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Split-spectrum amplitude decorrelation angiography for spectral-domain optical coherence tomography has enabled detailed, non-invasive assessment of vascular flow. This study evaluates choriocapillaris and retinal capillary perfusion density (CPD) in diabetic eyes using optical coherence tomography angiography (OCTA). Records of 136 eyes that underwent OCTA imaging at a single institution were reviewed. Eyes were grouped as non-diabetic controls (37 eyes), patients with diabetes mellitus (DM) without diabetic retinopathy (DM without DR, 31 eyes), non-proliferative diabetic retinopathy (NPDR, 41 eyes) and proliferative diabetic retinopathy (PDR, 27 eyes). Quantitative CPD analyses were performed on OCTA images for assessing perfusion density of the choriocapillaris and retinal plexus for all patients and compared between groups. Eyes with NPDR and PDR showed significantly decreased choriocapillaris CPD compared with controls, while DM eyes without DR did not show significant change. Choriocapillaris whole-image CPD was decreased by 8.3% in eyes with NPDR (p<0.01) and decreased by 7.1% in eyes with PDR (p<0.01). Choriocapillaris parafoveal CPD was decreased by 8.9% in eyes with NPDR (p<0.01) and decreased by 8.2% in eyes with PDR (p<0.01). Compared with controls, only eyes with PDR showed significantly decreased retinal CPD, as well as significantly increased foveal avascular zone (FAZ) area. In those patients, retinal whole-image CPD was decreased by 9.7% (p<0.01), retinal foveal CPD was decreased by 20.5% (p<0.01) and retinal parafoveal CPD was decreased by 11.4% (p<0.01). FAZ area was increased by 50.9% (p<0.01). Choriocapillaris and retinal CPD are reduced in diabetic retinopathy, while FAZ area is increased in eyes with PDR. Vascular changes captured by new imaging modalities can further characterise diabetic choroidopathy.

Autoimmune diseases (AIDs) are complex diseases characterized by persistent or recurrent inflammation, alteration of immune response, and production of specific autoantibodies. It is known that different AIDs share several susceptibility genetic loci. Tumor necrosis factor alpha inducible protein 3 (TNFAIP3) encodes the ubiquitin-modifying enzyme A20, which downregulates inflammation by restricting NF-κB, a transcription factor that regulates expression of various proinflammatory genes. Variants in TNFAIP3 gene have been described as associated with susceptibility to several AIDs. Here, we analyzed two TNFAIP3 polymorphisms in Italian patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and primary Sjogren’s syndrome (pSS), to verify if the genetic variability of TNFAIP3 gene is involved in genetic predisposition to AIDs also in the Italian population. We recruited 313 SLE patients, 256 RA patients, 195 pSS patients, and 236 healthy controls. Genotyping of rs2230926 and rs6920220 in TNFAIP3 gene was performed by an allelic discrimination assay. We carried out a case/control association study and a genotype/phenotype correlation analysis. A higher risk to develop SLE was observed for rs2230926 (P=0.02, OR=1.92). No association was observed between this SNP and the susceptibility to pSS or RA. However, the rs2230926 variant allele seems to confer a higher risk to develop lymphoma in pSS patients, while in RA patients, the presence of RF resulted significantly associated with the variant allele. Regarding the rs6920220 SNP, we observed a significant association of the variant allele with SLE (P=0.03, OR=1.53), pSS (P=0.016, OR=1.69), and RA (P=0.0001, OR=2.35) susceptibility. Furthermore, SLE patients carrying the variant allele showed a higher risk to develop pericarditis, pleurisy, and kidney complications. Our results support the importance of the TNFAIP3 gene variant role in the development of different autoimmune diseases in the Italian population and furtherly confirm a sharing of genetic predisposing factors among these three pathologies.

Optical coherence angiography (OCTA) is a noninvasive technique that has been introduced in recent years to detect ophthalmological pathology. The growing usage of OCTA to detect retinal abnormalities can be attributed to its advantages over the reference-standard fluorescein angiography (FA), although both of these techniques can be used in association. OCTA's advantages include its dye independency, its ability to produce depth-resolved images of retinal and choroidal vessels that yield images of different vascular layers of the retina, and the better delineation of the foveal avascular zone. OCTA's disadvantages include the lack of normalized patient data, artefactual projection issues, and its inability to detect low-flow lesions or pathologic conditions. Different OCTA platforms use unique algorithms to detect microvasculature, which are implemented in both spectral-domain (SD) and swept-source (SS) OCT machines. Microvascular changes in retinal vein occlusions (RVOs) are visible in both the superficial and deep capillary networks of the retina in OCTA. These visualizations include a decrease in foveal and parafoveal vascular densities, non-perfusion areas, capillary engorgement and telangiectasias, vascular tortuosity, microaneurysms, disruption of the foveal perivascular plexus, and formation of collateral vessels. The restricted field of view and inability to show leakage are important limitations associated with the use of OCTA in RVO cases. In this article, we present a brief overview of OCTA and a review of the changes detectable in different slabs by OCTA in RVO cases published in PubMed and Embase.

