced tryptophan catabolism can inhibit mitogen-induced CD4+ T cells activation without influencing CD8+ cells, but did not control CD25 nor Foxp3 expression in BM cells. Significantly higher expression of selected IDO-pathway genes was detected on peripheral monocytes isolated from RA as compared to healthy controls. Conclusion: This study sheds light on some immunosuppression aspects present or induced in BM. The potential of IDO-mediated pathways were confirmed in the periphery, what may represent the promising candidates for therapeutic strategies in RA.

Retinal Vessel Functionality Is Linked With ARMS2 A69S and CFH Y402H Polymorphisms and Choroidal Status in AMD Patients.

Purpose: We aimed to investigate the reactivity of retinal vessels to a flickering stimulus in patients with age-related macular degeneration (AMD) and healthy participants. We also assessed whether the parameters of retinal vessels are dependent on genetic predisposition. Methods: A total of 354 patients with AMD and 121 controls were recruited for the study. All participants underwent thorough ophthalmologic examination and static and dynamic retinal vessel analysis. AMD risk polymorphisms were genotyped in the CFH and ARMS2 genes. Results: We found no differences between the AMD group and controls in central retinal arteriolar equivalent (CRAE), central retinal venular equivalent (CRVE), arteriovenous ratio (AVR), dynamic analysis of arteries (DAAs), or dynamic analysis of veins (DAVs). Eyes with early AMD presented with significantly higher AVR values than eyes with late AMD. In the AMD group, DAA correlated positively with both choroidal thickness (Rs = 0.14, P = 0.00096) and choroidal volume (Rs = 0.23, P < 0.0001), and no such associations were observed in the controls. We found significantly lower DAA (1.47 ± 1.50) in TT homozygotes for the ARMS2 A69S polymorphism in comparison with GG homozygotes (2.38 ± 1.79) and patients with GG + GT genotypes (2.28 ± 1.84). We also observed less prominent DAV (3.24 ± 1.71) in patients with TC + CC genotypes in the CFH Y402H polymorphism compared with TT homozygotes (3.83 ± 1.68). Conclusions: Our findings suggest that retinal microcirculation appears to be associated with the genetic background, choroidal parameters, and clinical features of the patients with AMD.

Genetic factors associated with age-related macular degeneration: identification of a novel PRPH2 single nucleotide polymorphism associated with increased risk of the disease.

PURPOSE: Age-related macular degeneration (AMD) is associated with multiple environmental and genetic risk factors. Two main risk factors for AMD are variants in the CFH and ARMS2/HTRA1 genes. We investigated over 2000 variants in AMD patients and controls using high-throughput sequencing methods to search for variants associated with AMD. METHODS: A total of 296 AMD patients and 100 controls were enrolled in this study. Genetic analysis was performed with the Illumina NextSeq 500 system. RESULTS: Multivariate analysis of patients and controls, adjusted for age, sex and smoking status (pack-years), revealed that three SNPs were strong risk factors independently associated with AMD: CFH Y402H, ARMS A69S and PRPH2 c.582-67T>A (rs3818086). The TC genotype in CFH Y402H was associated with 1.90-fold higher odds, and the CC genotype was associated with 5.66-fold higher odds of AMD compared with the TT genotype. The GT genotype in ARMS A69S was associated with 2.40-fold higher odds, and the TT genotype was associated with 6.75-fold higher odds of disease compared with the GG genotype. In the case of rs3818086, the A allele could be considered a 'risk' allele, since AA + TA genotypes were associated with 2.33-fold higher odds of AMD compared with the TT genotype. CONCLUSIONS: Although PRPH2 mutations have been previously implicated in various forms of retinal degeneration, to the best of our knowledge, this study is the first to show that the rs3818086 variant increases the risk for AMD more than two times. Further studies on larger cohorts are required to elucidate how this variant affects protein structure.

Associations of microRNAs, Angiogenesis-Regulating Factors and CFH Y402H Polymorphism-An Attempt to Search for Systemic Biomarkers in Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) remains the leading cause of blindness in elderly people, but the pathophysiology of this disease is still largely unknown. We investigated the systemic expression of angiogenesis-regulating growth factors and selected miRNAs known to regulate angiogenesis in AMD patients. We also focused on possible correlations of their expression with the presence of CFH Y402H or ARMS A69S risk variants. A total of 354 AMD patients and 121 controls were enrolled in this study. The levels of angiogenesis-regulating factors were analyzed in plasma samples using Luminex technology. The expression of selected miRNAs was analyzed in peripheral blood plasma using real-time qPCR. The genetic analysis was performed with an Illumina NextSeq500 system. AMD was an independent factor associated with lower levels of angiogenin (ß = -0.29, p < 0.001), endostatin (ß = -0.18, p < 0.001), FGF-basic (ß = -0.18, p < 0.001), PlGF (ß = -0.24, p < 0.001), miRNA-21-3p (ß = -0.13, p = 0.01) and miRNA-155-5p (ß = -0.16, p = 0.002); and with higher levels of FGF-acidic (ß = 0.11, p = 0.03), miRNA-23a-3p (ß = 0.17, p < 0.001), miRNA-126-5p (ß = 0.13, p = 0.009), miRNA-16-5p (ß = 0.40, p < 0.001), miRNA-17-3p (ß = 0.13, p = 0.01), miRNA-17-5p (ß = 0.17, p < 0.001), miRNA-223-3p (ß = 0.15, p = 0.004), and miRNA-93 (ß = 0.11, p = 0.04). The expression of analyzed miRNA molecules significantly correlated with the levels of tested angiogenesis-regulating factors and clinical parameters in AMD patients, whereas such correlations were not observed in controls. We also found an association between the CFH Y402H polymorphism and miRNA profiles, whereby TT homozygotes showed evidently higher expression of miRNA-16-5p than CC homozygotes or TC heterozygotes (p = 0.0007). Our results suggest that the balance between systemic pro- and anti-angiogenic factors and miRNAs is vital in multifactorial AMD pathogenesis.

Studies on the effects of probiotic Lactobacillus mixture given orally on vaginal and rectal colonization and on parameters of vaginal health in women with intermediate vaginal flora.

OBJECTIVES: The vaginal microflora is composed of many bacterial species and plays a major role in maintaining the balance of this complex environment. This study was conducted in order to assess the degree and persistence of the colonization of vaginal epithelium by strains from an orally administered mixture of lactobacilli, containing Lactobacillus fermentum 57A, Lactobacillus plantarum 57B and Lactobacillus gasseri 57C. We also monitored its effects on parameters of vaginal health, especially total lactobacilli counts, vaginal pH and Nugent score. STUDY DESIGN: The patient group in this open study consisted of clinically healthy women with intermediate vaginal flora. Altogether 37 women were included in the study; 25 finished the full cycle consisting of 8 visits during 70 days. Lactobacillus mixture was administered as 1×10(8) c.f.u. once a day for 60 days. Lactobacillus isolates collected from vaginal and rectal samples from studied women during all visits were typed using molecular methods (PFGE for L. fermentum and L. gasseri and MLST for L. plantarum). Total lactobacilli counts, vaginal pH and Nugent score were also determined during the visits. RESULTS: We confirmed that the ingested strains were able to reach and colonize both sites within the third and eighth visits, i.e. between the 20th and 70th days of the study. Maximal colonization was recorded between the fifth and seventh visits (31st-60th days). Moreover, ingestion of the Lactobacillus mixture was related to normalization of vaginal parameters (within 28-60 days after the initiation of the treatment). This was demonstrated by a decrease of vaginal pH and Nugent score together with an increase of total numbers of lactobacilli in the vagina and rectum. No adverse events were noted during the course of the study. CONCLUSIONS: Oral application of the combination of the three probiotic strains derived from vaginal microbiota of healthy woman with high adherence abilities to both vaginal and colonic epithelium in vitro shows that both individual strains and their mixture can colonize vagina for some weeks, the effect of which is correlated with significant improvement of such parameters like pH and Nugent score values and total numbers of vaginal lactobacilli. This indicates that the mixture may be a good candidate for the planned double-blind, placebo-controlled randomized studies involving larger numbers of women.