Background Autophagy has emerged as a key mechanism in the survival and function of T and B lymphocytes, and its activation was involved in apoptosis resistance in rheumatoid arthritis (RA). To investigate whether the relationship between autophagy and apoptosis may impact the response to the therapy, we analyzed ex vivo spontaneous autophagy and apoptosis in patients with RA subjected to treatment with anti-tumor necrosis factor (TNF) drugs and in vitro the effects of TNFα and anti-TNF drugs on cell fate. Methods Peripheral blood mononuclear cells (PBMCs) from 25 RA patients treated with anti-TNF drugs were analyzed for levels of autophagy marker LC3-II by western blot and for the percentage of annexin V-positive apoptotic cells by flow cytometry. The same techniques were used to assess autophagy and apoptosis after in vitro treatment with TNFα and etanercept in both PBMCs and fibroblast-like synoviocytes (FLS) from patients with RA. Results PBMCs from patients with RA responsive to treatment showed a significant reduction in LC3-II levels, associated with an increased apoptotic activation after 4 months of therapy with anti-TNF drugs. Additionally, the expression of LC3-II correlated with DAS28. TNFα was able to induce autophagy in a dose-dependent manner after 24 h of culture in RA PBMCs and FLS. Moreover, etanercept caused a significant reduction of autophagy and of levels of citrullinated proteins. Conclusions Our results show how the crosstalk between autophagy and apoptosis can sustain the survival of immune cells, thus influencing RA progression. This suggests that inhibition of autophagy represents a possible therapeutic target in RA.

To determine the safety and efficacy of intravitreal aflibercept injection (IAI) in patients with diabetic retinopathy (DR) in the prevention of macular edema (ME) following cataract surgery. This phase 2, prospective, interventional, single-masked, randomized trial at a single academic center included 30 patients who were 18 years of age or older with nonproliferative DR and undergoing cataract surgery with phacoemulsification. Patients received 2 mg intravitreal aflibercept (0.05 mL) or sham injection during cataract surgery. Main outcome measures included treatment adverse events (AEs), best-corrected visual acuity (BCVA), and incidence of ME (defined as presence of cystoid abnormalities as detected by optical coherence tomography at any follow-up visit), a 30% or greater increase from preoperative baseline in central subfield macular thickness, or a BCVA decrease of more than 5 ETDRS letters from Day 7 due to retinal thickening. There were similar incidences of AEs between the two groups and no clinically serious ocular AEs in either group. The IAI group had fewer ME events at Day 14 (13% vs. 53%; P = .022), but there was no significant difference in ME events at Day 30 (27% vs. 60%; P = .057), Day 60 (27% vs. 60%; P = .057), or Day 90 (40% vs. 67%; P = .161). Compared to the study group, the control group had a significantly greater increase in central subfield thickness (CST) at Day 30 (50.05 μm vs. 7.95 μm; P = .040) and Day 60 (56.45 μm vs. 3.02 μm; P = .010). However, the difference in CST between groups was no longer significant at Day 90 (50.31 μm vs. 18.48 μm; P = .12). There were no significant differences in BCVA gains between the IAI and sham group at the end of the follow-up period (Day 90, ETDRS letters: 9.88 vs. 8.52; P = .66). Use of IAI in patients with DR for prevention of ME following cataract surgery showed no significant AEs. Although there were significant differences in ME incidence and retinal thickness at periods of time, there was no clinically meaningful benefit in terms of VA. Further larger trials are needed to validate these findings. [Ophthalmic Surg Lasers Imaging Retina. 2020;51:170-178.].

Background:The new classification criteria for antiphospholipid antibody syndrome (APS) have introduced many novelties, such as the presence of an entry criterion, the use of a scoring system, the introduction of previously “extra-criteria” manifestations, the weighting of thrombotic events according to thromboembolic and cardiovascular risk factors and the redefinition of obstetric APS (OAPS).Objectives:The aim of this study is to apply the latest classification criteria to a monocentric cohort of patients with diagnosis of APS and to compare it with the previous criteria.Methods:We retrospectively collected data from patients with clinical diagnosis of APS from the Lupus Clinic of Sapienza University of Rome and applied the new classification criteria and the previous 2006 “Sapporo” criteria.Results:In our cohort of 165 APS patients, 139 could be classified according to Sapporo criteria, while 106 patients fulfilled the new classification criteria. Among the 33 patients that could not be reclassified by the new criteria, 21 patients did not meet clinical domains (8 thrombotic APS patients did not reach enough points due to a high thromboembolic/cardiovascular risk and 13 OAPS patients due to the presence of poliabortivity or fetal death alone), while 18 lacked laboratory domains due to only IgM aPL positivity (Graph 1).Conclusion:In our cohort, the application of the new APS classification criteria identifies a smaller number of patients compared to the previous ones, confirming the reduced sensitivity of the former. The new criteria may lead to the exclusion of patients with specific clinical and laboratory characteristics, that require larger studies to be assessed.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of Interests:None declared.