Involvement of genetic factors in the response to a variable-dosing ranibizumab treatment regimen for age-related macular degeneration.

PURPOSE: To determine whether gene polymorphisms of the major genetic risk factor for age-related macular susceptibility 2 (ARMS2 A69S) and the complement factor H Y402H influence the response to a variable-dosing treatment regimen with ranibizumab for age-related macular degeneration. METHODS: This prospective cohort study included 90 patients (90 eyes) with exudative age related macular degeneration (AMD) treated with ranibizumab. Patients underwent a 1-year treatment as in the Study of Ranibizumab in Patients with Subfoveal Choroidal Neovascularization Secondary to Age-Related Macular Degeneration (Mitchell et al.). Injections were administered monthly when a patient lost five letters on the Early Treatment Diabetic Retinopathy Study chart or gained 100 µm in central subfield retinal thickness (CSRT). Genotypes (rs10490924 and rs1061170) were analyzed using gene sequence analysis. Best-corrected visual acuity (BCVA) and CSRT values were compared between ARMS2 and complement factor H genotypes. Multiple regression analysis was used to assess the statistical significance. RESULTS: Mean increase in visual acuity was 4.44±8.12 letters with a 103.63±94.7 µm decrease in CSRT. BCVA improvement was statistically significant in all genotype groups except in homozygous 69S in the AMRS2 gene. CSRT and BCVA changes were correlated (r=0.2521; 95% CI: 0.04746-0.4364, p=0.0165). Multiple regression analysis revealed a significant impact of 69S (p=0.015) on the change in BCVA. CONCLUSIONS: Visual acuity did not improve during the study in patients homozygous for ARMS2 69S, despite a decrease in CSRT. Further investigation is needed to confirm our findings and understand the mechanisms involved.

Elements regulating Potato leafroll virus sgRNA1 translation are located within the coding sequences of the coat protein and read-through domain.

Translation of viral proteins from subgenomic RNAs (sgRNAs) is a common strategy among positive-stranded RNA viruses. Unlike host mRNA, sgRNA of Potato leafroll virus (PLRV) does not possess a cap at its 5' end nor a poly(A) tail at the 3' terminus, both of which are known to be crucial for translation of RNA in eukaryotic cells. Here, we demonstrate, that in wheat germ extract (WGE) truncation of the sgRNA1 5' UTR increases translation efficiency, as it has previously been observed in rabbit reticulocyte lysate (RRL), whereas removal of the 3' UTR does not affect translation. We also describe two regulatory elements located within the coding sequence of the coat protein (CP) gene and its read-through domain (RTD) and are responsible for regulation of in vitro translation of the PLRV sgRNA1. The frst element is composed of the purine sequence AAAGGAAA located between the AUG codons of the CP and 17K genes. Deletion of this domain or its substitution by pyrimidines reduced by half the translation of both genes, whereas deletion of the RTD resulted in a 3.6-fold reduction in translation efficiency. This is the first report of translation regulatory elements of plant viruses located within a coding region.

Virus-like particles of potato leafroll virus as potential carrier system for nucleic acids.

Potato leafroll virus is a member of the polerovirus genus. The isometric virion is formed by a coat protein encapsidating single-stranded, positive-sense, mono-partite genomic RNA with covalently attached viral protein at the 5' end. The coat protein of the virus exists in two forms: i) a 23 kDa protein, the product of the coat protein gene, and ii) a 78 kDa protein, the product of the coat protein gene and an additional open reading frame expressed by read-through of the coat protein gene stop codon. The aim of this work was the expression of potato leafroll virus coat protein-based proteins that would be able to assemble into virus-like particles in insect cells. These modified particles were tested for their ability to encapsidate nucleic acids. Two types of N-terminally His-tagged coat protein constructs were used for the expression in insect cells: one, encoding a 23 kDa protein with the C-terminal amino-acid sequence corresponding to the wild type coat protein and the second with additional clathrin binding domain at the C-terminus. The expression of these two proteins by a recombinant baculovirus was characterized by Western immunoblotting with antibodies directed against potato leafroll virus. The protection or putative encapsidation of nucleic acids by these two coat protein derivatives was shown by DNase I and RNase A protection assays.