Background:It is reported that up to 90.4% of individuals diagnosed with Rheumatoid Arthritis (RA) seek medical assistance due to severe pain. The underlying causes of this pain are multifaceted, involving factors such as inflammation, secondary osteoarthritis, as well as central and peripheral sensitization. CGRP (calcitonin gene-related peptide) is a peptide exerting nociceptive and vasodilatory effects and a debated immunomodulatory effect; inflammatory arthropathies have been associated a local increase of CGRP release and CGRP seems to increase IL6 and IL8 secretion from fibroblast-like synoviocytes isolated from RA patients.Objectives:The aim of this prospective pilot study was to determine whether patients with RA have detectable levels of circulating CGRP, to investigate the correlation between CGRP and with pain levels reported by the patients and to assess the effect of baricitinib on pain and CGRP levels.Methods:We enrolled RA patients starting treatment with baricitinib for high-to-moderate active RA. At baseline and after 4 and 12 weeks of treatment we collected clinical data (number of tender and swollen joints), Erytrhosedimentation Rate (ESR), C Reactive Protein (CRP) levels, and patients reported outcomes [Patients Global Assessment (PGA) and pain on a 0-10 cm Visual Analogic Scale (VAS)]. CGRP serum levels were assessed in serum from RA patients at baseline and after 4 and 12 weeks of treatment with baricitinib using an ELISA kit. Data were expressed as mean ± standard deviation or median (IQR) according to distribution. Mann-Whitney and Spearman tests were performed for comparisons and p values < 0.05 were considered statistically significant.Results:We enrolled 43 patients (F:M =36:7, median age=58, IQR 11 years, median disease duration =144, IQR 150) starting baricitinib. We defined responders (R) patients those who achieved at least a EULAR moderate response (1.2 point reduction) of DAS28_CRP from baseline value and not-responders (NR) those who did not. At baseline pain VAS score did not differ significantly between R and NR. Already after 4 weeks of treatment all patients showed a significant reduction of pain (median pain score was 8(2) at baseline, 4(5) and 2(5) after 4 and 12 weeks, respectively; p=0.0014 and p<0.0001 vs baseline, respectively). R patients had lower pain VAS scores after 4 and 12 weeks of follow-up (p=0.0038 and p=0.0026) compared to NR. CGRP was significantly reduced in the whole cohort at 4 (p=0.0016) and 12 weeks (p=0.018) (Figure 1a). NR patients showed higher CGRP serum levels compared to R, after one month of baricitinib. Levels of CGRP significantly correlated with pain VAS (p=0.0152, r=0.22) (Figure 1b) and PGA (p=0.02, r=0.21), but not with ESR, CRP or disease activity (DAS28_CRP).Conclusion:With the same disease activity, we found higher levels of CGRP in active RA patients with higher levels of pain. Interestingly, CGRP was reduced to a greater extent in patients who responded to the JAK inhibitor. This preliminary result sheds light on a possible additional mechanism of baricitinib efficacy, linked to modulation of neurotransmission other than to control of inflammation. On the other hand, in NR patients, other mechanisms underlying pain may contribute to non-response to therapy.REFERENCES:[1] Walsh DA, et al. Nat Rev Rheumatol. Oct 2014;10(10):581-92.[2] Russell FA, et al. Physiol Rev. Oct 2014;94(4):1099-142.[3] Raap T, et al. J Rheumatol. Nov 2000;27(11):2558-65.Figure 1.a. Reduction of serum CGRP in patients treated with baricitinib, from baseline (T0) and after 1 and 3 months of treatment (T1, T3). b. correlation between levels of pain and serum CGRP.Acknowledgements:NIL.Disclosure of Interests:Cristina Garufi: None declared, Silvia Mancuso: None declared, Letizia Caruso: None declared, Fulvia Ceccarelli: None declared, Simona Truglia: None declared, Fabrizio Conti Eli Lilly, Abbvie, UCB, Pfizer, Galapagos, Francesca Romana Spinelli Eli Lilly, Abbvie, UCB, Galapagos

The venous system has been historically understudied despite its critical roles in blood distribution, heart function, and systemic immunity. This study dissects the microanatomy of upper arm veins at the single cell level, and how it relates to wall structure, remodeling processes, and inflammatory responses to injury. We applied single-cell RNA sequencing to 4 non-diseased human veins (3 basilic, 1 cephalic) obtained from organ donors, followed by bioinformatic and histological analyses. Unsupervised clustering of 20,006 cells revealed a complex ecosystem of endothelial cell (EC) types, smooth muscle cell (SMCs) and pericytes, various types of fibroblasts, and immune cell populations. The venous endothelium showed significant upregulation of cell adhesion genes, with arteriovenous zonation EC phenotypes highlighting the heterogeneity of vasa vasorum (VV) microvessels. Venous SMCs had atypical contractile phenotypes and showed widespread localization in the intima and media. MYH11 + DES lo SMCs were transcriptionally associated with negative regulation of contraction and pro-inflammatory gene expression. MYH11 + DES hi SMCs showed significant upregulation of extracellular matrix genes and pro-migratory mediators. Venous fibroblasts ranging from secretory to myofibroblastic phenotypes were 4X more abundant than SMCs and widely distributed throughout the wall. Fibroblast-derived angiopoietin-like factors were identified as versatile signaling hubs to regulate angiogenesis and SMC proliferation. An abundant monocyte/macrophage population was detected and confirmed by histology, including pro-inflammatory and homeostatic phenotypes, with cell counts positively correlated with age. Ligand-receptor interactome networks identified the venous endothelium in the main lumen and the VV as a niche for monocyte recruitment and infiltration. This study underscores the transcriptional uniqueness of venous cells and their relevance for vascular inflammation and remodeling processes. Findings from this study may be relevant for molecular investigations of upper arm veins used for vascular access creation, where single-cell analyses of cell composition and phenotypes are currently lacking.

The pathway of Janus tyrosine kinases (JAKs) has a central role in the pathogenesis of Rheumatoid Arthritis (RA) by regulating multiple immune functions and cytokine production. The JAK inhibitor tofacitinib is effective in RA patients not responding to methotrexate or TNF-inhibitors. Since hyperactive autophagy has been associated with impaired apoptosis of RA fibroblast-like synoviocytes (FLS), we aimed to investigate the role of tofacitinib in modulating autophagy and apoptosis in these cells. FLS isolated from RA biopsies were cultured with tofacitinib in presence of autophagy inducer rapamycin and in serum deprivation condition. Levels of autophagy, apoptosis, and citrullinated proteins were analyzed by western blot, flow cytometry, immunocytofluorescence, and Real-Time PCR. Rapamycin induced an increase in RA-FLS autophagy while the levels of autophagy marker LC3-II were reduced after in vitro treatment with tofacitinib. The analysis of autophagic flux by specific fluorescence dye confirmed the reduction of autophagy in RA FLS. The treatment with tofacitinib did not influence apoptosis of RA FLS. Modulation of the autophagic process by tofacitinib did not significantly change citrullination. The results of this study demonstrate that tofacitinib is able to modulate autophagy of FLS contributing to its effectiveness in RA patients.

Mitochondria are critical for cellular energy production and homeostasis. Oxidative stress and associated mitochondrial dysfunction are integral components of the pathophysiology of retinal diseases, including diabetic retinopathy (DR), age-related macular degeneration, and glaucoma. Within mitochondria, flavoproteins are oxidized and reduced and emit a green autofluorescence when oxidized following blue light excitation. Recently, a noninvasive imaging device was developed to measure retinal flavoprotein fluorescence (FPF). Thus, oxidized FPF can act as a biomarker of mitochondrial dysfunction. This review article describes the literature surrounding mitochondrial FPF imaging in retinal disease. The authors describe the role of mitochondrial dysfunction in retinal diseases, experiments using FPF as a marker of mitochondrial dysfunction in vitro, the three generations of retinal FPF imaging devices, and the peer-reviewed publications that have examined FPF imaging in patients. Finally, the authors report their own study findings. Goals were to establish normative reference levels for FPF intensity and heterogeneity in healthy eyes, to compare between healthy eyes and eyes with diabetes and DR, and to compare across stages of DR. The authors present methods to calculate a patient’s expected FPF values using baseline characteristics. FPF intensity and heterogeneity were elevated in diabetic eyes compared to age-matched control eyes, and in proliferative DR compared to diabetic eyes without retinopathy. In diabetic eyes, higher FPF heterogeneity was associated with poorer visual acuity. In conclusion, while current retinal imaging modalities frequently focus on structural features, functional mitochondrial imaging shows promise as a metabolically targeted tool to evaluate retinal disease.